基于苦楝转录组测序的SSR分子标记开发

doi:10.11707/j.1001-7488.20210609

Abstract

Abstract:

Objective: Based on transcriptome sequencing data,SSR markers of Melia azedarach were developed to provide theoretical and scientific basis for breeding,evaluation and genetic improvement of M. azedarach. Method: The transcriptome of M. azedarach leaves was sequenced by Illumina HiSeq^TM 2500. The transcriptome data was assembled,sequenced and analyzed by the Trinity and MISA software. Using the Primer 3 software,100 pairs of SSR primers were designed and synthesized. The 2% agarose gel electrophoresis and PAGE gel electrophoresis were used for preliminary screening and polymorphic screening. Result: A total of 20 077 unigenes were assembled and clustered based on the transcriptome sequences. The average length of unigenes was 1 431.82 bp and N50 was 1 955 bp. Simple Sequence Repeats (SSRs) were further analyzed in the sequences of unigenes,and 5 469 SSRs from 4 116 unigene sequences (20.50% of total) were identified. The average distribution distance of SSRs was 8.74 kb. The analysis of repetition types indicated that mononucleotide repetition was the main one,accounting for 50.23%,followed by dinucleotide (24.43%) and trinucleotide (22.11%). In SSR repeat units,6-10 repeats were the most,accounting for 51.27% of the total. 16 pairs of polymorphic primers were screened and amplified by PCR. A total of 56 alleles were detected with an average of 3.5 alleles and 2.31 effective alleles per locus. The average Shannon's diversity index (I) and the average polymorphism information content (PIC) were 0.861 and 0.507,respectively. Conclusion: The frequency of SSR loci,the distribution density and repetition frequency of SSR in transcriptome of M. azedarach were all at a high level. A total of 16 primer pairs developed in this study further enriched the existing SSR markers of M. azedarach,which are valuable for further studies on genetic diversity and molecular breeding of M. azedarach.

Key words: Melia azedarach, transcriptome sequencing, EST-SSR, primer

CLC Number:

S718.46

Jinfeng Cai,Xiaoming Yang,Wanwen Yu,Guibin Wang,Fuliang Cao. Development of SSR Molecular Markers Based on Transcriptome Sequencing of Melia azedarach[J]. Scientia Silvae Sinicae, 2021, 57(6): 85-92.

Figures/Tables 9

Table 1

Table 2

Table 3

Fig.1

Table 4

Table 5

Fig.2

Table 6

Characteristics of 16 polymorphic SSR primers of Melia azedarach"

引物编号 Primer number	重复基元 Repeat motif	引物序列 Primer sequence (5′—3′)	退火温度 T_m/℃	预计长度 Expected size/bp	等位基因数 Observed number of alleles(N_a)	有效等位基因数 Effective number of alleles(N_e)	Shannon 信息指数 Shannon’s information index(I)	多态信息含量 Polymorphic information content (PIC)
KL-P2-5	(AG)₁₀	F: CAACAGTCCTCCCTGCAAAT R: GTCACTCGCAATGATGTTGG	59	225	3	1.291	0.436	0.523
KL-P2-6	(GA)₂₇	F: CTCTTTGTGCCCTCATTCGT R: TGCTCATAATGCCATCCAAA	60	131	5	3.059	1.242	0.613
KL-P2-9	(AG)₁₁	F: CATTGTCCTTTCTTGCCGTT R: TGTCTGGTCAATGATGCGTT	59	206	5	2.866	1.191	0.653
KL-P3-2	(TTA)₈	F: ATTACACCCCCTCCCAAAAA R: AGGTGAGAATGGACGAATGG	60	244	5	2.551	1.073	0.641
KL-P3-7	(AGT)₁₁	F: CAACACGAGACGAGACCTGA R: TCGTTTTGAGGTGAGGAACC	60	176	5	2.398	1.054	0.598
KL-P3-8	(CCT)₅	F: CCTCTTTCTCCTTGTCCACG R: TGTGTTCCTCGTACACCCAA	60	106	5	4.420	1.535	0.654
KL-P3-11	(AAT)₇	F: CACCAAAGTGGCACAAGAGA R: GGACAAGAGAGGCAACAATCA	60	277	2	1.634	0.576	0.385
KL-P3-12	(GAA)₅	F: TTCCACAATGTAAGCCACGA R: ATGACTCGCGCAATATCAGA	58	115	2	1.840	0.649	0.423
KL-P4-2	(TAGC)₅	F: GAAACTGTCCGCAACAAACA R: GAACAAGAGGGATTCGTGGA	59	263	3	2.017	0.721	0.486
KL-P4-10	(TTTC)₅	F: CTCCCCTGCCTGTTATGGTA R: AGGGGCCAGAGCTGCTATAC	57	202	3	1.796	0.659	0.478
KL-P4-14	(AAAT)₅	F: CAAGGGCCCAACAAAAGTAA R: CCAGTAAAAATCCTGTCCCG	60	272	2	1.195	0.301	0.399
KL-P5-2	(TTTAT)₅	F: TTTGCAATTGAACACCTGGA R: TTCCACCTCTCTCCCAAGAA	60	266	5	4.193	1.482	0.692
KL-P5-4	(GACAT)₅	F: CTGAAACCCCGAAATTGAAA R: CGAAGACCATCATCTGGGAT	59	208	2	1.620	0.571	0.356
KL-P5-15	(CAATG)₅	F: AAAACAAAAGCCAAAAGGGG R: AGGCAGAGCATCAAACAACC	59	172	2	1.220	0.325	0.324
KL-P6-1	(AGGGTG)₅	F: TACGTGGAGGGAATACAGCC R: ATCTTCCTCGCGATTTTCCT	59	261	4	2.629	1.069	0.458
KL-P6-2	(GGCTGT)₅	F: CGTATTGTTCGTTTCTCCTGAA R: ACAGCAAGCGGGCTAGTTTA	60	265	3	2.236	0.886	0.421
均值Mean					3.5	2.310	0.861	0.507

Table 6

Fig.3

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种源 Provenances	样本数 Sample number
江苏南京Nanjing, Jiangsu	10
江苏邳州Pizhou, Jiangsu	16
江苏泰兴Taixing, Jiangsu	11
山东东明Dongming, Shandong	6
山东东营Dongying, Shandong	7
江西南昌Nanchang, Jiangxi	5
江西井冈山Jinggangshan, Jiangxi	4
河南信阳Xinyang, Henan	4
安徽合肥Hefei, Anhui	4
广东汕头Shantou, Guangdong	6
广西南宁Nanning, Guangxi	8
湖南长沙Changsha, Hunan	3
湖北荆州Jingzhou, Hubei	5
浙江临安Lin’an, Zhejiang	11
浙江乐清Yueqing, Zhejiang	6
合计Total	106

长度范围 Length range/bp	数量 Number	比例 Percentage(%)
0~500	5 188	25.84
501~1 000	4 391	21.87
1 001~1 500	3 285	16.37
1 501~2 000	2 691	13.40
2 001~2 500	1 780	8.87
2 501~3 000	1 099	5.47
3 001~3 500	631	3.14
3 501~4 000	414	2.06
4 001~4 500	249	1.24
＞4 500	349	1.74
合计Total	20 077	100

重复类型 Repeat type	SSR数量 Number of SSR	百分比 Percentage(%)	分布频率 Frequency(%)	平均距离 Average distance/kb	平均长度 Average length/bp
单核苷酸Mononucleotide	2 747	50.23	13.68	17.40	11.81
二核苷酸Dinucleotide	1 336	24.43	6.67	35.78	18.46
三核苷酸Trinucleotide	1 209	22.10	6.02	39.54	18.28
四核苷酸Tetranucleotide	117	2.14	0.58	408.59	21.18
五核苷酸Pentanucleotide	31	0.57	0.15	1 542.11	26.89
六核苷酸Hexanucleotide	29	0.53	0.14	1 648.47	33.46
总计Total	5 469	100	27.24	8.74	19.41

重复类型 Repeat type	基元种类数 Number of motif type	重复基元Repeat motif	数目Number	比例 Percentage(%)
单核苷酸 Mononucleotide	2	A/T	2 736	50.03
单核苷酸 Mononucleotide	2	C/G	11	0.20
二核苷酸 Dinucleotide	4	AG/CT	700	12.79
		AT/AT	497	9.09
		AC/GT	135	2.47
		CG/CG	4	0.07
三核苷酸 Trinucleotide	10	AAG/CTT	355	6.49
		AAT/ATT	178	3.25
		ATC/ATG	162	2.96
		AGC/CTG	154	2.82
		其他类型Other types	360	6.58
四核苷酸 Tetranucleotide	24	AAAG/CTTT	16	0.29
		AAAT/ATTT	20	0.37
		ACAT/ATGT	14	0.26
		其他类型Other types	67	1.23
五核苷酸 Pentanucleotide	25	AAGAG/CTCTT	3	0.05
		AAAAG/CTTTT	2	0.04
		AAATT/AATTT	2	0.04
		其他类型Other types	24	0.44
六核苷酸 Hexanucleotide	25	AAGATG/ATCTTC	3	0.05
六核苷酸 Hexanucleotide	25	其他类型Other types	26	0.48
总计Total	90		5 469	100

重复类型 Repeat type	重复次数Repeat number
重复类型 Repeat type	1~5	6~10	11~15	＞15
单核苷酸Mononucleotide	0	1 173	1 341	233
二核苷酸Dinucleotide	0	990	224	122
三核苷酸Trinucleotide	594	588	21	6
四核苷酸Tetranucleotide	84	33	0	0
五核苷酸Pentanucleotide	21	10	0	0
六核苷酸Hexanucleotide	19	10	0	0
总计Total	718	2 804	1 586	361
比例Percentage(%)	13.13	51.27	29.00	0.66

Development of SSR Molecular Markers Based on Transcriptome Sequencing of Melia azedarach

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