Transcriptomic sequencing analysis, development, and validation of EST-SSR markers in reed canary grass

Xuejie Jia; Yi Xiong; Yanli Xiong; Xiaofei Ji; Daxu Li; Shiqie Bai; Lijun Yan; Minghong You; Xiao Ma; Jianbo Zhang; Xuejie Jia; Yi Xiong; Yanli Xiong; Xiaofei Ji; Daxu Li; Shiqie Bai; Lijun Yan; Minghong You; Xiao Ma; Jianbo Zhang

doi:10.48130/GR-2023-0017

Figures (4) Tables (6)

Figure 1.
Characteristics of reed canary grass transcripts. (a) Distribution of transcripts lengths in Phalaris. (b) Functional annotation of transcripts based on Gene Ontology (GO) categorization. (c) Top 19 KEGG pathways containing the most transcripts. (d) Distribution of six SSR repeat types in different genic regions.
Figure 2.
Simple sequence repeats length distribution across different motif classification in reed canary grass.
Figure 3.
Unweighted Pair Group Method with Arithmetic (UPGMA) tree of the 19 accessions (the reliability of the clustering results is indicated by a bootstrap support value of more than 50% for each main branch of the clustering tree map) and genetic relationship among reed canary grass accessions using a Bayesian analysis.
Figure 4.
Principal coordinate analysis (PCoA) showing the relationships of the reed canary grass accessions.

Replicates	Read	Clean	Size of clean	Q20 (%)	Q30 (%)	GC (%)
Replicates	Length	Reads pairs	Base (bp)	Q20 (%)	Q30 (%)	GC (%)
Sample1	150	24,378,713	7,313,613,900	97	89.45	53.8
Sample2	150	22,716,853	6,815,055,900	97.55	91.1	53.7
Sample3	150	27,431,912	8,229,573,600	97.05	89.7	55.1
Mean	150	24,842,493	7,452,747,800	97.2	90.08	54.2

Table 1.

De novo transcriptome sequencing of reed canary grass.

Database Number of transcripts Percentage

Total 272,328 100%
KOG 46,697 17.15%
KEGG 59,324 21.78%
NR 158,464 58.19%
GO 110,631 40.62%
Swiss-Prot 106,768 39.21%
Unknown 113,24 41.58%

Table 2.
Annotation statistics of reed canary grass transcripts.

SSR mining	Number
Total number of sequences examined	272,328
Total size of examined sequences (bp)	351,691,355
Total number of identified SSRs	50,155
Number of SSR containing sequences	41,925
Number of sequences containing more than 1 SSR	6,779
Number of SSRs present in compound formation	1,936
Distribution of SSRs in different repeat types
Mono-nucleotide	22,859(45.58%)
Di-nucleotide	8,702(17.35%)
Tri-nucleotide	17,261(34.42%)
Tetra-nucleotide	824(1.64%)
Penta-nucleotide	318(0.63%)
Hexa-nucleotide	191(0.38%)

Table 3.

Statistics of SSRs identified in reed canary grass transcripts.

	TNB	NPB	PPB%	PIC	MI	Rp	H	I
SSR1	10	9	90	0.39	3.47	5.79	0.47	0.59
SSR2	16	16	100	0.38	6.01	9.05	0.49	0.62
SSR3	9	9	100	0.38	3.45	4.42	0.48	0.60
SSR4	6	6	100	0.39	2.31	2.21	0.47	0.49
SSR5	8	8	100	0.37	2.99	4.32	0.50	0.62
SSR6	7	7	100	0.37	2.62	4.00	0.50	0.61
SSR7	6	6	100	0.39	2.36	3.37	0.46	0.59
SSR8	7	7	100	0.39	2.70	3.26	0.47	0.59
SSR9	10	10	100	0.38	3.83	3.89	0.48	0.59
SSR10	7	7	100	0.38	2.63	3.68	0.49	0.63
SSR11	11	11	100	0.37	4.12	6.11	0.50	0.61
SSR12	7	7	100	0.38	2.64	4.21	0.49	0.67
SSR13	7	7	100	0.41	2.86	3.37	0.42	0.66
SSR14	9	9	100	0.38	3.45	4.32	0.48	0.6
SSR15	5	4	80	0.39	1.56	2.63	0.47	0.57
SSR16	6	6	100	0.37	2.24	1.05	0.50	0.56
SSR17	2	2	100	0.37	0.75	0.42	0.50	0.48
SSR18	5	5	100	0.41	2.07	1.16	0.41	0.63
SSR19	2	2	100	0.40	0.79	1.05	0.45	0.61
SSR20	3	3	100	0.39	1.17	1.26	0.47	0.59
SSR21	3	3	100	0.40	1.21	1.16	0.43	0.52
SSR22	3	3	100	0.37	1.12	1.37	0.50	0.62
SSR23	5	5	100	0.39	1.97	2.74	0.45	0.6
SSR24	3	3	100	0.39	1.18	2.11	0.46	0.72
SSR25	2	2	100	0.38	0.77	1.05	0.48	0.64
SSR26	2	2	100	0.38	0.76	0.95	0.49	0.61
SSR27	3	3	100	0.39	1.18	0.74	0.46	0.53
SSR28	2	2	100	0.38	0.76	1.58	0.49	0.66
SSR29	2	2	100	0.40	0.79	1.05	0.45	0.61
SSR30	4	4	100	0.39	1.57	2.11	0.46	0.56
SSR31	3	3	100	0.37	1.12	2.53	0.50	0.66
SSR32	2	2	100	0.41	0.83	1.16	0.41	0.72
SSR33	3	3	100	0.38	1.13	2.84	0.49	0.7
SSR34	2	2	100	0.40	0.79	1.05	0.45	0.56
SSR35	5	5	100	0.39	1.95	3.58	0.47	0.64
SSR36	3	3	100	0.38	1.14	1.58	0.49	0.62
SSR37	2	2	100	0.37	0.75	1.89	0.50	0.7
SSR38	2	2	100	0.38	0.75	1.05	0.49	0.64
SSR39	6	6	100	0.38	2.30	4.63	0.48	0.72
SSR40	5	5	100	0.43	2.13	1.79	0.38	0.52
SSR41	4	4	100	0.39	1.57	2.42	0.46	0.6
SSR42	2	2	100	0.38	0.76	1.58	0.49	0.71
SSR43	2	2	100	0.40	0.79	0.84	0.45	0.5
SSR44	2	2	100	0.38	0.76	1.68	0.49	0.67
SSR45	3	3	100	0.39	1.16	1.58	0.47	0.6
Total	218	216	99.08	0.37	80.74	114.63	0.50	0.61
Mean	4.84	4.80	99.33	0.39	1.85	4.98	0.47	0.61
MI, marker Index; Rp, resolving power; I, Shannon information index; H, heterozygosity.

Table 4.

Marker parameters calculated for each SSR primer combination used with reed canary grass accessions.

Geographical group	N	Na	Ne	I	He	P
NoA	11.000	1.955	1.577	0.512	0.341	96.53%
EU	4.000	1.495	1.432	0.358	0.244	62.38%
AS	2.000	0.866	1.168	0.144	0.098	23.76%
Pa	2.000	0.891	1.175	0.150	0.103	24.75%
N, Individual number of populations; Na, No. of different Alleles; Ne, No. of effective alleles; I, Shannon information index; He, Expected heterozygosity; P, Genetic variation.

Table 5.

Different genetic diversity estimates for four geographical groups of reed canary grass accessions.

Source of variation	df	SS	MS	Est. Var.	PMV (%)	Fst	P
Among pops	2	4.410	2.205	0.046	2%	0.023	0.143
Within pops	14	28.040	2.003	2.003	98%
Total	16	32.450		2.049	100%
df, degree of freedom; SS, square deviation; MS, mean square deviation; Est.Var, exist variance; Fst, coefﬁcient of genetic differentiation; PMV, Percentages of molecular variance.

Table 6.

Analysis of molecular variance (AMOVA) among and within geographical groups of reed canary grass accessions.

Database	Number of transcripts	Percentage
Total	272,328	100%
KOG	46,697	17.15%
KEGG	59,324	21.78%
NR	158,464	58.19%
GO	110,631	40.62%
Swiss-Prot	106,768	39.21%
Unknown	113,24	41.58%