EST-SSR markers development and application in an important medicinal plant, Schisandra chinensis (Schisandraceae)

Figures & data

Table 1. Schisandra chinensis accessions analyzed in this work.

No.	Name	Origin	No.	Name	Origin	No.	Name	Origin
1	Yanhong	Jilin	15	Changbai	Jilin	29	19-3-4	Jilin
2	BaiGuo	Jilin	16	84-7-2	Jilin	30	2012-7	Jilin
3	Zaohong	Jilin	17	158-11-6	Jilin	31	27-2-6	Jilin
4	Jinwuwei	Jilin	18	18-10-2	Jilin	32	A18-8-5	Liaoning
5	35-6-1	Jilin	19	8-1-1	Jilin	33	20-1-4	Jilin
6	168-1-6	Jilin	20	17-11-3	Jilin	34	19-3-5	Jilin
7	10-S8-N2	Jilin	21	130-9-6	Jilin	35	A71	Jilin
8	162-1-4	Jilin	22	2012-5	Liaoning	36	18-10-2	Jilin
9	17-N1-N1	Liaoning	23	20-28-1	Liaoning	37	94-9-2	Jilin
10	12-(-2)-1	Jilin	24	11-2-3	Liaoning	38	25-3-2	Jilin
11	25-6-10	Liaoning	25	14-3-1-5	Jilin	39	13-5-2	Jilin
12	50-5	Liaoning	26	106-43	Jilin	40	171-1-4	Jilin
13	137-3-3	Jilin	27	156-5-2	Heilongjiang	41	235-5-6	Jilin
14	99-8-7	Jilin	28	82-5-6	Jilin	42	11N1S1	Heilongjiang

Table 2. Functional annotation of S. chinensis unigenes.

Database	Unigene	Percentage
COG	7740	12.95%
GO	15625	26.13%
KEGG	9637	16.12%
KOG	14849	24.84%
Pfam	17203	28.77%
Swissprot	17998	30.10%
eggNOG	24329	40.69%
NR	26350	44.07%
All_Annotated	26827	44.87%

Figure 1. Distribution of the Blast hits in the NR database.

Table 3. Type, number and frequency of expressed sequence tag-simple sequence repeat markers in Schisandra chinensis.

Repeat type	Repeat number							Total	Percentage
	5	6	7	8	9	10	>10
Di		404	314	369	590	447	109	2233	64.54%
Tri	472	224	110	19			1	826	23.87%
Tetra	22	6	2			1		31	0.90%
Penta	4							4	0.12%
Hexa	8	2	2	1			1	14	0.40%
Compound								352	10.17%
Total	506	636	428	389	590	448	111	3460	100%
Percentage	8.57%	10.78%	7.25%	6.59%	10.00%	22.91%	33.90%

Table 4. Distribution of EST-SSR motifs in the transcriptome of S. chinensis.

Motif	Repeat number	Motif	Repeat number
GT/AC	520	CTC/GAG	153
TC/GA	9969	ATT/AAT	126
AG/CT	8131	AGC/GCT	261
CA/TG	833	CCG/CGG	55
AT	624	CGT/ACG	36
TA	471	ATG/CAT	149
CG	18	TCC/GGA	91
GC	6	ATC/GAT	195
Dinucleotide	20572	GTT/AAC	60
TGC/GCA	210	GAA/TTC	846
AAG/CTT	517	TGA/TCA	225
ACT/AGT	10	GCG/CGC	39
GGC/GCC	33	TGT/ACA	56
TAG/CTA	5	AGA/TCT	688
TCG/CGA	43	CAG/CTG	239
GAC/GTC	51	CCT/AGG	80
TAA/TTA	96	TGG/CCA	234
TAC/GTA	29	Trinucleotide	5100
CAA/TTG	89	Tetranucleotide	206
TAT/ATA	123	Pentanucleotide	27
GTG/CAC	210	Hexanucleotide	6
ACC/GGT	151

Figure 2. Frequency distribution of putative EST-SSRs identified from S. chinensis ESTs, according to motif type (a) and number of repeats (b).

Table 5. Information of 14 SSRs used for assessment of genetic diversity among 42 S. chinensis accessions.

No.	SSR name	Repeat motif	Forward primer sequence (5′→3′)	Tm (°C)	Reverse primer sequence (5′→3′)	Tm (°C)	Expected siz e (bp)
1	SSC00061	(TCT)6	GGCAAGCAGACACAGTCAAA	60.03	GTAGTGGAGGAGCGAAGACG	60.01	253
2	SSC00055	(CAG)6	AAGAAAGCCAGAACACGTCC	59.33	GCTTTTGTGACTTGGGGTGT	60.01	241
3	SSC00094	(AGA)6	TGTAAACTTTGAAGTCCAACCG	59.17	TTGCTTTCTGAAGCACGAGA	59.87	188
4	SSC00095	(ATT)6	CTGTCCCGCTTCTCTTTGAG	60.13	GTGGCCCGAGGTACTAACAA	59.99	123
5	SSC00082	(CAT)7	ATTACTACCACAGCCACCGC	60.02	GATGATCAGGTGTGGTCCCT	60.07	267
6	SSC00098	(GCG)7	CTCTCTCGCTTCATTCACCC	59.95	TCATCACTGTTCGATTCCCA	60.05	144
7	SSC00096	(TGGGCC)6	ACCATCTCCACTTTCGCAAC	60.03	ATCTGGAACTGTGATTGGGC	59.98	216
8	SSC00097	(CAT)7	CAAGCTTTCTTTCCCTCGTG	60.05	GATGATCAGGTGTGGTCCCT	60.02	267
9	SSC00035	(CAT)6	CAACTGGCTGGGCTGATATT	59.96	TGTTCGATTTGGTTGTTGGA	59.45	253
10	SSC00019	(ATC)7	GTGGAATTTCGATTCAGGGA	59.88	GGGAATTGGTCAATTGGGTA	59.86	186
11	SSC00041	(GTG)5	TAGTATGGGCTTGGAGGTGG	60.04	AATTTACTGCATTCGCTCGC	60.02	102
12	SSC00036	(CCA)6	ACTGCCATTCGCATTACTCC	59.63	TACGGATGGGAAGGTGAGAG	59.32	199
13	SSC00028	(CAT)5	TGAGGTCCCCATGAAGAAAG	59.84	CCGTTGGATGAAAGGAGTGT	59.86	137
14	SSC00062	(GTC)5	CACTTCAGAAATCTCGGCGT	60.40	CCAGCATGTCCAATCTTTCA	59.65	152

Table 6. Estimation of genetic diversity based on 14 SSRs among 42 S. chinensis.

No.	SSR name	Allele number	PIC	Effective allele number	Ho	He	SI	Amplicon range (bp)
1	SSC00061	2	0.2604	1.2654	0.7877	0.2098	0.3650	242–253
2	SSC00055	2	0.3741	1.9955	0.4951	0.4989	0.6920	240–255
3	SSC00094	4	0.5094	1.7890	0.5536	0.4410	0.8044	100–115
4	SSC00095	4	0.3749	1.9989	0.4943	0.4997	0.6929	192–203
5	SSC00082	3	0.3702	1.9192	0.5148	0.4790	0.6720	235–246
6	SSC00098	3	0.3456	1.6897	0.5869	0.4082	0.5983	270–283
7	SSC00096	2	0.5504	1.6156	0.6143	0.3810	0.5689	137–152
8	SSC00097	2	0.5701	1.9888	0.4965	0.4972	0.6903	128–139
9	SSC00035	3	0.5817	2.9132	0.3337	0.6567	1.0842	218–280
10	SSC00019	3	0.4635	1.9765	0.4998	0.4941	0.8562	140–155
11	SSC00041	2	0.3736	1.9836	0.4965	0.4959	0.6890	201–200
12	SSC00036	2	0.5817	1.2195	0.8177	0.1800	0.3251	181–195
13	SSC00028	3	0.3710	1.0241	0.9762	0.0235	0.4068	150–162
14	SSC00062	3	0.2865	1.3197	0.7546	0.2423	0.0646	188–202
	Mean	2.71	0.4295	1.7642	0.6016	0.3934	0.6078

Note: PIC, Polymorphism information content; He, expected heterozygosity; Ho, observed heterozygosity; SI, Shannon’s Information index.

Figure 3. Dendrogram of S. chinensis germplasms generated using the unweighted pair group method with arithmetic mean.

Availability of data and materials

The raw data of RNA-seq are deposited in Sequence Read Archives Database (http://www.ncbi.nlm.nih.gov/bioproject/609148) under accession number PRJNA609148. Other datasets supporting the conclusions of this article are included within the article (and its additional file).