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Molecular advances in St. Augustinegrass: from DNA markers to genome sequencing

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  • Received: 30 June 2024
    Revised: 09 August 2024
    Accepted: 13 August 2024
    Published online: 26 September 2024
    Grass Research  4 Article number: e021 (2024)  |  Cite this article
  • St. Augustinegrass (Stenotaphrum secundatum (Walt.) Kuntz) is one of the most important warm-season turfgrass species in the United States. Breeding efforts for this turfgrass have primarily focused on improving turf quality and increasing resistance to various biotic and abiotic stresses, including insects, diseases, drought, cold, and shade. While conventional breeding methods have been widely employed in St. Augustinegrass breeding programs, recent years have seen the integration of molecular tools and techniques such as molecular markers, linkage maps, quantitative trait loci (QTL) mapping, comparative genomics, and transcriptomics. Despite these efforts, genomic resources for St. Augustinegrass are still underdeveloped compared to other economically important crops. The recent establishment of a reference genome for St. Augustinegrass is a major milestone, opening new possibilities for genomics-enabled breeding of this important turfgrass. The use of modern genomic tools like genomic selection and marker-assisted selection (MAS) in breeding programs can enhance selection accuracy, shorten breeding cycles, improve trait incorporation, and significantly boost genetic gains, ultimately leading to the development of superior cultivars that meet industry demands. This review highlights recent advancements in genetics and genomics of St. Augustinegrass and identifies areas that require further research to bridge existing knowledge gaps.
  • The competition for consumer preference for fresh apples (Malus domestica) from exotic and tropical fruits is intense. Red-fleshed (RF) apple may not only provide a novel point of differentiation and enhanced visual quality, but also a source of increased concentration of potentially health-benefiting compounds within both the fresh fruit and snack/juice markets[1]. Two different types of RF apples have been characterised: Type 1 RF apple has red colouration not only in the fruit core and cortex, but also in vegetative tissues, including stems and leaves; Type 2 RF apples display red pigment only in the fruit cortex[1, 2]. To facilitate trade and lengthen the supply-window, harvested fruit are usually cold stored, which can induce a series of disorders, including physiological breakdown manifesting as a flesh browning disorder (FBD) in RF apples[3, 4]. FBD in RF apples can be caused by senescence, and there is also some evidence to suggest that a large proportion of RF apples are chilling-sensitive (Jason Johnston, Plant & Food Research Hawke's Bay, personal communication).

    Earlier studies suggested that Type 1 RF colour was determined by a promoter mutation of MdMYB10 that has a tandem replication of a myeloblastosis (MYB) binding cis-element (R6) within the promoter, resulting in autoregulation of MdMYB10[5]. However, van Nocker et al.[6] observed a large variation in the degree and pattern of red pigmentation within the cortex among the accessions carrying MdMYB10, and concluded that the presence of this gene alone was not sufficient to ensure the RF colour. A genome-wide association study (GWAS)[3], reported that, in addition to the MdMYB10 gene, other genetic factors (e.g. MdLAR1, a key enzyme in the flavonoid biosynthetic pathway) were associated with RF colour, too. Wang et al.[7] reported that many of the up-regulated genes in RF apples were associated with flavonoid biosynthesis (e.g., chalcone synthase (CHS), chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), UDP-glucosyltransferase (UGT) and MYB transcription factors). Recently, MdNAC42 was shown to share similar expression patterns in RF fruit with MdMYB10 and MdTTG1, and it interacts with MdMYB10 to participate in the regulation of anthocyanin synthesis in the RF apple Redlove®[8].

    Several transcription factor genes (e.g., MYB, WRKY, bHLH, NAC, ERF, bZIP and HSF) were reported to be differentially expressed during cold-induced morphological and physiological changes in 'Golden Delicious' apples[9]. A study by Zhang et al.[10] showed that ERF1B was capable of interacting with the promotors of anthocyanin and proanthocyanidin (PA) MYB transcription factors, and suggested that ethylene regulation and anthocyanin regulation might be linked in either direction. It was reported that ethylene signal transduction pathway genes or response genes, such as ERS (ethylene response sensor), EIN3 (ethylene-insensitive3) and ERFs (ethylene response factors), may play an important role in the regulatory network of PA biosynthesis[11].

    Espley et al.[12] observed no incidence of FBD in cold-stored fruit of 'Royal Gala', but over-expression of MdMYB10 in 'Royal Gala' resulted in a high rate of FBD in RF fruit, which was hypothesised to be caused by elevated fruit ethylene concentrations before harvest and more anthocyanin, chlorogenic acid (CGA) and pro-cyanidins in RF fruit. In addition, the MYB10 transcription factor was shown to elevate the expression levels of MdACS, MdACO, and MdERF106 ethylene-regulating genes[12]. To elucidate the mechanism regulating the FBD of RF apples, Zuo et al.[13] analysed the transcriptome of tree-ripe apples at 0, 0.5 and 4 h after cutting, and reported that the differentially expressed genes at different sampling points were mainly related to plant–pathogen interactions.

    GWAS is a powerful technique for mining novel functional variants. One of the limitations of GWAS, using SNP arrays, is that they require genotyping of large numbers of individuals, which may be expensive for large populations. DNA pooling-based designs (i.e., bulk segregant analysis) test differential allele distributions in pools of individuals that exhibit extreme phenotypes (XP) in bi-parental populations, large germplasm collections or random mating populations[1416]. In addition to reducing the number of samples to be genotyped, the use of whole genome sequencing (WGS)-based XP-GWAS has the potential to identify small-effect loci and rare alleles via extreme phenotypic selection.

    In this WGS-based XP-GWAS, we investigated the genetic basis of RF and FBD by sequencing the pools of individuals that exhibited extreme phenotypes for these two traits, and analysed the differences in allele frequencies between phenotypic classes. This method combines the simplicity of genotyping pools with superior mapping resolution. We also examined the transcriptome from transgenic apple fruit harbouring the R6:MYB10 promoter as a model for red flesh in apple. Differences in gene expression of a highly pigmented line were compared with expression in control fruit and these genes were then used for comparison with the seedling population. Understanding the genetic basis of the link between RF and FBD will help in design of strategies for selection against FBD in high-quality Type 1 RF apple cultivars.

    A snapshot of visual variation in FBD and WCI is presented in Fig. 1. The average WCI and FBD across all ~900 seedlings ranged from 0 to 7, and from 0% to 58%, respectively. Based on the MLM analysis, the estimated narrow-sense heritability (h2) of WCI and FBD was 0.57 (standard error = 0.18) and 0.09 (standard error = 0.05), respectively. The estimated genetic correlation between WCI and FBD was 0.58, and several fruit quality traits displayed unfavourable correlations with WCI (Supplemental Fig. S1). Seedlings with higher WCI scores were generally characterised by poor firmness and crispness, plus higher astringency and sourness. Estimated phenotypic and genetic correlations between all pairs of traits are listed in Supplemental Table S1.

    Figure 1.  Transverse cross-sections of apple slices showing range in (a) flesh colouration, and (b) flesh browning disorder for Type 1 red-fleshed apple. The weighted cortical intensity (WCI) scores (0−9 scale) and the proportion of the cortex area showing symptoms of flesh browning disorder are also displayed.

    A few seedlings had no red pigment in the cortex, but the average WCI score across all seedlings was 2.25. About two-thirds of the seedlings did not display any FBD symptoms, but among the remaining seedlings, FBD ranged between 1% and 58% (Fig. 2). The average WCI score for the 'low' and 'high' WCI pool was 0.45 and 5.2, respectively, while the average FBD was 0% and 20.6% for the 'low' and 'high' FBD pool, respectively (Supplemental Table S2). The average WCI score of the seedlings in the FBD pools was similar (Low: 5.5; High: 4.6), while the average FBD of the high- and low-WCI pools was 4.5% and 0.1%, respectively.

    Figure 2.  The distribution of weighted cortex intensity (WCI) scores (a) and the internal flesh browning disorder IFBD%; (b) in the population of ~900 apple seedlings. The green and red circles highlight the individuals used to form the 'low' and 'high' pools of samples.

    After filtering, about 204,000 SNPs were used and the average sequencing depth of SNP loci was similar for the two pools (42 vs 44). There was a near-perfect correlation between the Z-test statistics and G-statistics, so only the latter are discussed hereafter. A plot of the G' values, smoothed over 2 Mb windows, is shown for all 17 chromosomes (Chrs) in Supplemental Fig. S2. XP-GWAS identified genomic regions significantly associated with FBD on 12 out of the 17 Chrs (Fig. 3), and putative candidate genes within ±1.0 Mb distanceof the significant G' peaks were identified (Table 1). Additional genomic regions, which did not meet the significance threshold but displayed distinguished G' peaks, were also identified across all chromosomes (Supplemental Table S3).

    Figure 3.  G'-statistics across the linkage groups (LG) showing significant association with the flesh browning disorder (FBD) in apple The horizontal red lines indicate the significance threshold. The putative candidate genes (refer to Table 1) underpinning various G' peaks are also shown.
    Table 1.  A list of the genomic regions associated with internal flesh browning disorder (FBD) in apples. Putative candidate genes residing within these regions are also listed using GDDH13v1.1 reference genome assembly.
    ChrGenomic region (Mb)Putative genes functions
    221.9–23.5Ethylene-responsive element binding factor 13 (MdERF13: MD02G1213600);
    33.1–4.9cinnamate 4-hydroxylase (C4H) enzyme (MD03G1051100, MD03G1050900 and MD03G1051000); MdWRKY2: MD03G1044400; MdWRKY33 (MD03G1057400)
    38.2–9.6ascorbate peroxidase 1 (MdAPX1: MD03G1108200, MD03G1108300)
    314.7–16.6senescence-related MdNAC90 (MD03G1148500)
    336.5–37.5Ethylene response sensor 1 (MdERS1: MD03G1292200); flavonoid biosynthesis protein MdMYB12 (MD03G1297100); Heat shock protein DnaJ (MD03G1296600, MD03G1297000); pectin methylesterase (MdPME) inhibitor protein (MD03G1290800, MD03G1290900, MD03G1291000).
    411.0–13.0phenylalanine and lignin biosynthesis protein MdMYB85 (MD04G1080600)
    422.1–24.4MYB domain protein 1 (MD04G1142200); HSP20-like protein (MD04G1140600); UDP-glucosyltransferase (UGT) proteins UGT85A7 (MD04G1140700, MD04G1140900); UGT85A3 (MD04G1140800); UGT (MD04G1141000, MD04G1141300); UGT85A2 (MD04G1141400); UGT85A4 (MD04G1141500); DNAJ heat shock protein (MD04G1153800, MD04G1153900, MD04G1154100)
    427.6–29.6HCT/HQT regulatory genes MD04G1188000 and MD04G1188400
    629.8–31.7Volz et al. (2013) QTL for IFBD; anthocyanin regulatory proteins MdMYB86 (MD06G1167200); triterpene biosynthesis transcription factor MdMYB66 (MD06G1174200); Cytochrome P450 (MD06G1162600; MD06G1162700, MD06G1162800, MD06G1163100, MD06G1163300, MD06G1163400, MD06G1163500, MD06G1163600, MD06G1163800, MD06G1164000, MD06G1164100, MD06G1164300, MD06G1164400, MD06G1164500, MD06G1164700)
    713.9–15.9heat shock protein 70B (MD07G1116300)
    717.4–19.0Drought-stress WRKY DNA-binding proteins (MdWRKY56: MD07G1131000, MD07G1131400)
    723.6–25.2MdPAL2 (MD07G1172700); drought-stress gene NGA1 (MD07G1162400); DNAJ heat shock family protein (MD07G1162300, MD07G1162200), stress-response protein (MdNAC69: MD07G1163700, MD07G1164000)
    97.9–11.8MD09G1110500 involved in ascorbate oxidase (AO); MdUGT proteins (MD09G1141200, MD09G1141300, MD09G1141500, MD09G1141600, MD09G1141700, MD09G1141800, MD09G1142000, MD09G1142500, MD09G1142600, MD09G1142800, MD09G1142900, MD09G1143000, MD09G1143200, MD09G1143400) involved in flavonoids biosynthesis; heat shock proteins 89.1 (MD09G1122200) and HSP70 (MD09G1137300); Ethylene-forming enzyme MD09G1114800; Anthocyanin regulatory protein MdNAC42 (MD09G1147500, MD09G1147600)
    914.9–16.5triterpene biosynthesis transcription factor protein MdMYB66 (MD09G1183800);
    111.7–3.0ethylene response factor proteins (MdEIN-like 3: MD11G1022400)
    1138.6–40.6Senescence-related gene 1 (MD11G1271400, MD11G1272300, MD11G1272000, MD11G1272100, MD11G1272300, MD11G1272400, and MD11G1272500); Chalcone-flavanone isomerase (CHI) protein (MD11G1273600) and MdbHLH3 (MD11G1286900, MDP0000225680); cytochrome P450 enzyme (MD11G1274000, MD11G1274100, MD11G1274200, MD11G1274300, MD11G1274500, and MD11G1274600); heat shock transcription factor A6B (MdHSFA6B: MD11G1278900) – involved in ABA-mediated heat response and flavonoid biosynthesis.
    122.2–3.5heat shock protein 70-1 ((MD12G1025600, MD12G1025700 and MD12G1026300) and heat shock protein 70 (MD12G1025800 and MD12G1025900 and MD12G1026000); ethylene (MD12G1032000) and auxin-responsive (MD12G1027600) proteins.
    127.2–8.4DNAJ heat shock domain-containing protein (MD12G1065200 and MD12G1067400)
    1327.5–30.5MD13G1257800 involved in polyphenol 4-coumarate:CoA ligase (4CL)
    1337.5–39.5pectin methyl esterase inhibitor superfamily protein MdPMEI (MD13G1278600)
    1425.4–27.5Drought-stress response gene MdWRKY45 (MD14G1154500); chalcone synthase (CHS) family proteins (MD14G1160800 and MD14G1160900); triterpene biosynthesis transcription factor MdMYB66 (MD14G1180700, MD14G1181000, MD14G1180900); anthocyanin biosynthesis protein (MdMYB86: MD14G1172900); cytochrome P450 proteins (MD14G1169000, MD14G1169200, MD14G1169600, MD14G1169700)
    150–1.5Ethylene synthesis proteins (MD15G1020100, MD15G1020300 and MD15G1020500); dihydroflavonol reductase (DFR) gene (MD15G1024100)
    154.6–6.8MdMYB73 (MD15G1076600, MD15G1088000) modulates malate transportation/accumulation via interaction with MdMYB1/10; MdNAC52: MD15G1079400) regulates anthocyanin/PA; heat shock transcription factor B4 (MD15G1080700); stress-response protein MdWRKY7 (MD15G1078200)
    1553.5–54.9MdC3H (MD15G1436500) involved in chlorogenic acid biosynthesis; MdEIN3 (MD15G1441000) involved in regulating ethylene synthesis and anthocyanin accumulation
    168.9–10.9SAUR-like auxin-responsive protein (MD16G1124300) and MdNAC83: (MD16G1125800) associated with fruit ripening; MD16G1140800 regulates proanthocyanidin; MdPAE genes (MD16G1132100, MD16G1140500) regulates ethylene production.
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    The ethylene-responsive factor 13 (MdERF13: MD02G1213600) resided within the significant region (21.9–23.5 Mb) on Chr2, while the ascorbate peroxidase 3 (MdAPX3: MD02G1127800, MD02G1132200) resided in the prominent region between 9.45 and 11.28 Mb) (Fig. 3, Table 1). There were several genomic regions showing association with FBD on Chr3. The first region (8.2–9.5 Mb) flanked MdAPX1 (MD03G1108200, MD03G1108300), while MdERF3 (MD03G1194300) and heat shock protein 70 (HSP70: MD03G1201800, D03G1201700) resided within the prominent peak region (25.5–27.5 Mb). Another significant region (36.5–37.5 Mb) at the bottom on Chr3 harboured ethylene response sensor 1 (MdERS1: MD03G1292200), a flavonoid-biosynthesis related protein MdMYB12 (MD03G1297100), HSP DnaJ and pectin methyl esterase (PME) inhibitor proteins (Fig. 3, Table 1).

    The significant G' region (27.6–29.6 Mb) on Chr4 flanked the genes MD04G1188000 and MD04G1188400 involved in the biosynthesis of hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase (HCT/HQT), while the adjacent region (22.1–24.3 Mb) harboured a cluster of UDP-glucosyltransferase (UGT) proteins and HSP (Table 1). Another region between 11.0 and 13.0 Mb encompassed phenylalanine and lignin biosynthesis gene MYB85 (MD04G1080600)[17]. The only significant region associated with FBD on Chr6 spanned between 29.8 and 31.7 Mb, which included a SNP earlier reported associated with FBD[4]. This region also harboured several MYB proteins (MdMYB86: MD06G1167200; MdMYB98: MD06G1172900; MdMYB66: MD06G1174200) and a large cluster of cytochrome P450 proteins (Table 1).

    A significant FBD-associated region (13.9–15.9 Mb) on Chr7 encompassed the HSP 70B (MD07G1116300), while another significant region between 23.6 Mb and 25.2 Mb flanked the gene coding for phenylalanine ammonia lyase 2 (MdPAL2: MD07G1172700), a drought stress gene NGA1 (MD07G1162400), DNAJ HSP, and stress-response protein (MdNAC69: MD07G1163700, MD07G1164000) (Fig. 3, Table 1). A large significant region spanning between 7.9 and 11.8 Mb on Chr9 encompassed the gene MD09G1110500 putatively involved in ascorbate oxidase (AO), HSP (HSP70: MD09G1137300; HSP89.1: MD09G1122200), an ethylene-forming enzyme (MD09G1114800), and MdNAC42 (MD09G1147500, MD09G1147600). Another significant genomic region on Chr9 was between 14.9 and 16.5 Mb, which harbours the MYB domain protein MdMYB66 (MD09G1183800) (Table 1).

    A sharp G' peak region (1.7–3.0 Mb) on Chr11 associated with FBD encompassed ethylene insensitive 3 (MdEIN3: MD11G1022400) along with a cluster of UGT proteins, WD-40, and bHLHL proteins (Table 1). A significant region between 38.6 and 40.6 Mb at the bottom of Chr11 was dominated by clusters of senescence-related genes and cytochrome P450 enzymes. This genomic region also flanked a chalcone-flavanone isomerase (CHI) family protein (MD11G1273600) and a bHLH protein (MdbHLH3: MD11G1286900, MDP0000225680), along with the heat shock transcription factor A6B (HSFA6B: MD11G1278900) (Table 1, Supplemental Table S3).

    The region (2.2–3.3 Mb) associated with FBD on Chr12 flanked genes for the HSP 70 and 70-1, NAC proteins, ethylene and auxin-responsive proteins (Table 1). An adjacent significant region (7.2–8.4 Mb) flanked DNAJ HSP, along with bZIP, bHLH and WD-40 repeat-like proteins (Table 1; Supplemental Table S3). There was a large genomic region on Chr13 showing a significant association with FBD. In this region, the first G' peak (27.5–30.5 Mb) harboured MD13G1257800, which regulates polyphenol 4-coumarate: CoA ligase (4CL) synthesis. The second G' peak region (32.6–34.7 Mb) corresponded to an earlier mapped QTL for flesh browning in white-fleshed apples[18].

    The significant region (25.4–27.5 Mb) on Chr14 harboured various genes for proteins with different putative functions, such as AP2 proteins, WD-40 repeat family proteins, bHLH proteins, MdNAC83 (MD14G1150900), MdWRKY45 (MD14G1154500), chalcone synthase (CHS) family proteins (MD14G1160800 and MD14G1160900), cytochrome P450 proteins, and several MYB domain proteins (MdMYB86: MD14G1172900; MdMYB98: MD14G1179000; MdMYB66: MD14G1180700, MD14G1181000, MD14G1180900) (Fig. 3, Table 1, Supplemental Table S3). A sharp G' peak (15.2–17.2 Mb) on Chr14 did not meet the significance threshold corresponding to the FBD QTL in white-fleshed apples[18].

    The upper 1.5 Mb region on Chr15 associated with FBD encompassed several transcription factor families, including WD-40 repeats, bHLH, bZIP, ethylene synthesis proteins, and dihydroflavonol reductase (DFR) protein (MD15G1024100) (Fig. 3, Table 1; Supplemental Table S3). Another significant region on Chr15 (4.6−6.8 Mb) harboured HSF B4, MdMYB73 (which interacts with MdMYB1/10 to modulate malate transportation) and MdNAC52 (MD15G1079400), which regulates anthocyanin and PA synthesis by directly regulating MdLAR[19]. A significantly associated region at the bottom of Chr15 (53.5–54.9 Mb) harboured MdNAC35 (MD15G1444700), MdC3H (MD15G1436500) involved in the production of p-coumarate 3-hydroxylase (C3H) enzyme, which plays a role in chlorogenic acid biosynthesis, and MdEIN3 (MD15G1441000).

    The significant region between 8.9 and 10.9 Mb on Chr16 harboured a gene for SAUR-like auxin-responsive protein (MD16G1124300) and MdNAC83 (MD16G1125800), both of which have been reportedly associated with apple fruit ripening[20]. This region also encompassed MD16G1140800, which regulates PA[11], and a gene for the pectin acetyl esterase protein MdPAE10 (MD16G1132100) involved in ethylene production and shelf-life[21]. A sharp G' peak region (20.4–22.4 Mb) in the middle of Chr16 flanked MdERF1B (MD16G1216900) and MdMYB15 (MD16G1218000 and MD16G1218900), involved in altering anthocyanin and PA concentrations[10] (Fig. 3, Table 1, Supplemental Table S3).

    The average sequencing depth of the SNP loci (~160,000) retained for marker-trait association was similar for the two WCI pools (41 vs 44). The significant regions were located on Chrs 2, 4, 6, 7, 10, 15 and 16 (Fig. 4). The genomic intervals within ±1.0 Mb of the significant G' peaks, and the putative candidate genes within those intervals, are listed in Table 2. Additional genomic regions, which did not meet the significance threshold but displayed distinct G' peaks, were also identified across most chromosomes ( Supplemental Fig. S2, Supplemental Table S3). A significant region on Chr4 encompassed chalcone synthase (CHS) genes, along with an ERF (MD04G1009000) involved in regulating PA biosynthesis[11].

    Figure 4.  G’-statistics across the linkage groups (LG) showing significant association with the weighted cortex intensity (WCI) in apple. The horizontal red lines indicate the significance threshold. The putative candidate genes (refer to Table 2) underpinning various G' peaks are also shown.
    Table 2.  A list of the genomic regions significantly associated with the weighted cortex intensity (WCI) in apples. Putative candidate genes residing within these regions are also listed using GDDH13v1.1 reference genome assembly.
    ChrGenomic region (Mb)Putative genes functions
    211.2–13.2UDP-glucosyltransferase (UGT) proteins (MD02G1153000, MD02G1153100, MD02G1153200, MD02G1153300; MD02G1153400; MD02G1153500; MD02G1153700; MD02G1153800; MD02G1153900);
    40–1.2Chalcone synthase (CHS) genes (MD04G1003000; MD04G1003300 and MD04G1003400); DNAJ heat shock protein (MD04G1003500); MdERF (MD04G1009000) involved in regulating PA biosynthesis.
    69.5–11.0Ubiquitin protein (MD06G1061100); stress-response WRKY protein MdWRKY21 (MD06G1062800);
    612.1–13.6pectin methylesterase inhibitor superfamily protein (MdPME: MD06G1064700); phenylalanine and lignin biosynthesis gene (MdMYB85)
    616.0–17.6MD06G1071600 (MDP0000360447) involved in leucoanthocyanidin dioxygenase (LDOX) synthesis
    624.0–26.0UDP-glycosyltransferase proteins (MD06G1103300, MD06G1103400, MD06G1103500 and MD06G1103600); auxin response factor 9 (MdARF9: MD06G1111100) and heat shock protein 70 (Hsp 70; MD06G1113000).
    74.1–5.6Ethylene insensitive 3 protein (MdEIN3: MD07G1053500 and MD07G1053800) involved in proanthocyanidins (PA) biosynthesis; ubiquitin-specific protease (MD07G1051000, MD07G1051100, MD07G1051200, MD07G1051500, MD07G1051300, MD07G1051700 and MD07G1051800).
    1015.9–18.3bHLH proteins (MD10G1098900, MD10G1104300, and MD10G1104600); UGT protein (MD10G1101200); UGT 74D1 (MD10G1110800), UGT 74F1 (MD10G1111100), UGT 74F2 (MD10G1111000).
    1035.6–38.6Stress-response WRKY proteins (MdWRKY28: MD10G1266400; MdWRKY65: MD10G1275800). ethylene responsive factors (MdERF2: MD10G1286300; MdERF4: MD10G1290400, and MdERF12: MD10G1290900) and a NAC domain protein (MdNAC73: MD10G1288300); polyphenol oxidase (PPO) genes ((MD10G1298200; MD10G1298300; MD10G1298400; MD10G1298500; MD10G1298700; MD10G1299100; MD10G1299300; MD10G1299400).
    1524.8–26.8heat shock factor 4 (MD15G1283700), drought-stress response WRKY protein 7 (MdWRKY7: MD15G1287300), MdMYB73 (MD15G1288600) involved in ubiquitination and malate synthesis
    1531.7–34.2MYB domain protein 93 (MdMYB93: MD15G1323500) regulates flavonoids and suberin accumulation (Legay et al. 2016); ubiquitin -specific protease 3 (MD15G1318500),
    161.5–3.4MYB domain proteins (MD16G1029400) regulates anthocyanin; senescence-associated gene 12 (MD16G1031600); ethylene response factor proteins (MdERF118: MD16G1043500, and MD16G1047700 (MdRAV1); MdMYB62 ( MD16G1040800) flavonol regulation; malate transporter MdMa2 (MD16G1045000: MDP0000244249), Ubiquitin-like superfamily protein (MD16G1036000),
    165.4–7.4MdMYB88 (MD16G1076100) regulates phenylpropanoid synthesis and ABA-mediated anthocyanin biosynthesis; MdMYB66 (MD16G1093200) regulates triterpene biosynthesis, MdWRKY72 (MD16G1077700) mediates ultraviolet B-induced anthocyanin synthesis.
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    There were distinct G' peaks within a large genomic region (spanning between 9.0 and 18.0 Mb) significantly associated with WCI on Chr6 (Fig. 4). The G' peak region 12.1–13.6 Mb encompassed MdMYB85 (MD06G1064300), and a gene for a pectin methyl esterase

    (PME) inhibitor protein (MD06G1064700); while the region between 16.0 and 17.6 Mb flanked the genes involved in leucoanthocyanidin dioxygenase (LDOX) synthesis (Table 2). The auxin response factor 9 (MdARF9: MD06G1111100) and the HSP70 (MD06G1113000) resided in the significant region between 24.0 and 26.0 Mb on Chr6 (Table 2).

    The genomic region (4.1–5.6 Mb) with significant association with WCI on Chr7 flanked MdEIN3 (MD07G1053500, MD07G1053800), which plays an important role in the regulatory network of PA biosynthesis[11]. A significant region (35.6–38.7 Mb) on Chr10 encompassed gene clusters for bHLH and WRKY proteins along with an ethylene repressor factor (MdERF2: MD10G1286300) (Fig. 4, Table 2; Supplemental Table S3). This region also harboured MdERF4 (MD10G1290400), MdERF12 (MD10G1290900) and NAC domain genes (MdNAC73: MD10G1288300), which have been reported to be associated with fruit ripening[20]. In addition, there was a cluster of polyphenol oxidase (PPO) genes residing in this region (Table 2).

    On Chr15, the significant WCI-associated region spanning between 31.7 Mb and 34.2 Mb encompassed several TF families, including MdMYB93 (MD15G1323500) (Fig. 4, Table 2, Supplemental Table S3). A distinguished G' peak region (24.9–26.9 Mb) on Chr15 harboured genes for WD-40 repeat-like proteins, bHLH proteins, redox responsive transcription factor 1 (MD15G1283200), cytochrome P450 proteins, HSF4 (MD15G1283700), and MdWRKY7 (MD15G1287300). The MdMYB73 (MD15G1288600) residing in this region has been shown to interact with MdMYB1/10 and regulates several functions, including cold-stress response, ubiquitination and malate synthesis[22, 23].

    The significant 1.5–3.4 Mb region on Chr16 flanked an anthocyanin repressor MYB protein (MD16G1029400), senescence-associated gene 12 (MD16G1031600), malate transporter MdMa2 (MD16G1045000: MDP0000244249), and two ethylene response factor genes (MdERF118: MD16G1043500; MdRAV1: MD16G1047700), which interact in retaining flesh firmness[24]. The MdMYB62 (MD16G1040800) gene residing in this region is phylogenetically linked to MdMYB8 (MD06G1217200), which plays a major role in flavonoid biosynthesis[25]. Another significant region (5.4–7.4 Mb) on Chr16 harboured several bHLH genes, including MdMYB88 (MD16G1076100) involved in phenylpropanoid synthesis resulting in drought resistance[26] and ABA-mediated anthocyanin production[27]. The MdMYB66 (MD16G1093200) gene, which is involved in triterpene biosynthesis, and the MdWRKY72 (MD16G1077700) gene involved in anthocyanin synthesis, were also present in this region (Table 2, Fig. 4).

    The genomic regions tagged by XP-GWAS were enriched for regulatory functions, and several of these have a paralog (Table 3). For example, paralogues for a trio of genes (MdMYB86, MdMYB98 and Cytochrome 450) resided in the FBD-associated genomic regions on Chr6 and Chr14. Several pairs of genes resided together in the FBD-associated paralog regions; for example, MdNAC90 and germin-like protein 10 on Chr3 and Chr11; WRKY55 and WRKY70 on Chr1 and Chr7; and ERF1 and ERF5 on Chr4 and Chr6. The ethylene response factor MdERF1B had a paralog in the FBD-associated region on Chr13 and Chr16. Interestingly, three copies of MdNAC83 (Chrs 14, 16 17) and four copies of MdMYB66 (Chrs 4, 6, 9 and 14) resided in the FBD-associated regions. A pair of genes (RAV1 and MYB62) resided together in the WCI-associated paralog regions on Chrs13 and 16 (Table 3).

    Table 3.  A list of homologues genes/genomic regions associated with the internal flesh browning disorder (IFBD) and red flesh (WCI) in apples. Putative candidate genes residing within these regions are also listed using GDDH13v1.1 reference genome assembly.
    TraitChr (genomic region: Mb)Gene namePredicted gene IDPutative function
    IFBDChr6 (29.8–31.7 Mb)MdMYB66MD06G1174200Suberin/triterpene deposition
    IFBDChr14 (25.4–27.5 Mb)MdMYB66MD14G1180700, MD14G1181000, MD14G1180900Suberin/triterpene deposition
    IFBDChr4 (7.0–8.1 Mb)MdMYB66MD04G1060200Suberin/triterpene deposition
    IFBDChr9 (14.9–16.5 Mb)MdMYB66MD09G1183800Suberin/triterpene deposition
    IFBDChr6 (29.8–31.7 Mb)MdMYB86MD06G1167200Anthocyanin regulation
    IFBDChr14 (25.4–27.5 Mb)MdMYB86MD14G1172900Anthocyanin regulation
    IFBDChr6 (29.8–31.7 Mb)MdMYB98MD06G1172900Drought stress response
    IFBDChr14 (25.4–27.5 Mb)MdMYB98MD14G1179000Drought stress response
    IFBDChr6 (29.8–31.7 Mb)Cytochrome P450MD06G1162600, MD06G1162700, MD06G1162800, MD06G1163100, MD06G1163300, MD06G1163400, MD06G1163500, MD06G1163600, MD06G1163800, MD06G1164000, MD06G1164100, MD06G1164300, MD06G1164400, MD06G1164500, MD06G1164700Flavonoid and triterpenic metabolism
    IFBDChr14 (25.4–27.5 Mb)Cytochrome P450MD14G1169000, MD14G1169200, MD14G1169600, MD14G1169700Flavonoid and triterpenic metabolism
    IFBDChr3 (14.7–16.6 Mb)MdNAC90MD03G1148500Senescence-related
    IFBDChr11 (16.5–18.5 Mb)MdNAC90MD11G11679000Senescence-related
    IFBDChr14 (25.4–27.5 Mb)MdNAC83MD14G1150900Senescence/ripening-related
    IFBDChr16 (8.9–10.9 Mb)MdNAC83MD16G1125800Senescence/ripening-related
    IFBDChr17 (0–0. 7 Mb)MdNAC83MD17G1010300Senescence/ripening-related
    IFBDChr3 (14.7–16.6 Mb)Germin-like protein 10MD03G1148000Polyphenol oxidase
    IFBDChr11 (16.5–18.5 Mb)Germin-like protein 10MD11G1167000, MD11G1167100, MD11G1167400, MD11G1169200Polyphenol oxidase
    IFBDChr1 (26.9–28.5 Mb)MdWRKY55MD01G1168500Drought stress response
    IFBDChr7 (30.3–31.9 Mb)MdWRKY55MD07G1234600Drought stress response
    IFBDChr1 (26.9–28.5 Mb)MdWRKY70MD01G1168600Drought stress response
    IFBDChr7 (30.3–31.9 Mb)MdWRKY70MD07G1234700Drought stress response
    IFBDChr4 (7.0–8.1 Mb)MdERF1MD04G1058000Ethylene responsive factor
    IFBDChr6 (5.5–7.3 Mb)MdERF1MD06G1051800Ethylene responsive factor
    IFBDChr4 (7.0–8.1 Mb)MdERF5MD04G1058200Ethylene responsive factor
    IFBDChr6 (5.5–7.3 Mb)MdERF5MD06G1051900Ethylene responsive factor
    IFBDChr13 (18.0–20.0 Mb)MdERF1BMD13G1213100Ethylene response factor 1
    IFBDChr16 (20.4–22.4 Mb)MdERF1BMD16G1216900Ethylene response factor 1
    WCIChr13 (2.6–4.4 Mb)MdRAV1MD13G1046100Ethylene responsive factor
    WCIChr16 (1.5–3.4 Mb)MdRAV1MD16G1047700Ethylene responsive factor
    WCIChr13 (2.6–4.4 Mb)MdMYB62MD13G1039900Flavonol biosynthesis
    WCIChr16 (1.5–3.4 Mb)MdMYB62MD16G1040800Flavonol biosynthesis
    IFBDChr9 (7.9–11.8 Mb)MdNAC42MD09G1147500, MD09G1147600Anthocyanin accumulation
    WCIChr17 (11.4–12.4 Mb)MdNAC42MD17G1134400Anthocyanin accumulation
    IFBDChr9 (7.9–11.8 Mb)HSP 70MD09G1137300Heat stress response
    WCIChr17 (11.4–12.4 Mb)HSP 70MD17G1127600Heat stress response
    IFBDChr4 (11.0–13.0 Mb)MdMYB85MD04G1080600Phenylalanine and lignin biosynthesis
    WCIChr6 (12.1–13.6 Mb)MdMYB85MD06G1064300Phenylalanine and lignin biosynthesis
    IFBDChr15 (13.2–14.2 Mb)MdEBF1MD15G1171800Ethylene inhibition
    WCIChr8 (15.2–17.2 Mb)MdEBF1MD08G1150200Ethylene inhibition
    IFBDChr15 (53.5–54.9 Mb)MdEIN3MD15G1441000Ethylene insensitive 3 protein
    WCIChr8 (30.3–31.6 Mb)MdEIN3MD08G1245800Ethylene insensitive 3 protein
    IFBDChr11 (1.7–3.0 Mb)MdEIN3MD11G1022400Ethylene insensitive 3 protein
    WCIChr7 (4.1–5.6 Mb)MdEIN3MD07G1053500, MD07G1053800Ethylene insensitive 3 protein
    IFBDChr15 (4.6–6.8 Mb)MdMYB73MD15G1076600, MD15G1088000Cold-stress response & malate accumulation
    WCIChr15 (24.8–26.8 Mb)MdMYB73MD15G1288600Cold-stress response & malate accumulation
    IFBDChr15 (4.6–6.8M b)MdWRKY7MD15G1078200Anthocyanin accumulation
    WCIChr15 (24.8–26.8 Mb)MdWRKY7MD15G1287300Anthocyanin accumulation
    IFBDChr15 (43.5–45.5 Mb)MdMYB93MD15G1369700Flavonoid & suberin accumulation
    WCIChr15 (31.7–34.2 Mb)MdMYB93MD15G1323500Flavonoid & suberin accumulation
     | Show Table
    DownLoad: CSV

    Paralogs of several regulatory functions were also found in the regions associated with either FBD or WCI; for example, a significant region (7.9–11.8 Mb) harbouring a gene trio (MdNAC42, HSP70 and HSP89.1) on Chr9 was associated with FBD, but the paralogs of this trio also resided in a distinct G' region associated with WCI on Chr17 (Table 3, Supplemental Table S3). Some other examples included MdMYB85 (Chr4 for FBD, and Chr6 for WCI), MdEBF1 (Chr8 for WCI, and Chr15 for FBD), MdEIN3 (Chr8 for WCI, and Chr15 for FBD), and EIN3 (Chr7 for WCI, and Chr11 for FBD). A pair of genes (MdMYB73 and MdWRKY7) resided together in the separate regions associated with FBD (4.6–6.8 Mb) and WCI (24.8–26.8 Mb) within Chr15 (Table 3).

    The red pigmentation in fruit flesh differed amongst transgenic lines, with fruit from line A10 presenting the most deeply pigmented tissues (Supplemental Fig. S3), while those from lines A2 and A4 were similar in having a lower intensity of pigmentation. No pigmentation was observed in the flesh of control 'Royal Gala' (RG) fruit. RNAseq analysis of a representative transgenic line (A2) compared with RG revealed that a total of 1,379 genes were differentially expressed (log 2-fold), with 658 genes upregulated and 721 genes downregulated. This list was then assessed for commonality with the genomic regions and candidate genes from the XP-GWAS.

    Genes that contained mis-sense SNPs (which would result in a change in predicted protein) and that were also differentially expressed between A2 red-fleshed transgenic line and white-fleshed 'Royal Gala' (control) apples included anthocyanin-related flavanone 3-hydroxylase, chalcone synthase and dihydroflavonol 4-reductase (Table 2 & 4). Many more upstream DNA variants (e.g. in potential promoter-controlling elements) were seen in this group of differentially expressed genes (DEGs) that were also in regions underlying WCI or FBD. Genes encoding enzymes that may be involved in FBD, such as a Rho GTPase activating protein, peroxidase, lipoxygenase 1, and ethylene-forming enzyme (ACO4), were DEGs and showed mis-sense SNPs, including a potential stop codon in the Rho GTPase-activating protein (Table 4). This intersection between DNA change and differential expression warrants further research to evaluate the functions of these genes.

    Table 4.  List of candidate genes associated with WCI or FBD in the XP-GWA and R6:MdMYB10 apple datasets.
    Mutations in GWAS apple populationPredicted gene functionExpression in R6 and ‘Royal Gala’ apples
    GeneMissense
    SNPs
    SNP
    Stop
    Upstream
    variants
    DatasetLocusAnnotation and TAIR IDAverage
    RPKM
    R6 flesh
    Average RPKM
    WT flesh
    log2Fold
    Change
    MD02G11322001FBD Supplemental Table S3Chr02:9450615-11289014flavanone 3-hydroxylase
    (F3H, TT6, F3'H) AT3G51240
    387752.47
    MD02G11336003FBD Supplemental Table S3Chr02:9450615-11289014fatty acid desaturase 5
    (FAD5) AT3G15850
    18109.98
    MD02G11537001WCI Table 2Chr02:11278254-13234556UDP-Glycosyltransferase, lignin related AT2G1856010385341.03
    MD03G10592002FBD Supplemental Table S3Chr03:3177994-4910866Peroxidase AT5G053402514.21
    MD03G1143300422FBD Table 1Chr03:14797661-16611554bZIP transcription factor (DPBF2, AtbZIP67) AT3G44460114852-2.80
    MD03G1147700121FBD Table 1Chr03:14797661-16611554Rho GTPase activating protein AT5G22400205891.29
    MD04G1003400331WCI Table 2Chr04:1-1284894Chalcone synthase (CHS, TT4) AT5G1393014433122.34
    MD04G120410063FBD Table 1Chr04:27629536-29612282lipoxygenase 1 (LOX1, ATLOX1) AT1G550205462681.09
    MD06G1160700129FBD Table 1Chr06:29862341-31737341peptide met sulfoxide reductase AT4G251302121254562.04
    MD06G116140014FBD Table 1Chr06:29862341-31737341Pectin lyase-like protein AT5G63180614129530-2.22
    MD07G1240700126FBD Supplemental Table S3Chr07:30357332-31979025Fe superoxide dismutase 2 AT5G511001051826-3.99
    MD07G13069006FBD Supplemental Table S3Chr07:34607570-36531467UDP-glucosyl transferase 78D2 AT5G170506771112.78
    MD08G12491002WCI Supplemental Table S3Chr08:30486569-31607516HSP20-like chaperone (ATHSP22.0) AT4G102503186456.12
    MD09G111400032FBD Table 1Chr09:7999212-11883202fatty acid desaturase 5 (FAD5) AT3G158507608.68
    MD09G11468001FBD Table 1Chr09:7999212-11883202PHYTOENE SYNTHASE (PSY) AT5G1723038456160901.32
    MD10G132810045FBD Supplemental Table S3Chr10:40235258-41736791ethylene-forming enzyme (ACO4) AT1G050108769033910331.21
    MD15G10236001FBD Table 1Chr15:1-1487288jasmonic acid carboxyl methyltransferase (JMT) AT1G19640472111552.07
    MD15G10241008FBD Table 1Chr15:1-1487288dihydroflavonol 4-reductase (DFR, TT3, M318) AT5G428008723091.63
    MD17G11334004WCI Supplemental Table S3Chr17:11422073-12433463PHYTOENE SYNTHASE (PSY) AT5G172306001721.87
    MD17G12606002FBD Supplemental Table S3Chr17:31098955-32776972dehydroascorbate reductase 1 (DHAR3) AT5G167101867−1.78
     | Show Table
    DownLoad: CSV

    Several gene families (e.g. chalcone synthase, UGT, anthocyanin synthase, HSP, PAL, ERF, WRKY proteins, ABA, and bZIP transcription factors) residing in WCI-associated genomic regions have been reported to be associated with RF in apple[7, 12, 28]. Several of these genes are implicated in stress responses, suggesting that flavonoids and anthocyanin biosynthesis could also be associated with stress (e.g., drought, water loss) tolerance of RF apples[7].

    MdMYB73, which may play a role in the cold-stress response[23, 29], resided in the WCI-associated regions. Ethylene is among the various modulators of environmental stresses induced by factors such as drought and cold temperatures[9, 28]. Several ERF proteins (e.g. MdERF1B, MdERF3) interact with the promoters of MYB domain proteins to regulate anthocyanin and proanthocyanidin (PA) accumulation in apple[10, 11, 30]. The WCI-associated genomic regions in our study flanked several ERFs previously reported to have roles in anthocyanin and PA regulation. MdERF4 (MD10G1290400), one of the three ERFs residing in the WCI-associated region on Chr10, has high phylogenetic similarity with MdERF38, which interacts with MdMYB1 to regulate drought-related anthocyanin biosynthesis[31]. Some ERFs (e.g. MdERF2: MD10G1286300; MdERF4: MD10G1290400, and MdERF12: MD10G1290900) and MdNAC73 residing in the WCI-associated regions have been reported to be associated with fruit maturation[20] – suggesting an interaction between ethylene production and RF colour[12].

    The WCI-associated region on Chr15 included a gene MdMYB73 (MD15G1288600) involved in malate acid synthesis. Previous studies have reported that MdMYB1 regulates both anthocyanin and malate accumulation, and perhaps MdBT2 regulates MdMYB73-mediated anthocyanin accumulation[22, 23]. The malate transporter gene MdMa2 and the MdMYB7 gene, involved in the regulation of anthocyanin and flavanols in RF apple[32], resided together in the WCI-associated upper region of LG16. Taken together, these results support the hypothesis that there is interplay between anthocyanin and malate accumulation in the RF apple. There was a cluster of ubiquitin-specific proteases underpinning the WCI-associated regions on Chrs 7 and 15, which is supported by earlier reports suggesting that ubiquitin-specific proteases respond to auxin and might suppress anthocyanin biosynthesis proteins[33]. The auxin response factor 9 (MdARF9: MD06G1111100), which has also been shown to suppress anthocyanin biosynthesis in RF callus samples[33], also resided in the WCI-associated genomic region on LG7.

    Seedlings in both the low- and high-FBD pools carried the MdMYB10 gene, which suggests that MdMYB10 itself is not the causal factor of FBD in RF apples. Long-term cold storage generally results in senescence-related flesh breakdown, and several transcription factor genes (e.g. MYB, WRKY, NAC, ERF, cytochrome P450, and HSP) have been shown to express differentially during long-term cold storage[9]. The FBD-associated genomic regions in our study harboured several ERFs, suggesting that ethylene synthesis proteins may be contributing to cell wall disassembly, allowing PPO enzymes to come into contact with phenolic compounds and potentially leading to FBD symptoms[18].

    Pectin methyl esterase (PME) genes resided in the FBD-associated significant regions on Chrs 3, 13 and 17. Volatile generation and senescence degradation have been suggested to be bio-markers of FBD, and the expression levels of methyl esters were found to be associated with FBD and senescence in 'Fuji' apples after cold storage[34]. The MdPME2 gene has also been reported to be associated with apple flesh firmness and mealiness[21, 35]. The co-location of genes encoding cell wall-degrading enzymes (MdPME) and QTLs for FBD has been reported in apple[18, 36].

    The clusters of cytochrome P450 enzymes and senescence-related genes resided in some of the FBD-associated regions in this study. There are no earlier reports of the involvement of P450 enzymes in apple FBD expression, but some genes related to cytochrome P450 were found to be upregulated during litchi fruit senescence[37]. Pericarp browning in litchi is mainly attributable to the degradation of anthocyanin, and the ABA-initiated oxidation of phenolic compounds by PPO[37]. Flavonoids are among the major polyphenols in RF apples[5], and cytochrome P450 is part of the regulatory mechanism for flavonoid metabolism[38]. Association of FBD with the genomic region harbouring P450 would suggest its role in enhanced polyphenol synthesis causing FBD. The co-occurrence of a senescence-related gene MdNAC90 (MD03G1148500; MD11G11679000) and the PPO regulator germin-like proteins (in the FBD-associated paralog regions on Chrs 3 and 11) lends support to an interplay between senescence and oxidation of phenolics and anthocyanins.

    A significant region on Chr9 encompassed a cluster of UGT proteins, which play a role in the regulation of flavonoids and phenolic compounds, as well as converting phloretin to phloridzin[39]. Cytochrome P450, which resided within several FBD-associated regions, is reportedly involved in flavonoid metabolism, such as chlorogenic acid (CGA) acid and phloridzin, which have also been positively associated with suberin production and cell wall disassembly[40, 41].

    Reactive oxygen species (ROS) play an important role in regulating physiological processes in plants, such as senescence[37]. Legay et al.[42] suggested that MdMYB93 (MD15G1369700), found residing in the FBD-associated regions in this study, plays a critical role in remobilisation of flavonoid/phenolic compounds, which can be utilised for detoxification of ROS in the case of oxidative stress. However, flavonoid biosynthesis has also been linked with suberin production causing cuticle cracks in apples[42, 43], and the development of cuticular cracks could accelerate flesh browning as a result of an enhanced oxidative process[44].

    Several HSP (e.g. HSP70, HSP70-1, HSP60, HSP89.1, and HSP DNAJ) and HSF (e.g. HSF4, HSFB4, HSFA6B) resided in the FBD-associated genomic regions. Ferguson et al.[45] showed that, during summer, apple flesh temperature could reach as high as 43 °C, and that an increase in the expression of HSP in apples was associated with high daily flesh temperatures, suggesting a role of HSP to counter heat stress. HSPs have been reported to interact with AP2/ERFs and to play a role in flavonoid biosynthesis and drought tolerance in apple[46]. Heat stress affects lignin accumulation and its substrate, O-phenols, and has been reported to play role in enzymatic browning[47]. Additionally, HSF that regulate HSP expression have also been reported to be regulated by cold stress to generate heat-induced cold tolerance in banana[48]. HSF1 was shown to transcriptionally regulate the promoters of HSP to enhance chilling tolerance in loquat fruit[49]. Activity of the enzymes (PAL, C4H, 4CL) of the phenylpropanoid pathway was positively correlated with loquat fruit lignification, whilst suppression of their expression by heat shock treatment and low-temperature conditioning significantly reduced fruit lignification[50].

    Wang et al.[7] showed that ascorbate peroxidase (APX) was among the genes that were upregulated in RF apple compared with in white-fleshed apples. Several genes (e.g. MdAPX1, MdAPX3, MdAPX4, and MdDHAR1) involved in ascorbate synthesis resided in the genomic regions associated with FBD. Co-localisation of MdDHAR and ascorbic acid (AsA) synthesis genes in the FBD-linked genomic regions have been reported[51], suggesting that the low AsA content increases fruit susceptibility to FBD[52]. It has been shown that the expression of MdMYB1 and MdDHAR genes was strongly correlated in RF apples, and that AO and APX were upregulated by anthocyanin regulatory genes[31].

    There were several genes associated with CGA biosynthesis residing in the FBD-associated regions on various linkage groups, including the genes MYB19 (MD07G1268000) and MdC3H (MD15G1436500). Higher concentrations of CGA were reported in transgenic apple lines carrying MdMYB10[12], suggesting a role of CGA metabolism in the expression of FBD[53]. Interestingly, some of the FBD-associated regions (e.g. Chrs 9, 11, 13, 14 and 17) reported here in RF apples coincide with those reported earlier for FBD in white-fleshed apples[18, 36, 53], suggesting some common underlying genetic mechanisms.

    As discussed above, ERFs have been reported to be involved in the accumulation of anthocyanin and PA biosynthesis, while ethylene synthesis proteins also contribute to cell membrane breakdown, allowing the PPO enzyme to come into contact with phenolic compounds, potentially leading to FBD symptoms[12, 18]. We observed clusters of anthocyanin biosynthesis proteins (bHLH), ERFs (MdERF2, MdERF4, MdERF12) and PPO genes together in the genomic region associated with WCI on Chr10. The co-occurrence of these gene families perhaps facilitates potential interactions that contribute to the genetic correlation between WCI and FBD. We noted that genes involved in flavonoid regulation and ethylene synthesis occurred together in the FBD-associated regions on several chromosomes (e.g. MD16G1140800 and MdPAE10: MD16G1132100; MdERF1B: MD16G1216900 and MdMYB15: MD16G1218000, MD16G1218900) – suggesting these genes could be in linkage disequilibrium and this would contribute to the expression of WCI and FBD.

    MYB7 (MD16G1029400) resided in the WCI-associated upper region of Chr16, and the expression level of MYB7 was shown to be correlated with that of LAR1 in peach fruit[54]. The MdLAR1 protein (MDP0000376284), which is located about 1.2 Mb upstream of MYB7 (MD16G1029400), was reported to be associated with WCI and FBD in apple[3]. Mellidou et al.[36] reported that 4CL (MD13G1257800 in the FBD-linked region on Chr13), which catalyses the last step of the phenylpropanoid pathway, leading either to lignin or to flavonoids, was upregulated in browning-affected flesh tissues. The gene MdMYB85, involved in the regulation of flavonoid and lignin biosynthesis, resided in the WCI-associated region on Chr6 and FBD-associated paralogous region on Chr4. Metabolic interactions between anthocyanin and lignin biosynthesis have been reported for apple[55] and strawberry[56], while flesh lignification and internal browning during low-temperature storage in a red-fleshed loquat cultivar was shown to be modulated by the interplay between ERF39 and MYB8[57].

    The co-localisation of MdMYB66 and cytochrome P450 proteins, along with the anthocyanin regulatory protein MdMYB86, in paralogous FBD-associated genomic regions on Chr6 and Chr14 suggests that these genes interact as a 'hub' contributing to the WCI-FBD genetic link. The paralogs of some other genes were found to be residing in the regions associated with either WCI or FBD. For example, MdNAC42 and HSP70 co-localised in the FBD-associated region on Chr9, but this same pair of genes also resided in the most prominent region associated with WCI on Chr17. Similarly, paralogs of MdEIN3 resided in the WCI-associated region on Chr7 (MD07G1053500, MD07G1053800) and FBD-associated region on Chr11 (MD11G1022400). Interestingly, the paralogs of MdMYB73 and MdWRKY7 co-localised in the WCI- (24.6–26.8 Mb) and FBD-associated (4.6–6.8 Mb) regions on Chr15. The WCI-associated region at the bottom of Chr11 hosted a cluster of senescence-related genes, along with the anthocyanin biosynthesis gene MdbHLH3 (MD11G1286900), suggesting they might interact in the genetic nexus between FBD and WCI.

    FBD in RF apples can be caused by senescence, injury via extreme temperature exposure (chilling or heat), or enzymatic (cut fruit) reaction. Genes reported to be connected to all three factors were located in various FBD-associated genomic regions in this study. Postharvest strategies that both delay senescence and limit exposure to low temperatures may be needed to manage FBD. We also hypothesise that high ascorbic acid content could help to minimise expression of FBD in Type-1 RF cultivars. The adverse genetic correlation between WCI and FBD appears to arise from dual and/or interactive roles of several transcription factors, which would pose challenges for designing a conventional marker-assisted selection strategy. The use of bivariate genomic BLUP to estimate breeding values to simultaneously improve adversely correlated polygenic traits (e.g. WCI and FBD), could be an alternative approach[58, 59].

    A population of 900 apple seedlings composed of 24 full-sib families was generated in 2011 by selected crossings between six red-leaved pollen parents and six white-fleshed female parents. All six pollen parents inherited their red-leaf phenotype from the same great-grandparent 'Redfield'[1]. Each pollen parent was involved in four crosses, and the female parents were involved in three to six crosses each. Foliage colour of young seedlings is a phenotypic marker for Type 1 RF apple. The main purpose of this trial was to understand the flesh colour variation and FBD in the Type 1 RF seedlings, so only the seedlings with red foliage (i.e. carrying MdMYB10) were kept for this trial. The number of seedlings per family varied from 10 to 95. The seedlings were grafted onto 'M9' rootstock and were planted in duplicate at the Plant & Food Research orchard in Hawke's Bay, New Zealand (39°39′ S, 176°53′ E) in 2015.

    Phenotyping for RF and FBD was conducted over two consecutive fruiting seasons (2017 and 2018). Fruit were harvested once, when judged mature, based on a change in skin background colour from green to yellow, and when the starch pattern index (SPI) was between 1.0 and 2.0 (on a scale of 0 to 7). In each season, six fruit were harvested from each plant and stored for 70 d at 0.5 °C, followed by 7 d at 20 °C before fruit evaluation. Fruit were cut in half across the equator and the proportion of the cortex area (PRA) that was red in colour, and the intensity of the red colour (RI) (= 1 (low) to 9 (high)) was scored. A weighted cortical intensity (WCI) was then calculated (PRA × RI) as an estimation of the amount of red pigment in the fruit. The proportion of the cortex area showing symptoms of FBD was also recorded. WCI and FBD were averaged over all fruit for a particular seedling. Fruit were also assessed for the following eating quality traits on a 1 (lowest) to 9 (highest) scale: firmness, crispness, juiciness, sweetness, sourness and astringency, to understand the genetic correlations of eating-quality traits with WCI and FBD.

    The binary vector pSA277-R6:MYB10 was transferred into Agrobacterium tumefaciens strain LAB4404 by electroporation. Transgenic 'Royal Gala' plants were generated by Agrobacterium-mediated transformation of leaf pieces, using a method previously reported[5]. Wild-type 'Royal Gala' and three independent transgenic lines (A2, A4 and A10) of R6:MdMYB10 were grown under glasshouse conditions in full potting mix with natural light. The resulting fruit were assessed for flesh colour phenotypes at harvest (around 135 d after full bloom). Fruit peel and cortex from three biological replicates were collected and frozen in liquid nitrogen, with each replicate compiled from five pooled mature fruit for each transgenic line or wild-type control.

    Total RNA of 36 samples (3 R6:MYB10 lines and 1 wild type control, 3 time points, 3 biological replicates) was extracted, using Spectrum Plant Total RNA Kit (SIGMA). Removal of genomic DNA contamination and first-strand cDNA synthesis were carried out using the mixture of oligo (dT) and random primers according to the manufacturer's instructions (QuantiTect Reverse Transcription Kit, Qiagen). Real-time qPCR DNA amplification and analysis was carried out using the LightCycler 480 Real-Time PCR System (Roche), with LightCycler 480 software version 1.5. The LightCycler 480 SYBR Green I Master Mix (Roche) was used following the manufacturer's method. The qPCR conditions were 5 min at 95 °C, followed by 45 cycles of 5 s at 95 °C, 5 s at 60°C, and 10 s at 72 °C, followed by 65 °C to 95 °C melting curve detection. The qPCR efficiency of each gene was obtained by analyzing the standard curve of a cDNA serial dilution of that gene. The expression was normalized to Malus × domestica elongation factor 1-alpha MdEF1α (XM_008367439) due to its consistent transcript levels throughout samples, with crossing threshold values changing by less than 2.

    Individual fruit measurements were first averaged for each seedling. As the phenotyping was repeated over two years, we used a mixed linear model (MLM) accounting for this 'permanent environmental effect', as previously described[58]. Pedigree-based additive genetic relationships among seedlings were taken into account for estimation of genetic parameters using ASReml software[60]. Product-moment correlations between best linear unbiased predictions (BLUP) of breeding values of all seedlings for different traits were used as estimates of genetic correlation among traits.

    A selective DNA pooling procedure was adopted to construct DNA pools. A high-pool and a low-pool were constructed separately for the two traits (WCI and FBD). Genomic DNA was extracted from the leaves of selected seedlings, and quantified by fluorimetry using the picogreen reagent (Cat#P11496, Thermo). The low and high pools consisted of 35 seedlings each, and normalised amounts (~300 ng) of DNA from individuals were pooled. The pools were dried down with DNA Stable reagent (Cat#93021001, Biomatrica) in a centrifugal evaporator and shipped for sequencing. Each DNA pool was sequenced using paired-end 125 bp reads on the Illumina HiSeq 2500 platform. The quality of raw sequence reads was checked with FastQC/0.11.2 and MultiQC/1.2. Based on the quality control reports, the reads were aligned to the published apple reference genome GDDH13 v1.1[61] using the program bowtie2/2.3.4.3[62] with trimming from both ends before alignments and aligning in full read length ("-5 6 -3 5 –end-to-end"). The mapping results were marked for duplicate alignments, sorted, compressed and indexed with samtools/1.12[63]. Based on the alignment of binary alignment map (BAM) files of the high and low pools, single nucleotide polymorphism (SNP) identification was performed using samtools/1.12 ('samtools mpileup') and bcftools/1.12 ('bcftools call –mv')[63, 64]. Variant sites with missing genotype in any of the pools, or having the same genotype between the pools, were discarded. To minimise the influence of sequencing quality on association analysis, the identified SNPs were further filtered according to the following criteria: 1) a Phred-scaled quality score > 20; and 2) the read depth in each pool was neither < 35, nor > 500.

    The allele frequencies between each pair of bulk DNAs (low versus high WCI; low versus high FBD) were compared at each SNP locus. Differences in the allele frequencies between the low and high pool were expected to be negligible for unlinked SNP markers, but allele frequency differences would be larger for SNPs closely linked to the underlying quantitative trait loci (QTLs) contributing to the extreme phenotypes. A nonparametric test (G-statistic = 2 × Σni ln(ni/nexp), where ni (i = 1 to 4) represented counts of reference and alternate alleles at a particular SNP generated from sequencing of the low and high pool, and nexp was the expected allele count assuming no allele frequency divergence between the two DNA pools[65].

    We then calculated a modified statistic (G'), which took into account read count variation caused by sampling of segregants as well as variability inherent in short-read sequencing of pooled samples[65]. Using R package QTLseqr[66], firstly a G-statistic was calculated for each SNP marker, and then a weighted average using Nadaraya-Watson kernel was obtained to yield a G' statistic for a sliding genomic window of 2 Mb size. The Nadaraya-Watson method weights neighbouring markers' G-values by their distance from the focal SNP so that closer SNPs receive higher weights. The 95th percentile value of G' was used as a threshold to identify significant hotspots and to identify the putative candidate genes residing within the ±1 Mb region around the G' peak. For comparison purposes, a standard two-sided Z-test[14] was also performed to determine the significance of allele frequency differences at SNP loci between the pools for each trait.

    The GDDH gene models intersecting with the XP-GWA hotspots were pulled out with bedtools/2.30.0 ("bedtools intersect -wo -nonamecheck"). The selected genes were further blasted to TAIR10 ("-evalue 1e-5") and the annotated functions from Arabidopsis genes with the best blast score, the highest % identity, and the longest aligned length, were used. Then the expressions of genes located in the GWA hotspots were extracted from the RNAseq analysis of the R6:MdMYB10 representative transgenic line, and the log 2-fold change between the R6:MdMYB10 and 'Royal Gala' apples were calculated.

    This research was funded in 2017/18 by the Strategic Science Investment Fund of the New Zealand Ministry of Business, Innovation and Employment (MBIE) and from 2019 by the Plant & Food Research Technology Development – Pipfruit programme. We thank our colleague Jason Johnston for providing some pictures of the flesh browning disorder in red-fleshed apples. Richard Volz and Jason Johnston provided constructive comments and suggestions on the manuscript.

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Gaire S, Yu X, Milla-Lewis SR. 2024. Molecular advances in St. Augustinegrass: from DNA markers to genome sequencing. Grass Research 4: e021 doi: 10.48130/grares-0024-0017
    Gaire S, Yu X, Milla-Lewis SR. 2024. Molecular advances in St. Augustinegrass: from DNA markers to genome sequencing. Grass Research 4: e021 doi: 10.48130/grares-0024-0017

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Molecular advances in St. Augustinegrass: from DNA markers to genome sequencing

Grass Research  4 Article number: e021  (2024)  |  Cite this article

Abstract: St. Augustinegrass (Stenotaphrum secundatum (Walt.) Kuntz) is one of the most important warm-season turfgrass species in the United States. Breeding efforts for this turfgrass have primarily focused on improving turf quality and increasing resistance to various biotic and abiotic stresses, including insects, diseases, drought, cold, and shade. While conventional breeding methods have been widely employed in St. Augustinegrass breeding programs, recent years have seen the integration of molecular tools and techniques such as molecular markers, linkage maps, quantitative trait loci (QTL) mapping, comparative genomics, and transcriptomics. Despite these efforts, genomic resources for St. Augustinegrass are still underdeveloped compared to other economically important crops. The recent establishment of a reference genome for St. Augustinegrass is a major milestone, opening new possibilities for genomics-enabled breeding of this important turfgrass. The use of modern genomic tools like genomic selection and marker-assisted selection (MAS) in breeding programs can enhance selection accuracy, shorten breeding cycles, improve trait incorporation, and significantly boost genetic gains, ultimately leading to the development of superior cultivars that meet industry demands. This review highlights recent advancements in genetics and genomics of St. Augustinegrass and identifies areas that require further research to bridge existing knowledge gaps.

    • Among the seven species of the genus Stenotaphrum, S. secundatum (Walt.) Kuntze (family Poaceae, subfamily Panicoideae, tribe Paniceae) is the only species commercially used as turfgrass in the United States[1]. Commonly known as St. Augustinegrass, this warm-season turfgrass is native to tropical and subtropical climates. It is predominantly grown in coastal regions and is one of the most adaptable warm-season turfgrasses under shading. St. Augustinegrass is a robust perennial grass known for its dense canopy and aggressive growth, which make it resistant to weed infestation[2]. Additionally, it exhibits relatively lower input requirements (water, fertilizer, pesticides) compared to cool-season turfgrasses like tall fescue (Festuca arundinacea Shreb.). Moreover, it thrives well in various soil conditions and high temperatures[2,3]. Despite that, the sensitivity of St. Augustinegrass to low-temperature stress has limited its adaptability and marketability in the transitional climatic zone of the US. Improving its freeze tolerance could expand its geographical distribution and adaptability to colder environments where this species is not currently grown.

      St. Augustinegrass has a base chromosome number of nine with five different ploidy levels reported including diploid (2x = 2n = 18), triploid (3x = 2n = 27), aneuploid (2n = 28 − 32), tetraploid (4x = 2n = 36), and hexaploid (6x = 2n = 54)[4,5]. Chromosomal disparities across St. Augustinegrass ploidy levels are closely correlated with adaptive and morphological differences[2,5,6]. The species' genetic diversity is categorized into distinct races and groups contingent on ploidy levels, geographic distribution, and morpho-agronomic traits[48]. The existence of genetic variation in St. Augustinegrass is evident across various physiological, morphological, and adaptive traits, encompassing cold, shade, and drought tolerance, disease resistance, turf quality, leaf attributes, growth rate, and stomatal density[2,5,6].

      Most of the active breeding endeavors in St. Augustinegrass have been directed toward the improvement of turfgrass quality[911] and increasing tolerance to several biotic and abiotic stresses including cold[9,10,1214], drought[15], shade[16], the southern chinch bug (Blissus insularis Barber)[1720], and gray leaf spot disease (Pyricularia oryzae Cavara)[21]. While significant phenotypic variation has been reported in St. Augustinegrass polyploid germplasm[5,15,16,22], their exploitation in breeding programs has been offset by challenges such as diminished pollen viability, sterility concerns, and compromised seed set or development attributed to imbalanced chromosomal compositions, as well as uncertainties regarding the origins of higher-ploidy cultivars[5,22].

      Although conventional breeding methods have been widely applied by St. Augustinegrass breeding programs, genomic-based breeding is becoming more popular due to its efficiency in achieving higher genetic gains. However, compared to other popular turfgrass species and other economically important commercial crops, genomic resources for St. Augustinegrass are still in development. Challenges like limited historical sequencing data and molecular markers, inadequate access to a high-quality reference genome, complex regulation of polygenic traits, and funding limitations for St. Augustinegrass research have impeded significant progress in St. Augustinegrass genomics. This review aims to spotlight the recent advancements in genetics and genomics in this species, while also pinpointing areas that require further exploration to bridge knowledge gaps.

    • To date, only clonally propagated commercial cultivars of St. Augustinegrass are available in the market[23]. These cultivars are difficult to differentiate morphologically, particularly under high soil fertility conditions[24]. The first application of molecular markers in St. Augustinegrass was the use of isoenzyme markers for the identification of clonally propagated cultivars. Green et al.[24] were able to distinguish 28 clones of St. Augustinegrass into several groups based on quantitative differences, but no qualitative differences were observed. While the usefulness of isoenzyme variation was demonstrated in this study, the integration of highly reproducible and polymorphic molecular markers was needed to enhance the efficiency of breeding and selection.

      The first report on the use of DNA markers in St. Augustinegrass was slightly more than a decade ago by Genovesi et al.[25] who used 144 expressed sequence tags-simple sequence repeats (EST-SSR) for the identification of true hybrids recovered via embryo rescue. These hybrids were developed from interploidy crosses between the aneuploid cultivar 'Floratam' and five diploid cultivars. As DNA sequence for St. Augustinegrass was not available at the time, EST-SSR markers derived from buffalograss cDNA sequence data were adopted for the study. These markers not only identified true hybrids but also revealed levels of genetic variation present among analyzed cultivars[25].

      After this milestone, several other DNA marker studies were undertaken for molecular taxonomy and genetic diversity exploration[5,8,2629]. The study by Milla-Lewis et al.[5] was a pioneering effort to comprehensively assess genetic diversity in Stenotaphrum germplasm, utilizing both molecular and cytological approaches, primarily focusing on its implications for breeding purposes. Amplified fragment length polymorphism (AFLP) markers were used to fingerprint 40 St. Augustinegrass cultivars and plant introductions, supporting previously known morphological classifications, which were predominantly based on morphological and performance traits[4,6,7]. In this study, grouping accessions by ploidy levels yielded more distinct differentiation than by germplasm type. Furthermore, a clear distinction between secundatum and dimidiatum accessions as well as between diploid and polyploid accessions was found. The clustering of diploids into two distinct races (Breviflorous and Longicaudatus races) suggested possible dual origins for the species, and the distinct clustering of S. secundatum polyploids into the Bitterblue and Floratam groups hinted at either chromosome doubling (followed by chromosome loss), or interspecific hybridization. The authors also pointed out the need for additional investigation into the genome origins of polyploids to identify more usable accessions and to manage potential challenges related to sexual incompatibility.

      The results from Milla-Lewis et al.[5] were later supported by Mulkey et al.[8,28]. This study[28] utilized 12 AFLP primer combinations to assess genetic diversity in the University of Florida's St. Augustinegrass germplasm collection, one of the largest in the US, and compared it with publicly available cultivars and plant introductions from the National Plant Germplasm System (NPGS). The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster and principal component analysis (PCA) identified two major subgroups that aligned well with ploidy levels, with most diploid accessions in one group and polyploids in the other. Meanwhile, triploid and higher ploidy segregation was less distinct[28]. These studies showed that AFLP markers were valuable tools for evaluating genetic diversity and ploidy levels in Stenotaphrum germplasm[5,28]. In a subsequent study, Mulkey et al.[8] used Illumina sequencing data to develop the first set of simple sequence repeat (SSR) markers specifically designed for the species. Ninety-four SSR primer pairs were subsequently used for Stenotaphrum germplasm evaluation. Results aligned with previous molecular diversity studies[5,28]. Furthermore, through cluster and PCA, the germplasm collection was classified into six subpopulations. Although the transferability of the markers developed to other turfgrass species was low indicating poor applicability outside of St. Augustinegrass, the study provided valuable insights into genetic relationships and population structure in Stenotaphrum and underscored their potential for better parental selection, cultivar development, and strategic germplasm utilization.

      Using Roche 454 pyrosequencing technology, Wang et al.[29] also isolated and characterized 33 SSR primer pairs specifically for Stenotaphrum Trin grasses, with a particular emphasis on St. Augustinegrass. Of the total sequencing reads produced from this technique, only 4.56% (2,614) of them contained SSRs. The identified primers were deposited in GenBank (accession number: KT036573—KT036605). High genetic diversity among the 18 St. Augustinegrass accessions evaluated was observed with 92% of the scorable bands (161) being polymorphic. These accessions were clustered into three distinct major groups that appeared to be associated with ploidy levels which was consistent with other molecular studies[5,8,28]. The findings of Mulkey et al.[8] and Wang et al.[29] highlight the reliability of high-throughput sequencing for efficient identification and isolation of SSR sequences. The SSR markers developed in these studies could be used for several applications, including assessing genetic diversity within St. Augustinegrass populations, constructing linkage maps, verifying the purity of clonal cultivars, and assisting breeding programs aimed at improving specific traits in St. Augustinegrass through marker-assisted selection (MAS).

      The use of molecular markers in St. Augustinegrass has also been reported for cultivar identification[8,26,27]. In the study led by Kimball et al.[26], AFLP markers were used to examine identity preservation in samples of 'Raleigh', a cultivar released publicly by North Carolina State University in the early 1980s. 'Raleigh' samples were collected from production fields across the southern United States and compared to the original stock at NC State. With the analysis of the 143 polymorphic AFLP markers, the study found that samples of 'Palmetto', a modern patented cultivar, maintained higher genetic similarity to its original stock (0.97) compared to samples of 'Raleigh', which exhibited a broader range of similarity values (0.24 to 1). The study concluded that molecular makers can be a valuable tool for protecting clonally propagated turfgrass cultivars. In a subsequent study by Kimball et al.[27], AFLP markers were used to identify the true identity of off-types within 'Captiva', a cultivar released by the University of Florida in 2007[30], production fields. The study examined 72 samples collected from various sod farms across Florida and compared them to seven reference St. Augustinegrass cultivars, including 'Captiva', 'Bitterblue', 'Floratam', 'Floraverde', 'Palmetto', 'Raleigh', and 'Sapphire'. Results indicated that many off-type samples had the highest genetic similarity to 'Palmetto' (49%), suggesting potential contamination during commercial production of 'Captiva'. Mulkey et al.[8] evaluated 94 SSR markers for varietal identification, as morphological methods have limitations in discriminating closely related materials. SSR markers, being easy to use and highly polymorphic, successfully identified multiple cultivars with unique allele combinations. A set of five SSR markers could uniquely identify 20 out of 22 commercial cultivars offering practical benefits for varietal purity maintenance and breeder selection. These studies underscored the importance of using molecular markers in assessing genetic integrity and identifying contaminants to preserve genetic purity in clonally propagated turfgrass species, benefiting both producers and consumers[8,26,27].

    • Improvement of complex quantitative traits, which are controlled by multiple genes, can be a challenge in St. Augustinegrass breeding. Traditional breeding methods have limitations in this aspect. Thus, the utilization of advanced marker technologies and statistical approaches is needed. Constructing genetic linkage maps using appropriate populations and markers is critical for quantitative trait loci (QTL) analysis. Linkage mapping requires the creation of genetic maps based on recombination frequencies among markers, enabling the determination of the relative positions of markers in linkage groups. Drawing on these linkage maps, QTL analysis establishes connections between genotypic markers and phenotypic traits. Although QTL mapping studies in St. Augustinegrass have been limited, investigations into this species have yielded a few high-density linkage maps and have played a crucial role in pinpointing QTL and molecular markers linked to both abiotic and biotic stress factors. The primary emphasis has been on responding to environmental stresses such as drought[3133], freezing temperatures[10], and diseases[34,35], as well as physiological and morphological parameters[10,32,36]. However, these QTL still need to be validated in different populations and environments before they can be applied in marker-assisted selection.

      Mulkey[34] constructed the first linkage map for St. Augustinegrass using a combination of 107 AFLP and 36 SSR markers in a pseudo-F2 population of the cultivar 'Raleigh' × Plant Introduction 410353 (PI 410353). However, the relatively small population size and number of markers utilized resulted in a partial linkage map with low coverage. A higher number of linkage groups (LGs) than the number of chromosomes in each haplotype of St. Augustinegrass (2n = 2x = 18) were obtained: 13 LGs for the Raleigh map and 12 for the PI 410353 map. Using these partial linkage maps, the authors identified four potential QTL associated with gray leaf spot (GLS, causal agent Pyricularia oryzae Cavara) resistance; three related to the area under the disease progress curve (AUDPC) and one to the area under the lesion expansion curve (AULEC). However, the limitations in population size and number of markers could pose the issue of overestimating QTL effects. While the initial St. Augustinegrass linkage map required further improvement in its coverage and accuracy for a comprehensive understanding of environmental influences on variances and improved QTL analysis, it laid the groundwork for future genetic mapping efforts.

      Kimball et al.[10] constructed the first complete linkage map covering all nine haploid chromosomes of the St. Augustinegrass genome using 160 SSR markers in a pseudo-F2 mapping population of 'Raleigh' × 'Seville'. The linkage map was used for QTL analysis of field winter survival, laboratory-based freeze tolerance, and turfgrass quality traits. The study identified multiple QTL associated with these traits including overlapping QTL on LG 3 (99.21 cM) for winterkill and spring green-up; on LG 3 (68.57–69.50 cM) for turfgrass quality, turfgrass density, and leaf texture; and on LGs 1 (38.31 cM), 3 (77.70 cM), 6 (49.51 cM), and 9 (34.20 cM) for surviving green tissue and regrowth. Additionally, QTL from the field- and laboratory-based freeze testing co-localized on LG3[10]. This indicated the potential for identifying true candidate genes for freeze tolerance in those regions.

      The same population as in Kimball et al.[10] was later used in several other studies for linkage mapping and QTL analysis. Yu et al.[36] developed the first high-density linkage maps for the species using 2,871 genotyping-by-sequencing (GBS)-derived single nucleotide polymorphism (SNPs) markers in combination with 81 SSR markers. This integrated map (named LG1–LG9) covered a total distance of 1,241.7 cM with an average marker distance of 0.4 cM, making it the most comprehensive genetic map for St. Augustinegrass at the time. Maps were also developed for each parental genotype (named RLG1-RLG9 for the 'Raleigh' map and SLG1–SLG9 for the 'Seville' map) and covered a total distance of 1,238.7 cM and 914.2 cM for the 'Raleigh' and 'Seville' maps, respectively. Additionally, these maps were also used to map QTL associated with turfgrass quality traits. A total of 48 potential QTL were identified, with three hot spot regions showing overlap between different traits on LG3 and LG8 of the integrated map. Through annotation, these QTL regions were found to contain genes related to leaf development[36].

      The high-density genetic maps by Yu et al.[36] provided a powerful foundation for molecular studies in St. Augustinegrass. A comprehensive multi-year, multi-environment analysis was conducted to detect QTL associated with drought-related traits, including relative water content, chlorophyll content, leaf firing, leaf wilting, percent green cover, and normalized difference vegetative index (NDVI) evaluated in both field and greenhouse settings[31]. The study identified a total of 70 QTL associated with these traits. Overlapping QTL were found in LGs RLG1, RLG4, RLG6, RLG7 and SLG2. Notably, a hotspot region in RLG6 contained five overlapped QTL for multiple traits including leaf wilting, leaf firing, leaf relative water content across both experimental settings. Sequence analysis in overlapped regions in these LGs (RLG1, RLG4, RLG6, RLG7, and SLG2) revealed the presence of nine drought response genes including ZHD and WRKY transcription factors, ethylene-insensitive protein, cold-responsive protein kinase, OBERON-like protein, light-harvesting complex-like protein (OHP2), Magnesium-chelatase subunit (ChlD), Osmotin-like protein and LRR receptor-like serine/threonine-protein kinase (GSO1). This study was further expanded to incorporate QTL mapping of morphological characteristics to understand their potential correlation with drought tolerance[32]. This was the first study to perform QTL analysis for morphological traits, namely leaf blade width, leaf blade length, canopy density, and shoot growth orientation. Co-localization of QTL associated with morphological and drought-related traits was reported in the study. Two previously reported drought-related QTL[31] for relative water content and percent green cover overlapped with QTL for leaf length and leaf width on SLG3. Meanwhile, no overlapping regions were found between canopy density and shoot growth orientation, and drought-related QTL. However, overlapping QTL for shoot growth orientation and leaf length were found on RLG1, and overlapping QTL for canopy density, leaf length, and leaf width were identified on SLG3. These findings provided evidence of the potential influence of morphological traits on drought stress responses. Within QTL intervals related to drought tolerance and morphological traits, three key genes associated with plant growth and development [Gibberellin 2-beta-dioxygenase (GA2oxs), F-box/LRR-repeat protein (D3), S-adenosylmethionine decarboxylase proenzymes (SAMDCs), two water stress response genes (E3 ubiquitin ligases (PUB22 and PUB23), BAM1 (Beta-amylase)], and two genes contributing to drought tolerance through root system maintenance [GSO1 (Gene controlling primary root growth), Root phototropism protein 2 (RPT2) and Periodic tryptophan protein 2 (PWP2)] were identified.

      To address the limitations encountered in Mulkey[34] in mapping QTL for GLS resistance, Yu et al.[35] further expanded the study by increasing the population size to 153 hybrids and using a high number of SNP markers (2,257 and 511 for parents 'Raleigh' and PI 410353, respectively). With these improvements, the authors were able to improve the coverage of both parental linkage maps and detect more putative QTL. Twenty QTL associated with GLS resistance were identified, with three prominent hotspots located in LGs P2 and P5. Notably, two significant QTL, glsp2.3 and glsp5.2, which collectively resulted in a 20.2% reduction in disease incidence, were identified. These results suggested the potential use of these favorable alleles via marker-assisted selection in St. Augustinegrass breeding to effectively enhance GLS resistance. However, the lack of available genomic information for St. Augustinegrass at the time limited access to gene information within the QTL intervals. The study resulted in two candidate genes, XM_025948638.1 and XM_004968938.4, that code for β-1,3-glucanases, recognized as pathogenesis-related (PR) proteins, being identified within both glsp2.3 and glsp5.2. While these PR protein genes showed the potential for improving GLS resistance in St. Augustinegrass, a better understanding of their potential role was essential. Additionally, both studies were performed under controlled conditions, and to date, there has been no QTL research to validate these findings in field settings. Further investigations involving multiple environments are essential to elucidate the practical applications of these QTL and underlying genes in breeding programs.

      Rockstad et al.[33] developed a new population to validate the results from Yu et al.[31] by crossing breeding lines XSA 10098 and XSA 10127, the most contrasting genotypes in terms of drought response from the 'Raleigh' × 'Seville' population utilized by Kimball et al.[10] and Yu et al.[31]. The study used a draft of the first St. Augustinegrass reference genome[37] for alignment in the SNP calling pipeline, which resulted in the densest linkage map to date using four times as many markers (12,269) compared with 2,952 in Yu et al.[36] and 2,257 in Yu et al.[35]. Among the 24 QTL regions uncovered in this study, 16 were observed to overlap with regions identified in prior studies for drought tolerance[31] and morphological characteristics linked to drought tolerance[32]. These overlapping regions were found on chromosomes 3, 4, 6, and 9. Of particular interest was the co-localization of QTL for percent recovery from drought, percent green cover, leaf wilting, relative water content, and area under the leaf wilting curve in this study with relative water content in Yu et al.[31], which occurred within the QTL region on chromosome 3.

      Using the population and linkage map developed by Rockstad et al.[33], Weldt et al.[38] conducted a field evaluation to validate previously identified QTL associated with drought and drought-related traits[3133]. Weldt et al.[38] addressed the need to validate QTL in different mapping populations and under varying environmental conditions by employing a different mapping population from Yu et al.[31,32] and environments different from those used by Rockstad et al.[33]. The study identified 22 QTL on five linkage groups, with 19 overlapping with QTL from previous studies[3133] on LGs 1, 2, 4, and 9. Although the same mapping population was used in both the greenhouse evaluation by Rockstad et al.[33] and the field evaluation by Weldt et al.[38], only two QTL in LG1 and LG9 were found to overlap between the studies, highlighting the influence of environmental factors on QTL localization and expression. These QTL could be used in investigating drought avoidance and tolerance traits under field and greenhouse conditions.

    • Yu et al.[36] used the first high-density linkage map to perform comparative genome analyses between St. Augustinegrass and three other grass species (foxtail millet: Setaria italica (L.) P. Beauv., sorghum: Sorghum bicolor [L.] Moench, and rice: Oryza sativa). This is the only comparative genomics study that has been carried out on St. Augustinegrass to date. The study revealed chromosomal rearrangements and fusion events post-divergence from their common ancestor[36]. St. Augustinegrass and foxtail millet exhibited high synteny and collinearity. However, several inter-chromosomal rearrangements and inversions differentiated their genomes. In sorghum, comparative genomics revealed high collinearity. Notably, an event of nest chromosome fusion was identified, indicating a fusion between sorghum chromosomes Chr8 and Chr9, leading to the formation of an R(S)LG3 in St. Augustinegrass. Using rice as a reference, the study identified three pairs of fused rice chromosomes in St. Augustinegrass, highlighting evolutionary changes among these species. Overall, the study provided insights into the genomic relationships, conservation, and rearrangements between St. Augustinegrass and these model grass species, increasing our understanding of the evolutionary history of the grass family.

      The first endeavor to characterize gene expression in St. Augustinegrass at the molecular level was that of Jo et al.[39], who utilized tools and genetic resources from rice to assess the transcriptomic response of St. Augustinegrass to M. grisea. Utilizing large-scale EST screening through reverse northern hybridization, 30 rice EST clones, showing differential expression in St. Augustinegrass, were selected and their putative functions were categorized. The findings revealed a conserved response to M. grisea infection between rice and St. Augustinegrass. This study not only provided insights into the identification and characterization of defense-related genes in turfgrass, but also highlighted the potential for leveraging rice genetic resources in understanding the molecular response of St. Augustinegrass to fungal pathogens[39].

      Schoonmaker[37] developed the first-ever reference genome for St. Augustinegrass using a combination of PacBio CCS, Illumina, and Hi-C technologies. Two haplotype assemblies were created for the freeze-tolerant diploid cultivar 'Raleigh', with the primary assembly being more complete (465.41 MB in 631 scaffolds) than the secondary one (401.52 MB in 539 scaffolds). Both haplotype assemblies were close to the expected genome size, accounting for 95.2% and 82.1% of the expected haplotype genome size, respectively. Compared to previously published turfgrass assemblies for African bermudagrass (Cynodon transvaalensis Burtt Davy)[40] and zoysiagrass (Zoysia japonica Steud.)[41], these assemblies had higher contig and scaffold lengths, meeting reference genome quality requirements. A total of 67,805 genes were annotated, and a standardized pipeline was developed for consistent annotation across warm-season turfgrasses. The study also successfully quantified 'Raleigh's' heterozygosity using two haploid assemblies, revealing it to be a hybrid with high levels of heterozygosity. Accurate quantification of heterozygosity in S. secundatum had not been previously done[37]. This study not only facilitated the development of crucial tools for future investigations but also provided insights into St. Augustinegrass genetics.

      With the availability of a reference genome, Rockstad et al.[33] and Weldt et al.[38] investigated differentially expressed genes (DEGs) in leaves and roots, respectively, under drought stress conditions compared to normal watering, focusing on both tolerant (XSA10098) and sensitive ('Raleigh') genotypes of St. Augustinegrass. In leaves, the drought-sensitive genotype showed changes in the expression of a greater number of genes (either up- or down-regulated) compared to the drought-resistant genotype[33]. Similar results were observed in the roots of St. Augustinegrass[38], indicating a lack of effective regulatory mechanisms such as a homeostatic system to counteract the effects of water deprivation in sensitive genotypes. Stress response-related plant hormone signal transduction pathways such as the abscisic acid metabolic process was upregulated in both leaves[33] and roots[38] and photosynthetic genes were down-regulated in leaves of both genotypes[33]. Tolerant genotypes showed upregulation of secondary metabolite pathways in leaves, while both genotypes had downregulation in roots. Mitogen-activated protein kinase (MAPK) signaling pathway genes showed complex patterns across genotypes and tissue, with downregulation in the sensitive genotype's leaves and mixed regulation in leaves of the tolerant genotype and roots of both genotypes[33,38].

      In addition, Rockstad et al.[33] combined leaf transcriptomic data with a QTL mapping study[31,32] for the first time in St. Augustinegrass, which was possible due to the availability of a St. Augustinegrass reference genome[37]. The findings revealed 12 co-localized candidate genes involved in cell wall organization, photorespiration, zinc ion transport, regulation of reactive oxygen species, channel activity, and regulation in response to abiotic stress. In a subsequent study, Weldt et al.[38] identified 21 candidate genes through the integration of root transcriptomics data with prior QTL studies[3133], revealing a similar pathway and gene involvement. A notable colocalization of DEGs encoding for putative LRR receptor-like serine/threonine-protein kinase, and Cysteine-Rich Peptide Family on chromosome 6 in both leaf and root tissue showed potential functional significance in relation to drought response in St. Augustinegrass[33,38]. These findings are valuable for understanding the mechanisms underlying drought tolerance in St. Augustinegrass and may inform breeding efforts aimed at developing more resilient cultivars by narrowing down the confidence interval of significant QTL and identifying reliable candidate genes within those to be used in MAS.

      While the studies summarized here have increased our understanding of the genetic control of some of the most economically important St. Augustinegrass traits and have laid the foundation for the identification of key genes that could be incorporated in breeding programs, most of these studies were conducted in only one population or a single environment, except for the drought studies. Validating the identified QTL in different environments and/or populations is needed before they can be implemented in MAS to increase selection accuracy compared to traditional, phenotype-based selection methods. However, it is imperative to note that MAS may not always be practical, especially for traits governed by multiple minor genes such as stress tolerance-related traits. Integrating genomic selection into turfgrass breeding programs can offer a more suitable alternative with numerous advantages, including increased selection accuracy, accelerated breeding cycles, improved trait incorporation, enhanced genetic gains, and resource optimization. This approach overcomes the challenges associated with MAS for polygenic traits and enables more efficient selection for complex traits like stress tolerance. By leveraging genomic information, breeders can expedite the development of superior turfgrass cultivars that meet the evolving needs of the industry and end-users. Furthermore, the availability of the recently generated reference genome will be instrumental in supporting turfgrass breeding research as it facilitates accurate mapping of DNA markers like SNPs in linkage maps, a crucial step in identifying QTL controlling traits of interest. Furthermore, a reference genome enables the integration of meta-QTL[42], genome-wide association (GWAS) and -omics studies, as well as cross-species comparative genomic and transcriptomic analyses. Integration of these approaches in discovering genomic regions and candidate genes associated with traits of interest can expand the molecular tools available for improved selection accuracy and increased efficiency in the breeding pipeline, marking the beginning of the genomics-enabled breeding era for St. Augustinegrass.

    • The authors confirm contribution to the paper as follows: study conception and design: Milla-Lewis SR, Gaire S, Yu X; data collection: Gaire S; analysis and interpretation of results: Gaire S; draft manuscript preparation: Milla-Lewis SR, Gaire S, Yu X. All authors reviewed the results and approved the final version of the manuscript.

    • Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

      • The authors wish to thank The North Carolina State University Center for Turfgrass Environmental Research and Education for funding provided to support the PhD student working on this project.

      • The authors declare they have no conflict of interest. Susana R. Milla-Lewis is the Editorial Board member of Grass Research who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and the research groups.

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
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    Gaire S, Yu X, Milla-Lewis SR. 2024. Molecular advances in St. Augustinegrass: from DNA markers to genome sequencing. Grass Research 4: e021 doi: 10.48130/grares-0024-0017
    Gaire S, Yu X, Milla-Lewis SR. 2024. Molecular advances in St. Augustinegrass: from DNA markers to genome sequencing. Grass Research 4: e021 doi: 10.48130/grares-0024-0017

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