基于RNA-seq数据的栽培种花生SSR位点鉴定和标记开发

doi:10.3864/j.issn.0578-1752.2020.04.003

摘要/Abstract

摘要：

【目的】鉴定花生RNA-seq数据中的SSR位点,明确转录组中SSR位点的分布和结构特点,开发与花生基因相关联的SSR标记,为花生重要功能基因的挖掘、等位变异研究和分子标记辅助育种奠定基础。【方法】根据栽培种花生全生育期中22种不同类型的组织RNA-Seq数据,使用MISA软件分析SSR位点分布及特征,采用Primer 3设计基因关联的SSR引物,并利用电子PCR软件对引物的质量进行检测,随机合成38对引物,进行多态性检测。【结果】从52 280条转录本中共鉴定19 143个SSR位点,分布于14 084条转录本,发生频率为26.94%。重复单元类型为单核苷酸—五核苷酸,以单核苷酸和三核苷酸为重复单元的SSR位点数最多,分别占位点总数的39.24%和38.40%。各重复单元优势基序类型分别为A/T、AG/CT、AAG/CTT、AAAG/CTTT和AACAC/GTGTT,占所在重复单元中的比例分别为97.62%、72.01%、30.96%、24.59%和16.67%。重复单元的重复次数为5—47次,单个SSR位点的长度的分布范围为10—47 bp,基序长度主要集中在10—14 bp;复合SSR位点的长度范围为21—249 bp,以31—40 bp为主。鉴定的SSR位点中共有13 477个SSR位点可以进行引物设计,其中5 020条转录本序列对应到特定的基因,共包含5 859个可进行引物设计的SSR位点,这些SSR位点在A基因组和B基因组共20条染色体上不均匀分布,其中B03染色体上SSR位点最多,为484个。对特定基因SSR引物进行电子PCR检测,在A.duranensis、A.ipaensis、A.hypogaea基因组中有效扩增位点分别为4 468、4 929和10 188个,有效引物数分别为3 968（67.74%）、4 232（72.25%）和5 174（88.33%）对,在A. hypogaea基因组中,SSR引物扩增位点主要以2个位点为主,其中,有1 477对引物单位点扩增。根据SSR引物扩增位点在栽培种花生基因组中的位置信息绘制了SSR位点的物理图谱。在38对SSR引物中,共有35对（92.1%）SSR引物可以扩增出清晰的条带,其中,有11对（28.9%）SSR引物在2个品种间扩增出差异条带。【结论】鉴定了13 477个可进行标记开发的SSR位点,开发、检测了5 859个基因相关SSR标记,在栽培种花生基因组中具有较高的扩增效率,并构建了基因相关SSR位点的物理图谱。

关键词: 花生, RNA-seq数据, SSR位点, 基因关联SSR标记, 物理图谱

Abstract:

【Objective】 This study aimed to identify SSRs in peanut RNA-seq data,clarify their distribution and structural characteristics,and develop gene-associated SSR markers. The study may lay the foundation for the excavation of important functional genes of peanut, the study of isometric variation and molecular markers assisted breeding. 【Method】From 22 different cultivated peanut tissue types and ontogenies that represent its full development, the reported RNA-Seq data, were used to analyze the distribution and characteristics of SSR using MISA software. Gene-associated SSR primers were designed by Primer 3.0 and its quality were detected by e-PCR 2.3.9. 38 pairs of primers were randomly synthesized for polymorphism testing. 【Result】A total of 19 143 SSRs were identified from 52 280 transcripts, distributed in 14 084 transcripts, with a frequency of 26.94%. The dominant SSR repeat unit types were mononucleotide and trinucleotide in mononucleotide to pentanucleotide, accounting for 39.24% and 38.40% of the total SSR locus. Dominant motif types of each repeat unit were A/T, AG/CT, AAG/CTT, AAAG/CTTT, AACAC/GTGTT, accounting for 97.62%, 72.01%, 30.96%, 24.59%, and 16.67% in the corresponding repeat units, respectively. The repetition of repeat units was 5-47 times, and the length distribution range of single SSR site was 10-47bp, mainly concentrated at 10-14 bp. The length range of compound SSR locus was 21-249 bp, mainly concentrated at 31-40 bp. Among all the SSR,13 477 SSR could be used to develop SSR markers, of which 5 020 transcript sequences were annotated to specific genes, containing 5 859 SSR markers locus. These SSRs were unevenly distributed on the 20 chromosomes of A and B genomes, and chromosomes B03 had the most SSR locus of 484. Using electronic PCR, 4 468, 4 929 and 10 188 effective loci were amplified in the genome of A. duranensis, A. ipaensis, A. hypogaea, with 3 968 (67.74%), 4 232 (72.25%) and 5 174 (88.33%) effective markers, respectively. In the genome of A. hypogaea, SSR primers amplified mainly with 2 loci, while 1 477 pairs of SSR primers were single-locus markers. And the physical map of amplified SSR loci was drawn according to the loci position in cultivated peanut genome. Among the randomly synthesized primers, 35 pairs (92.1%) of SSR primers amplified stable and clear bands in two peanut varieties, among which 11 pairs (28.9%) of SSR primers amplified different band. 【Conclusion】 In this study, 13 477 potential primer design SSRs were identified, 5 859 gene-associated SSR markers were developed and detected,with high amplification efficiency in cultivated peanut genome,and the physical map of gene-associated SSR were constructed.

Key words: peanut, RNA-seq data, SSR loci, gene-associated SSR markers, physical map

徐志军, 赵胜, 徐磊, 胡小文, 安东升, 刘洋. 基于RNA-seq数据的栽培种花生SSR位点鉴定和标记开发[J]. 中国农业科学, 2020, 53(4): 695-706.

ZhiJun XU, Sheng ZHAO, Lei XU, XiaoWen HU, DongSheng AN, Yang LIU. Discovery of Microsatellite Markers from RNA-seq Data in Cultivated Peanut (Arachis hypogaea)[J]. Scientia Agricultura Sinica, 2020, 53(4): 695-706.

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重复单元类型 Repeat unit type	基序种类 Motif	SSR位点数量 SSR number	比例 Ratio (%)	分布频率 Distribution frequency (%)	优势基序 Dominating motif
重复单元类型 Repeat unit type	基序种类 Motif	SSR位点数量 SSR number	比例 Ratio (%)	分布频率 Distribution frequency (%)	基序类型 Motif type	数量 Number	比例 Ratio (%)
单核苷酸 Mononucleotide	4	7513	39.24	14.37	A/T	7334	97.62
二核苷酸 Dinucleotide	12	3926	20.51	7.51	AG/CT	2827	72.01
三核苷酸 Trinucleotide	60	7351	38.40	14.06	AAG/CTT	2276	30.96
四核苷酸 Tetranucleotide	87	305	1.59	0.58	AAAG/CTTT	75	24.59
五核苷酸 Pentanucleotide	39	48	0.25	0.09	AACAC/GTGTT	8	16.67
总计 Total	202	19143		36.62

重复单元类型 Repeat unit type	重复次数Repetition times
重复单元类型 Repeat unit type	5	6	7	8	9	10	11	12	13	14	≥15
单核苷酸Mononucleotide						2985	1519	943	657	468	941
二核苷酸 Dinucleotide		1446	896	640	475	340	115	14
三核苷酸 Trinucleotide	4352	2108	761	130
四核苷酸 Tetranucleotide	246	59
五核苷酸 Pentanucleotide	48
总计 Total	4646	3613	1657	770	475	3325	1634	957	657	468	941
分布频率 Distribution frequency(%)	24.2	18.87	8.66	4.02	2.48	17.37	8.54	5.00	3.43	2.44	4.92

染色体 Chromosome	SSR位点数Number of SSR loci			对应基因数Number of genes			平均SSR位点密度 Average density of SSR loci
染色体 Chromosome	单一位点 Single loci	复合位点 Compound loci	总计 Total	单位点基因 Gene contain single loci	多位点基因 Gene contain multiple loci	总计 Total	平均SSR位点密度 Average density of SSR loci
A01	252	9	261	136	37	217	1.21
A02	170	15	185	145	13	172	1.08
A03	380	17	397	220	53	335	1.19
A04	222	18	240	160	24	212	1.13
A05	314	13	327	179	48	277	1.18
A06	229	11	240	160	26	213	1.13
A07	175	11	186	110	24	160	1.16
A08	255	13	268	146	36	225	1.19
A09	265	16	281	168	35	242	1.16
A10	198	18	216	148	22	193	1.12
B01	273	16	289	177	34	250	1.16
B02	264	15	279	157	36	236	1.18
B03	451	33	484	328	48	430	1.13
B04	323	21	344	169	49	281	1.22
B05	291	15	306	201	33	270	1.13
B06	293	16	309	152	45	253	1.22
B07	281	20	301	190	31	261	1.15
B08	249	15	264	135	39	219	1.21
B09	339	17	356	188	52	298	1.19
B10	309	17	326	182	44	276	1.18
全基因组 Whole genome	5533	326	5859	3451	729	5020	1.17

DNA模板 DNA template	扩增位点 Amplified loci	有效扩增位点 Effective amplified loci	引物扩增位点统计 Primer amplified loci statistics				有效引物数 Number of effective primer pairs
DNA模板 DNA template	扩增位点 Amplified loci	有效扩增位点 Effective amplified loci	1	2	3	>3	有效引物数 Number of effective primer pairs
A.duranensis	4760	4468 (93.86%)	3721 (93.75%)	152 (3.83%)	47 (1.18%)	49 (1.23%)	3969 (67.74%)
A.ipaensis	5264	4929 (93.64%)	3866 (91.33%)	249 (5.88%)	52 (1.23%)	66 (1.56%)	4233 (72.25%)
A.hypogaea	10818	10188 (94.17%)	1477 (28.54%)	3221 (62.24%)	252 (4.87%)	225 (4.35%)	5175 (88.33%)