徐志军¹, 赵胜², 徐磊¹, 胡小文¹, 安东升¹, 刘洋^,1 1 中国热带农业科学院湛江实验站/广东省旱作节水农业工程技术研发中心,广东湛江 524013
2 中国农业科学院农业基因组研究所, 广东深圳 518120

Discovery of Microsatellite Markers from RNA-seq Data in Cultivated Peanut (Arachis hypogaea)

XU ZhiJun¹, ZHAO Sheng², XU Lei¹, HU XiaoWen¹, AN DongSheng¹, LIU Yang^,1 1 Zhanjiang Experiment Station, Chinese Academy of Tropical Agricultural Sciences/Guangdong Engineering Technology Research Center for Dryland Water-saving Agriculture, Zhanjiang 524013, Guangdong
2 Shenzhen Agricultural Genome Research Institute, Chinese Academy of Agricultural Sciences, Shenzhen 518120, Guangdong

通讯作者: 刘洋,E-mail： lyfull@163.com

责任编辑: 李莉
收稿日期:2019-07-28接受日期:2019-10-21网络出版日期:2020-02-16

基金资助:

中国热带农业科学院科技创新团队专项资金.1630102017002

Received:2019-07-28Accepted:2019-10-21Online:2020-02-16
作者简介 About authors
徐志军,E-mail：zhijunxu1990@163.com。








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.
Keywords：peanut;RNA-seq data;SSR loci;gene-associated SSR markers;physical map


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本文引用格式
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 doi:10.3864/j.issn.0578-1752.2020.04.003
XU ZhiJun, ZHAO Sheng, XU Lei, HU XiaoWen, AN DongSheng, LIU Yang. Discovery of Microsatellite Markers from RNA-seq Data in Cultivated Peanut (Arachis hypogaea)[J]. Scientia Acricultura Sinica, 2020, 53(4): 695-706 doi:10.3864/j.issn.0578-1752.2020.04.003

0 引言

【研究意义】栽培种花生（Arachis hypogaea）是中国主要的油料与经济作物之一,在保证中国食用油和植物蛋白供给、促进农民增收方面具有重要作用。伴随花生基因组学和分子生物学的发展,大量基于分子标记的花生品种鉴定^[1,2]、种质资源遗传多样性^[3,4,5]、重要性状的QTL定位^[6,7,8,9]、基因挖掘和育种研究^{[10,11,12,13,14]}得以开展。然而,由于花生遗传基础狭窄,SSR标记多态性较低,基因组较大（约2.7 GB）,且结构复杂,现有的SSR标记,特别是用于高密度遗传图谱构建和重要功能基因挖掘的标记还较为缺乏,限制了花生重要功能基因的解析和应用。【前人研究进展】近年来,SSR标记因其分布广泛、共显性、多态性好、分辨率高、重复性好等优点而被广泛应用于花生遗传研究和育种实践中。利用基因组文库（如BAC文库）^{[15,16,17,18,19,20,21,22,23,24]}、表达序列标签（expressed sequence tags,EST）文库^{[25,26,27,28,29,30,31,32]}、转录组数据^[33,34,35]、全基因组数据^{[9, 36-37]}和近缘物种（如大豆）^[38]中的SSR序列进行花生SSR标记的开发,并应用于花生的研究。任小平等^[2]利用筛选到的60对核心SSR标记构建了100份花生品种的指纹图谱;张照华等^[39]在高油酸育种中利用62对SSR标记对10个BC₄F₂特定基因型株系进行遗传背景评估,筛选出与亲本中花16最优的近等基因系材料。除此之外,利用基于SSR标记的遗传连锁图谱和关联图谱,鉴定出与栽培种花重要农艺性状紧密连锁的QTL,如株高等株型相关性状^[6,40-42]、荚果和种子相关性状^[7-8,43-44]、抗旱^[45]、叶斑病等抗病性^[46,47]。在水稻、大豆、玉米等作物上的研究还表明,功能基因中的特异性SSR标记（或紧密连锁标记）还可以用于分析基因的等位变异和功能变异^[48,49,50]。【本研究切入点】目前,尽管一批栽培种花生重要性状相关QTL被鉴定出来,但是进入育种应用的还较少,最主要的原因是构建的遗传图谱标记密度还不够高,鉴定的QTL的遗传距离还较大,标记与基因间的连锁还不够紧密,在实际运用中基因丢失的风险较高。因此,有必要利用现有资源开发更多SSR标记,对这些重要功能基因进行精细定位和图位克隆。利用野生花生基因组,JOSH等^[51]对栽培种花生全生育期中22种不同类型的组织进行RNA-seq测序,组装了栽培种花生的转录组图谱,包含了花生正常生育条件下最多的基因转录本数据,这些转录本数据中包含了大量还未利用的SSR标记资源,且来自转录组的SSR直接与功能基因的表达相关,是开发功能基因特征标记的潜在资源。【拟解决的关键问题】本研究拟利用已发表的栽培种花生RNA-seq数据,鉴定SSR位点、开发与基因相关联的SSR标记,进一步丰富花生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。

1 材料与方法

1.1 试验材料

栽培种花生的RNA-seq组装数据、注释信息来源于花生基因组数据库（https://peanutbase.org/）。

1.2 RNA-seq数据处理

根据转录组数据注释信息,将组装序列与基因名称、染色体定位、基因功能等信息进行一一对应。

1.3 SSR位点挖掘及基因SSR引物设计

使用MISA软件（microsatellite identification tool,http://pgrc.ipk-gatersleben.de/misa/）搜索栽培种花生转录组unigene中的简单重复序列,并对SSR重复基序类型进行特征分析。查找标准：单核苷酸基序至少重复次数为10,而2、3、4、5和6核苷酸基序最少重复次数分别为6、5、5、5和5。

使用Primer3.0软件对SSR位点进行引物设计,每个SSR位点分别设计3组引物,并且满足以下的特征：（1）长度在15—25 bp;（2）PCR扩增产物长度为100—400 bp;（3）退火温度（Tm值）在50℃—60℃;（4）GC的含量在40%—60%;（5）避免出现发夹结构及引物二聚体。

1.4 e-PCR引物质量检测

使用e-PCR Version: 2.3.9对设计的引物进行电子PCR检测,参数设置按照DENG等^[52]方法进行。分别分析来源于栽培种花生转录组的SSR引物在A.duranensis、A.ipaensis和栽培种花生（Tifrunner）全基因组（https://peanutbase.org/）中的扩增情况,统计并记录引物在基因组上的扩增次数,剔除扩增产物长度小于100 bp或大于500 bp及特异性不好的引物。使用Tbtools^[53]软件对扩增位点在基因组上的位置进行可视化。

1.5 花生DNA提取及PCR检测

选取花生幼嫩叶片,采用改良CTAB法提取DNA,用1%的琼脂糖凝胶电泳检测DNA浓度与纯度,于-20℃保存备用。38对随机选取的基因SSR引物由生工生物工程（上海）股份有限公司合成（电子附表1）。PCR反应体系和PCR程序按照HUANG等^[6]方法进行。

2 结果

2.1 栽培种花生RNA-seq SSR位点分布及结构特点

栽培种花生22个组织RNA-seq深度测序共组装获得52 280条转录本（总长度75 821 219 bp）,其中33 293条转录本注释到相应基因（注释到A基因组16 222个基因,B基因组17 071个基因）。利用MISA软件从全部转录本中共鉴定出19 143个SSR位点,其中有1 494个SSR位点以复合位点的形式存在。在全部转录本中共有14 084条转录本含有SSR位点,发生频率为26.94%,平均每3.96 kb出现一个SSR;3 606条（6.90%）转录本中含有≥2个SSR位点,大部分转录本含有2—4个位点,单条转录本中最多含7个SSR位点。在所有鉴定的SSR位点中,大部分SSR位点分布于转录本序列5′端300 bp或3′端300 bp的区域,包括UTR、内含子和CDS区域。

栽培种花生转录组重复单元类型丰富,单核苷酸到五核苷酸均存在,各重复单元组成的SSR数量上存在着较大差异（表1）。其中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SSR位点总数的39.24%和38.40%,分布频率为14.37%和14.06%,其中以五核苷酸为重复单元的SSR位点数最少,仅为48个。在SSR位点基序种类方面,不同重复单元在基序种类上存在着丰富的多样性,单核苷酸到五核苷酸基序种类分别为4、12、60、87和39种,共202种,从单核苷酸到四核苷酸基序种类随着重复单元碱基数增加而增加（表1）。

Table 1
表1
表1栽培种花生RNA-seq SSR位点分布特征
Table 1Distribution of RNA-seq SSR locus characteristics in cultivated peanut

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
					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

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各重复单元优势基序类型随着重复单元增加,在所属重复单元SSR位点中所占的比例呈下降趋势（表1）。在鉴定出的SSR位点中,单核苷酸重复单元中,优势基序类型为A/T,为7 334个,占所有以单核苷酸为重复单元的SSR位点的97.62%;二核苷酸重复单元中,优势基序类型为AG/CT,占该重复单元位点的72.01%;三核苷酸到五核苷酸重复单元中,优势基序类型依次为30.96%、24.59%和16.67%。

2.2 栽培种花生RNA-seq SSR重复次数和基序长度

鉴定的SSR位点各重复单元的重复次数和SSR位点长度存在着明显的差异（表2和图1）。重复单元的重复次数为5—47（单核苷酸）次,随着重复次数的增加,同一重复单元类型的SSR位点数逐渐减少。其中,单核苷酸重复次数主要集中在10—12次;二核苷酸重复次数主要集中在6—8次;三核苷酸重复次数主要集中在5—6次;四核苷酸和五核苷酸重复次数主要集中在5次。从整体上看,重复单元的重复次数主要集中在5、6和10次,在所有SSR位点中的分布频率分别为24.24%、18.87%和17.37%。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,其中,长度为10和12 bp的基序数量最多,分别为2 985和2 389个;复合SSR位点的长度范围为21—249 bp,其中,以长度为31—40 bp的复合位点最多,随着基序长度增加,复合SSR位点数呈逐步减少的趋势。

Table 2
表2
表2栽培种花生SSR各重复单元重复次数及分布频率
Table 2Repetition times and distribution frequency of each SSR repeat unit in cultivated peanut

重复单元类型 Repeat unit type	重复次数Repetition times
	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

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图1

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图1SSR基序长度分布

Fig. 1Distribution of SSR motif length

2.3 栽培种花生SSR标记的开发

利用primer3.0软件,对14 084条转录本中的SSR位点进行引物设计,发现共有13 477个SSR位点可以进行引物设计,其中,可以进行引物设计的单个SSR 位点有12 515个,复合SSR位点962个。不能进行引物设计的SSR位点共4 172个,其中,单个SSR位点3 797个,复合SSR位点375个;这些SSR位点中共有1 771个SSR位点位于序列5′端50 bp以内,2 179个SSR位点位于3′端100 bp区域内,SSR位点位于序列起始端和末端,位点一端序列过短或无序列是造成这些位点不能进行引物设计的主要原因。在可以进行引物设计的序列中,共有5 661条转录本未注释到基因,这些序列中共包含7 305个SSR位点,有1 235条序列含有多个位点,单条序列最多含有7个SSR位点。

根据序列注释信息,共有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（单一位点和复合位点）,基因的平均SSR位点密度为1.17（表3）,共设计出17 574对特定基因SSR引物（每个位点设计3对引物）。与基因对应的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（表3）,其中B03染色体上SSR位点最多,为484个;单一位点范围为170（A02）—451（B03）,共5 533个;复合位点范围为9（A01）—33（B03）,共326个。这些包含SSR位点的基因主要以单位点基因的形式存在（68.75%）,单条染色体基因数目为160（A07)—430（B03）,其中单位点基因范围为110（A07）—328（B03）,多位点基因范围为13（A02)—49（B04）。

Table 3
表3
表3可进行引物设计的特异基因SSR位点统计
Table 3Statistics of primer design specific gene-associated SSR

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
	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
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

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2.5 e-PCR引物质量检测分析

利用一组特定基因SSR引物（5 859对）,分别以A.duranensis、A.ipaensis和栽培种花生基因组为模板进行电子PCR。结果（表4）表明,栽培种花生特定基因SSR引物在A.duranensis、A.ipaensis和栽培花生基因组中都具有较高的扩增效率,在3个基因组中的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。且这些SSR引物,在栽培种花生基因组中具有更高的多态性：在A.duranensis和A.ipaensis基因组中,引物扩增位点主要以1个位点为主,在有效引物中的比例分别为93.75%和91.33%;而在栽培种花生基因组中,SSR引物扩增位点主要以2个位点为主（62.24%）,其次是1个位点（28.54%）,且扩增3个及以上位点的SSR引物数要显著高于A.duranensis和A.ipaensis。在所有检测的引物中,共有3 250（55.47%）对引物在3个基因组中均可有效扩增,716（12.22%）对可在A.duranensis和栽培种花生基因组中扩增,978对可在A.ipaensis和栽培种花生基因组中扩增,231（3.94%）对仅在栽培种基因组中扩增（图2）。根据SSR标记在栽培种花生基因组中的扩增情况和扩增位点信息,绘制了SSR位点物理图谱（图3）。根据QTL两端标记在基因组上的位置,利用SSR位点物理图谱,可以为QTL精细定位提供SSR标记信息。如图4所示,80个基因关联SSR标记可用于QTL qBWRB02.1的区间加密。

Table 4
表4
表4基因关联SSR引物e-PCR扩增位点统计
Table 4Statistics of gene-associated SSR primer amplified in peanut genome by e-PCR

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
			1	2	3	>3
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%)

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图2

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图2基因关联SSR标记在基因组中的扩增分布

Fig. 2Amplification site distribution of gene-associated SSR markers in peanut genome

图3

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图3花生SSR标记位点物理图谱

红线表示基因,蓝线表示正义链上的SSR位点,绿线表示负义链上的SSR位点
Fig. 3Physical map of SSR markers in peanut genome

The red line represents for peanut genes, the blue line represent for SSR locus located on the positive chain, the green line represent for SSR locus located on the negative chain

图4

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图4花生青枯病抗性相关QTL定位

红线表示基因,蓝线表示正义链上的SSR位点,绿线表示负义链上的SSR位点
Fig. 4QTL analysis of bacteria wilt resistance in peanut

The red line represent for peanut genes, the blue line represent for SSR locus located on the positive chain, the green line represent for SSR locus located on the negative chain

2.6 SSR标记扩增及多态性分析

随机合成了38对SSR引物在栽培种花生基因组中进行扩增验证,在远杂9102和花育910基因组中共有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,有3个SSR标记为显性标记,在2个花生品种中表现为条带有无的多态性（图5）。表明开发的基因关联SSR引物在花生基因组中具有较高的扩增效率和较好的多态性。

图5

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图5随机SSR引物在花生品种远杂9102和花育910中的扩增情况（部分）

M：Marker;Y：远杂9102;H：花育910。13—24：SSR引物A05D8UNY-1-1、A0686JNN-1-1、A06R0Z7V-1-1、A06RB83Y-1-1、A076T298-1-1、A07F0Y1Z-1-1、A07F4DXF-1-1、A08648HW-2-1、A089H31H-1-1、A08A0WFX-1-1、A091GS41-1-1、A09D2340-1-1
Fig. 5Randomly SSR markers amplification in Yuanza 9102 and Huayu 910 (part)

M: Marker; Y: Yuanza 9102; H: Huayu 910. 13-24: SSR marker A05D8UNY-1-1, A0686JNN-1-1, A06R0Z7V-1-1, A06RB83Y-1-1, A076T298-1-1, A07F0Y1Z-1-1, A07F4DXF-1-1, A08648HW-2-1, A089H31H-1-1, A08A0WFX-1-1, A091GS41-1-1, A09D2340-1-1

3 讨论

栽培种花生（2n=AABB）来源于数百万年前野生花生A.duranensis（2n=AA）和A. ipaensis（2n=BB）间的自然杂交、加倍事件,已有研究表明野生花生A基因组和B基因组具有高度的共线性和一致性^[54]。本研究开发的基因关联SSR引物中,共有62.24%（3 221对）的有效引物具有2个有效扩增位点,且大部分引物扩增的位点分别分布于栽培种花生A基因组和B基因组的同源染色体上,如引物A0101UKP-1-1的2个扩增位点,定位到A01和B01染色体上的一对等位基因Aradu.01UKP.1和Araip.K30076.1的基因序列上;且这些引物可以同时在野生花生A、B基因组上有效扩增。栽培种花生SSR标记的这些特性也进一步印证了花生A、B基因组的高度同源性。

SSR标记已广泛的应用于作物的遗传多样性分析、指纹图谱构建、杂种鉴定、遗传图谱构建、基因挖掘和育种实践中。来源于转录组的SSR直接与功能基因的表达相关,鉴定转录组中与基因直接关联的SSR位点,开发与基因关联的SSR标记,对于研究基因的等位变异及变异对功能的影响、重要性状关联的基因挖掘和精细定位具有重要意义^[55]。本研究鉴定了13 477个可进行引物设计的SSR位点,并对5 020条基因转录本中5 859个SSR位点进行了引物设计和电子PCR检测,进一步丰富了花生SSR标记,为基于分子标记的花生研究提供了可利用的资源。其中有1 147个基因关联SSR标记e-PCR检测只有一个扩增位点,具有一定的特异性,这些SSR标记经进一步鉴定,有可能开发成基因的特征标记,应用于基因在不同种质中的等位变异研究和基因的功能变异研究。

当前,尽管一批栽培种花生重要性状相关QTL被鉴定出来,为花生分子标记辅助育种奠定了基础;但实际上,进入育种应用分子标记的还较少,最主要的原因是鉴定的QTL的遗传距离还较大,标记与基因间的连锁还不够紧密,在实际运用中存在目标性状丢失的风险^[36]。利用SSR位点物理图谱和QTL两端标记在基因上的位置,可以获得对区间加密的SSR标记信息。作者前期利用花生抗、感青枯病亲本远杂9102×徐州68-4构建的RIL群体,在B02连锁群上鉴定出一个稳定的QTL qBWRB02.1（或qBWRB02.4,标记区间为AGGS1592-AHTE0775）,表型变异解释率为6.91%—18.68%^[56]。根据标记AGGS1592、GM2196（AHTE0775相邻连锁标记）在基因组上的位置信息,将qBWRB02.1定位于B02染色体5.53 Mb区域（包含402个基因）,根据SSR位点物理图谱,在此区间包含80个基因关联的SSR标记（图4）。利用这些SSR标记可对该位点区间进行加密,对花生青枯病抗性基因的进行精细定位,从而大大减少候选基因的数量,为目的基因的图位克隆奠定基础。

4 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。

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