基于苦楝转录组测序的SSR分子标记开发

doi:10.11707/j.1001-7488.20210609

摘要/Abstract

摘要：

目的: 基于转录组测序数据开发苦楝SSR标记，为苦楝种质资源的选育、评价和遗传改良提供理论基础和科学依据。方法: 利用Illumina HiSeq^TM 2500平台对苦楝叶片进行高通量测序，分别利用Trinity、MISA软件对转录组数据进行拼接组装、序列检索及SSR分布类型和特征分析。采用Primer 3软件设计合成100对SSR引物，分别用2%琼脂糖凝胶电泳和PAGE凝胶电泳进行初步筛选和多态性筛选。结果: 共获得Unigenes 20 077条，平均长度为1 431.82 bp，N50为1 955 bp；对Unigenes进行SSR位点的发掘和分析，发现4 116条Unigenes序列中含有5 469个SSR位点，SSR的发生频率为20.50%，平均分布距离为8.74 kb；单、二、三核苷酸重复单元分别占总SSR的50.23%、24.43%和22.11%；SSR重复单元以6~10次重复的最多，占总数的51.27%。筛选出16对多态性引物并进行PCR扩增，共检测到56个等位基因片段，平均每个位点含3.5个，平均有效等位基因数为2.31个，平均Shannon多样性指数I为0.861，平均多态性信息含量PIC为0.507。结论: 苦楝转录组SSR位点具有较高的出现频率和分布密度，基元类型和重复次数相对较高，具有高多态性的潜能，可以进行有目的的引物设计和开发。开发的16对SSR引物在苦楝上可检测到较高的多态信息含量，进一步丰富了楝科现有SSR标记数据库资源，可为楝科植物在基因组水平上的遗传多样性分析和分子辅助育种等提供参考。

关键词: 苦楝, 转录组, EST-SSR, 引物

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

中图分类号:

S718.46

蔡金峰,杨晓明,郁万文,汪贵斌,曹福亮. 基于苦楝转录组测序的SSR分子标记开发[J]. 林业科学, 2021, 57(6): 85-92.

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.

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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