Genetic diversity analysis and fingerprint construction for 45 Chinese Zoysia germplasm collections

As a perennial warm-season grass, Zoysia (Zoysia spp.) is recognized for its low maintenance requirements as well as relatively high tolerance to drought, disease, and traffic, and is widely cultivated for athletic fields, home lawns, and other recreational sites, particularly in east Asia^[1]. With the rapid development of the turf industry, it is extensively recognized that germplasm resources are hugely important. Therefore, a large number of researches are focussed on the collection^[2−5], evaluation^[6−8], and breeding^[9−11] of Zoysia spp.

Zoysia species exhibit a high rate of outcrossing and are prone to interspecific hybridization, resulting in a wide range of genetic variation among Zoysia plants^[12]. Kimball et al.^[13] identified interspecific hybridization and hypothesized that these hybrids were the result of introgression between species through common breeding methods of Zoysia, including directed hybridization of selected parents and cross-pollination in open cross areas. Genetic background analysis of abundant germplasm is an important prerequisite for identifying parents and breeding Zoysia varieties^[14]. Traditionally morphological identification has several drawbacks due to the susceptibility to environmental factors and plant growth period as well as the limited morphological indexes. Furthermore, some germplasm are difficult to differentiate based on phenotype alone^[15]. Molecular markers are an effective means to grasp the genetic information of germplasm. Among several common molecular markers, simple sequence repeats (SSRs) are widely used molecular markers in plant genetics and breeding, due to their multiallelic, codominant inheritance and extensive genome coverage^[16]. Sequence-related amplified polymorphism (SRAP) was developed by Li & Quiros^[17] in Brassica. This method is advantageous due to its simplicity, reasonable throughput rate, disclosure of numerous codominant markers, and targeting of open reading frames (ORFs)^[18]. Both marker systems have been applied to a range of fields, including the analysis of genetic diversity of germplasm^[19−22], the identification of cultivar and marker-trait association^[23]. SSR and SRAP have been reported to be effectively utilized to examine the genetic diversity^[4], analyze genetic similarity among cultivars^[24], and identify molecular markers linked with quantitative trait loci for biotic and abiotic stress tolerance in Zoysia^{[3, 25]}. These markers also serve as a potent tool for constructing DNA fingerprints^{[7, 26, 27]}.

The evaluation and improvement of germplasm are of great significance for the effective utilization of these resources. Previous researchers investigated the genetic variation of some Zoysia germplasm. Anderson^[28] measured inflorescence traits, morphological characteristics, and restricted fragment length polymorphisms (RFLPs) to evaluate the genetic and morphological variations in 11 species of Zoysia. Li et al.^[29] reported a linkage map of Zoysia matrella based on SSR markers, combined the previously reported SSR linkage maps of different Zoysia species (Z. japonica and Z. matrella) and different mapping populations (F1, S2, and F2)^[30] to construct a complete SSR genetic linkage map. Xie et al.^[31] conducted a study to analyze the genetic diversity and relationships of 84 Zoysia germplasms using inter-simple sequence repeat (ISSR) markers. Moore et al.^[32] analyzed changes in levels of allelic diversity at the gene and population levels in 40 Zoysia cultivars released between 1910 and 2016 using SSR markers.

In this study, 45 Chinese Zoysia germplasm collections were analyzed by SSR and SRAP markers. The results of these analyses were used to clarify the genetic relationship, analyze the genetic diversity of 45 Chinese Zoysia germplasm collections, and construct the fingerprint to distinguish breeding lines from the two commercially available cultivars at the molecular level. The aim of this study was to provide a theoretical basis for the development, evaluation, and breeding of Zoysia germplasm.

Forty-five Chinese Zoysia germplasm collections were used for the study, among which 38 were collected from the provinces of Liaoning, Anhui, Zhejiang, and Hainan in China, five were breeding lines from radiation mutagenesis and two commercial Z. japonica cultivars, 'Lanyin No. 3' and 'Qingdao' (Table 1). Forty-five Zoysia germplasm collections were grown in the resource nursery of Turf Experiment Station of Nanjing Agricultural University, located at 119°14′38″ east longitude and 31°49′46″ north latitude, with a subtropical monsoon climate and an average annual temperature of 15.2 °C. A randomized block design was utilized with each germplasm planted in an area of 3 m × 3 m with three replicates and plots spaced 2 m apart in August, 2020. About 100 stolons were planted evenly in each plot with 2−3 stem nodes for each stolon. Plants were watered twice weekly and fertilized once a month. Plants were not trimmed in 2021 in order to measure morphological indexes, and trimmed once a week in 2022 to evaluate turf density.

Morphological characteristic data collection

Morphological data were collected on the 15^th in June and September 2021. Six morphological characteristics of 45 Chinese Zoysia germplasm collections were determined as follows: leaf length and leaf width were obtained by randomly measuring the length and middle width of the third fully expanded leaf from the top, respectively. Measurements were repeated 10 times and the average was calculated as the final value for each replicate. Ten healthy stolons were randomly selected and the length and diameter of the fourth section were measured as internode length and stem diameter, respectively. The turf height, which is the natural height of plant growth, was measured using the five-point method^[33]. Turf density was determined by counting the number of tillers in a 5 cm × 5 cm wire frame and each plot was counted three times.

Genomic DNA extraction

Genomic DNA was extracted from young Zoysia leaves (0.1 g) using the modified CTAB method^[34]. The quality of DNA was verified by 0.8% agarose gel electrophoresis, and the DNA concentration was determined by NanoReady. The DNA of 45 samples was diluted to 50 ng·μL⁻¹ using purified water and stored in the refrigerator at 4 °C or −20 °C for later use.

SSR and SRAP analysis

Forty SSR primer pairs from Röder et al.^[35] and Tsuruta et al.^[36] were used in this study. SSR-PCR and SRAP-PCR was performed in a total volume of 20 μL containing 1 μL genomic DNA, 10 μL of 2 × Mix (Yeasen Biotechnology Co., Ltd., Shanghai, China), 1 μL of 10 μmol·L⁻¹ each PCR primer and 7 μL purified water. SSR-PCR reactions were performed in a Bio-Rad thermal cycler (Bio-Rad Inc., Hercules, CA, USA), DNA amplifications were performed with an initial step at 94 °C for 3 min, followed by 35 cycles of 50 s at 94 °C, 30 s at 55 °C, 1 min at 72 °C, and a 10 min final extension step at 72 °C.

According to the SRAP primer design method published by Li & Quiros^[17], 10 forward primers and five reverse primers were randomly selected. SRAP-PCR reactions were performed with an initial step at 94 °C for 4 min and five cycles of 60 s at 37 °C and 60 min at 72 °C. Then 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 10 s; and then a final extension at 72 °C for 10 min.

The amplifications were performed in Applied Biosystems Veriti^® thermal cycler. Amplification products were stored at 4 °C before being electrophoresed through 10% non-denatured polyacrylamide gels in 1 × TBE (pH 8.0) buffer running at 120 V constant voltage for 1.5 h and then the gels were stained with fast silver stain^{[37, 38]}. According to the silver staining results, 10 SSR primer pairs and nine SRAP primer pairs were initially screened against 45 Chinese Zoysia germplasm collections (Tables 2 & 3).

Data analysis

Gel images from all accessions were visually evaluated and coded as either a '1' for the presence or '0' for the absence of a band for each marker. Based on this method, matrices of '0' and '1' were obtained for 45 germplasm collections based on the amplification of each pair of primers. Popgene 3.2 software was used to calculate the number of alleles (Na), effective number of alleles (Ne), Shannon information index (I) and Nei's gene diversity (H). Genetic distances were calculated for the 45 Chinese Zoysia germplasm collections according to Dice^[39] using NTSYS-pc. Genetic similarity coefficient (GS) was calculated, and dendrograms was constructed using unweighted pair-group method with arithmetic averages (UPGMA) by the NTSYS-pc computer program package. The confidence probability of the fingerprint was calculated based on the probability formula P = 1/2ⁿ, where n is the number of alleles, i.e., the number of polymorphic bands for each primer pair.

A total of 395 bands were amplified with 19 pairs of primers, among which 380 were polymorphic bands, with a polymorphism ratio of 96.20% (Table 4). Ten SSR primer combinations amplified a total of 231 polymorphic bands with a polymorphism ratio of 97.18%. The number of bands scored per SSR primer combination ranged from 16 to 36, with a mean of 23.70. A total of 149 polymorphic bands were amplified by nine pairs of SRAP primers, and the polymorphism ratio was 93.43%. The number of bands scored per SRAP primer combination ranged from 13 to 30, with a mean of 17.56. Based on the amplification of SSR and SRAP primers, the effective alleles ranged from 1.192 to 1.556 with an average of 1.332, Shannon information index ranged from 0.236 to 0.510 with an average of 0.350, and Nei's gene diversity index ranged from 0.146 to 0.338, and the mean value is 0.219. The results showed that there was a high level of genetic diversity among the tested germplasm collections, and the SSR and SRAP primers screened were suitable for amplification detection of Zoysia.

Genetic similarities

NTSYS-pc software was used to calculate the value of GS between any two materials according to Dice's coefficient, which was based on the combined amplification results of SSR and SRAP (Table 5). The GS of the 45 germplasm collections ranged from 0.623 to 0.856, with an average value of 0.727 and a variation of 0.233. ZG048 was most genetically similar to ZG053 (GS = 0.856), and ZG025 was most genetically dissimilar to ZG032 (GS = 0.632). Furthermore, two commercial cultivars, Lanyin No. 3 and Qingdao, were found to be highly genetically similar (GS = 0.815). The least genetically similar to Lanyin No. 3 was ZG040 (GS = 0.651), while the least genetically similar to Qingdao was ZG008 (GS = 0.661).

Cluster analysis

Based on the results of SSR and SRAP molecular markers, the Dice genetic similarity coefficients were used to cluster 45 Chinese Zoysia germplasm collections. The cophenetic correlation for the UPGMA clustering was high (r = 0.75), suggesting that the cluster analysis strongly represented the similarity matrix. A UPGMA cluster was constructed based on combining data from both markers, which divided the 45 accessions into six major clusters at a similarity index value of 0.71 (Fig. 1). Cluster I contained 32 germplasm collections, comprised of 17 germplasm collections from Anhui, six germplasm collections from Zhejiang, five germplasm collections from Hainan, one material from Liaoning, one radiation mutagenic material and two commercial cultivars. Cluster II comprised of three germplasm collections from Anhui. Cluster III consisted of four accessions, comprising of one from Jiangsu, two from Anhui and one breeding germplasm collections. Cluster IV included two germplasm collections from Zhejiang. Cluster V included only one germplasm from Hainan, ZG046, indicating that ZG046 had some genetic differences from other germplasm collections from the same region. Cluster VI contained three radiation mutagenic materials. Using a genetic similarity coefficient of 0.73, the samples of Cluster I were differentiated into three subgroups: A, B, and C. Subgroup A consisted of Lanyin No. 3, Qingdao, and five germplasm collections from Anhui and Liaoning. Subgroup B contained 10 from Zhejiang and Anhui and one from Hainan (ZG050). Subgroup C included one radiation mutagenic material, nine germplasm collections from Zhejiang and Anhui, and four germplasm collections from Hainan.

Figure 1. 

Unweighted pair-group method with arithmetic averages dendrogram of the 45 Chinese Zoysia germplasm collections constructed using simple sequence repeats (SSR) and sequence-related amplified polymorphism (SRAP) markers.

Cluster I contained most of the germplasm collections and had the most diverse source locations, including four sources, indicating that there was genetic similarity among the germplasm from different sources. For example, ZG003 from Liaoning and ZG004 from Anhui preferentially clustered together and then aggregated with the rest of the germplasm collections in Cluster I. The genetic similarity coefficient of the two germplasm collections was 0.853, which was higher than the average. Aside from Cluster I, the other accessions preferentially clustered together according to their source location, such as Clusters II, IV, and VI.

In terms of morphological characteristics of the major clusters (Table 6), germplasm collections in subgroup A of Cluster Ⅰ were mainly presented with long and wide leaves, long internodes, large diameter of above ground stems and low density. Germplasm collections of subgroup B was tall and dense, making them suitable for vegetative propagation in ecological restoration or green space construction in parks. Germplasm collections in Subgroup C and Clusters II and III exhibited intermediate morphological characteristics, belonging to the intermediate type germplasm collections. Germplasm collections from Cluster IV (ZG032 and ZG040) were tall with low density which may not be suitable for utilizing as turf. ZG046 was the sole material in Cluster V with short leaves and short internodes, superior traits for sports field turf. Germplasm collections in Cluster VI were mainly characterized by narrow leaves and fine texture, suggesting potential to be used for ornamental lawn in urban green spaces.

Fingerprints of 45 Zoysia germplasms

The selected SSR and SRAP primers were used to amplify the DNA from 45 Chinese Zoysia germplasm collections, and the results of amplified bands were stable and reproducible. Fewer primers are preferred if they are able to distinguish among varieties. In this study, one SSR primer set (Xgwm234-5B) and two SRAP primer sets (Me3-Em1 and Me3-Em2) were selected to construct SSR and SRAP fingerprints of 45 germplasm collections, respectively, by considering the clarity and percentage of polymorphic of amplified bands for each primer pair (Table 7). The primer pairs Xgwm234-5B, Me3-Em1, and Me3-Em2 amplified 21, 30, and 12 polymorphic bands, respectively. The fingerprint detection probability formula P = 1/2ⁿ was used of which n was 21 and 42 for SSR and SRAP fingerprints, respectively. The confidence probability of both SSR and SRAP fingerprints were more than 99.999%. These results indicated that the fingerprint obtained in this study could be used to distinguish among the 45 Chinese Zoysia germplasm collections.

Many Zoysia species, such as Z. sinica, Z. japonica, and Z. matrella, were utilized as turfgrass with relatively substantial genetic diversity. Weng et al.^[40] analyzed 131 Zoysia plants collected from Taiwan with random amplified polymorphic DNA (RAPD) and isoenzymes and determined that these germplasms exhibit high genetic variation at the DNA level. Kimball et al.^[41] selected 50 pairs of SSR primers to amplify 62 DNA samples from Zoysia cultivars and accessions, and the genetic similarity obtained from their analysis ranged from 0.29 to 0.51. In our study, the genetic similarity coefficient of 45 germplasm collections ranged from 0.623 to 0.856 indicating that the diversity of 45 germplasm collections was lower. The level of genetic diversity and similarity within and between natural populations is determined by the interaction of climate differences and gene flow, with climate being a major driving force for organisms to adapt to the environment and generate heritable mutations^[42]. Zoysia has gained popularity and been widely utilized around the world due to its remarkable heat tolerance, cold tolerance, and saline-alkali tolerance, which results in the complex genetic variation of Zoysia plants.

In this study, the 45 germplasm collections were divided into six clusters by UPGMA. The geographic origin of Cluster I was relatively complex, comprising of germplasm collections from Anhui (17 samples), Zhejiang (six samples), Hainan (five samples), Liaoning (one sample), one radiation mutagenic material, and two commercial cultivars. Notably, the two control cultivars of Lanyin No. 3 and Qingdao are both in subgroup A of cluster I, yet their origins are quite distinct. Lanyin No. 3 was introduced from the United States by Gansu Ecology Institute in 1988, while Qingdao was collected in Jiaozhou Bay, Shandong Province in 1990, and subsequently cultivated and domesticated. Similarly, it has been reported that the Zoysia cultivars Empire, JaMur, and Atlantic were difficult to be differentiated by 40 SSR markers^[24]. Numerous warm-season turfgrass cultivars are highly genetically similar despite reported pedigrees or place of collection^{[43, 44]}. Utilizing other types of markers such as single nucleotide polymorphisms (SNPs) or whole genome sequencing of these cultivars might be able to provide additional information to uncover genotypic diversity. Cluster V was comprised of a single ZG046 from Hainan, while the other five (ZG047, ZG048, ZG049, ZG050, and ZG053) from Hainan were placed in Cluster I. Additionally, these five germplasm collections exhibited significant differences from ZG046 in leaf length and leaf width. Results showed that the genetic distance between ZG046 and the other five germplasm collections was significant. Therefore, ZG046 can be used as a parent material to expand the genetic basis of the breeding population and increase the genetic diversity of breeding material, which would be beneficial for further Zoysia breeding improvement.

In this study, 45 Chinese Zoysia germplasm collections were characterized and analyzed based on their morphological traits and molecular clustering results. The morphological traits of Zoysia were found to be influenced by various factors, such as origin and genetic inheritance, as well as environmental aspects such as adaptability to the planting environment, centralized management of breeders and extensive cultivation, which have led to increased gene exchange among the Zoysia populations^[45]. Liu et al.^[46] conducted genetic diversity analysis of 42 seashore paspalum (Paspalum vaginatum Sw.) based on morphology and SRAP molecular markers, and the results of the molecular marker clustering showed that germplasms from the same region were more likely to be clustered together, while the morphology clustering showed that the germplasms clustered together often originated from distinct geographical locations.

DNA fingerprints generated using molecular markers can offer more precise genetic information^[47]. Molecular markers have been utilized to construct fingerprints to identify plant varieties, including radish and cucumber^{[48, 49]}. Accurate identification of cultivars is essential for cultivation and breeding^[50]. This study demonstrates that both SSR and SRAP are effective methods for constructing fingerprints of Zoysia germplasm. The 21 polymorphic bands amplified by one pair of SSR primer Xgwm234-5B were able to distinguish all 45 germplasm collections, while the SRAP molecular marker required a total of 43 bands amplified by two pairs of primers Me3-Em1 and Me3-Em2 to generate the fingerprints of the 45 germplasm collections. As Zoysia breeding advances, the number of varieties will continue to increase. To ensure the accuracy and reliability of the fingerprint of increasing number of new varieties, additional primers need to be developed, and it is essential to expand the fingerprint database of Zoysia. In this study, 45 Zoysia lines were divided into six clusters based on both SSR and SRAP markers and fingerprints of those plants were constructed. The findings of this study provided a theoretical basis for the subsequent evaluation and identification of Zoysia germplasm as well as new variety development.