CN111690761B - 香葱est-ssr分子标记及其应用 - Google Patents
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Abstract
本发明公开了一种香葱EST‑SSR分子标记,所述香葱EST‑SSR分子标记具有如编号为Cluster‑10188.0;Cluster‑11228.0;Cluster‑11382.0;Cluster‑11719.0;Cluster‑12571.0;Cluster‑13488.0;Cluster‑13953.0;Cluster‑14907.0;Cluster‑1547.0;Cluster‑1574.0;Cluster‑168.0;Cluster‑17176.0;Cluster‑18261.0;Cluster‑18284.1;Cluster‑18905.0;Cluster‑19764.0;Cluster‑19959.3;Cluster‑22324.0;Cluster‑22847.0;Cluster‑24908.0;Cluster‑2567.0;Cluster‑25807.1;Cluster‑2608.10612;Cluster‑2608.1143;Cluster‑2608.11633;Cluster‑2608.12824;Cluster‑2608.13377;Cluster‑2608.18176;Cluster‑2608.19044;Cluster‑2608.19078;Cluster‑2608.19327;Cluster‑2608.19412;Cluster‑2608.19628;Cluster‑2608.19820;Cluster‑2608.20126;Cluster‑2608.2015;Cluster‑2608.22133所示的序列。本发明方法首次使用EST‑SSR标记对香葱进行遗传多样性进行分析,操作简单,能对香葱种质进行有效区分,填补香葱SSR分子标记开发的空白。通过这些EST‑SSR标记的开发不仅丰富了香葱的分子标记数量,而且为其种质资源的遗传分析提供了理论依据。
Description
技术领域
本发明涉及植物鉴别与育种技术领域,特别涉及香葱EST-SSR分子标记及其应用。
背景技术
香葱别名四季葱、菜葱、冬葱等,香葱一般株高约20-30公分,叶绿色,圆筒形,中空,先端渐尖,花白绿色,聚生成团,鳞茎基部易连生,群生状。香葱的外形就好比缩小的大葱,葱白长,叶子尖细,因为它确实是大葱的变种。属百合科葱属一年生或者多年生草本植物,因其鲜食、加工、园艺观赏及药用兼用,是日常生活中不可或缺的特色蔬菜品种之一。广西香葱年产值亿元,在促进地区经济进步与农民增收方面发挥着巨大的作用。然而,栽培葱属有着巨大的基因组,育种中存在缺乏遗传变异、遗传基础非常狭窄严重的近交系衰退的困扰。因此,开展香葱遗传育种研究具有重大意义。
遗传变异对促进植物育种和保护策略起到重要作用。通过研究可以了解物种的群体遗传结构和多态性水平,为研究物种起源、品种分类、亲本选配、品种保护等提供依据,是研究、保护和利用现有种质资源的重要基础。
分子标记是分析遗传变异的有用工具,为表型和基因型变异之间的联系提供了有效的手段。SSR分子标记检测的是基因组简单重复序列信息,因其呈共显性标记,多态性丰富,重复性好,已广泛应用于许多作物的基因定位、遗传连锁图谱构建、辅助育种、遗传多样性研究等方面。由于国内关于葱属作物遗传多样性研究主要集中在洋葱、大葱、韭菜、大蒜等葱属作物,使用的标记技术主要有形态标记、RAPD、AFLP、ISSR、SRAP等,说明SSR分子标记可有效进行葱属栽培品种的遗传多样性研究,但未见有香葱遗传多样性EST-SSR标记开发。与其他蔬菜作物相比,香葱分子育种技术应用研究相对较少,差距较大,尤其是分子标记技术在遗传育种上的应用亟待加强。
基于上述现有存在的问题,针对在香葱遗传多样性SSR标记开发的技术空白,本发明提供了一种香葱遗传多样性SSR标记。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
本发明的目的在于提供一种香葱EST-SSR分子标记及其应用,不仅能丰富香葱的分子标记数量,还能使该分子标记在香葱种质资源分钟的有效利用。
为实现上述目的,本发明提供了一种香葱EST-SSR分子标记,所述香葱EST-SSR分子标记具有如编号为Cluster-10188.0;Cluster-11228.0;Cluster-11382.0;Cluster-11719.0;Cluster-12571.0;Cluster-13488.0;Cluster-13953.0;Cluster-14907.0;Cluster-1547.0;Cluster-1574.0;Cluster-168.0;Cluster-17176.0;Cluster-18261.0;Cluster-18284.1;Cluster-18905.0;Cluster-19764.0;Cluster-19959.3;Cluster-22324.0;Cluster-22847.0;Cluster-24908.0;Cluster-2567.0;Cluster-25807.1;Cluster-2608.10612;Cluster-2608.1143;Cluster-2608.11633;Cluster-2608.12824;Cluster-2608.13377;Cluster-2608.18176;Cluster-2608.19044;Cluster-2608.19078;Cluster-2608.19327;Cluster-2608.19412;Cluster-2608.19628;Cluster-2608.19820;Cluster-2608.20126;Cluster-2608.2015;Cluster-2608.22133所示的序列。
上述EST-SSR分子标记可采用如下引物对扩增得到:
Cluster-10188.0:SEQ ID NO.1-2;Cluster-11228.0:SEQ ID NO.3-4;Cluster-11382.0:SEQ ID NO.5-6;Cluster-11719.0:SEQ ID NO.7-8;Cluster-12571.0:SEQ IDNO.9-10;Cluster-13488.0:SEQ ID NO.11-12;Cluster-13953.0:SEQ ID NO.13-14;Cluster-14907.0:SEQ ID NO.15-16;Cluster-1547.0:SEQ ID NO.17-18;Cluster-1574.0:SEQ ID NO.19-20;Cluster-168.0:SEQ ID NO.21-22;Cluster-17176.0:SEQ IDNO.23-24;Cluster-18261.0:SEQ ID NO.25-26;Cluster-18284.1:SEQ ID NO.27-28;Cluster-18905.0:SEQ ID NO.29-30;Cluster-19764.0:SEQ ID NO.31-32;Cluster-19959.3:SEQ ID NO.33-34;Cluster-22324.0:SEQ ID NO.35-36;Cluster-22847.0:SEQID NO.37-38;Cluster-24908.0:SEQ ID NO.39-40;Cluster-2567.0:SEQ ID NO.41-42;Cluster-25807.1:SEQ ID NO.43-44;Cluster-2608.10612:SEQ ID NO.45-46;Cluster-2608.1143:SEQ ID NO.47-48;Cluster-2608.11633:SEQ ID NO.49-50;Cluster-2608.12824:SEQ ID NO.51-52;Cluster-2608.13377:SEQ ID NO.53-54;Cluster-2608.18176:SEQ ID NO.55-56;Cluster-2608.19044:SEQ ID NO.57-58;Cluster-2608.19078:SEQ ID NO.59-60;Cluster-2608.19327:SEQ ID NO.61-62;Cluster-2608.19412:SEQ ID NO.63-64;Cluster-2608.19628:SEQ ID NO.65-66;Cluster-2608.19820:SEQ ID NO.67-68;Cluster-2608.20126:SEQ ID NO.69-70;Cluster-2608.2015:SEQ ID NO.71-72;Cluster-2608.22133:SEQ ID NO.73-74。
针对EST-SSR分子标记,本发明提供香葱的引物组合物,包括SEQ ID NO.1-SEQ IDNO.74所示的引物组合对。
上述如SEQ ID NO.1-SEQ ID NO.74所述的引物对依次分别用于扩增上述37个EST-SSR分子标记。
进一步地,本发明提供所述香葱的引物组合物试剂盒。
在此基础上,本发明提供所述的试剂盒在香葱EST-SSR分子标记或所述引物组合物或所述的试剂盒的如下任一应用:
(1)在香葱品种鉴定中的应用;
(2)在香葱遗传图谱或分子身份证构建中的应用;
(3)在香葱遗传育种中的应用;
(4)在香葱亲缘关系或遗传多样性分析中的应用。
本发明还提供香葱种质资源遗传多样性分析的方法,包括以下步骤:
(1)提取香葱的基因组DNA;
(2)以所述基因组DNA为模板采用所述引物组合物进行PCR扩增;
(3)数据分析:对电泳结果进行读带和进行聚类分析。
优选地,上述技术方案中,步骤(2)中引物组合物进行PCR扩增包括:PCR扩增总体积为14μL:含DNA聚合酶Mix 5μL、上下游引物10μmol/L各1μmol,模板DNA为2μL中含有60ng,加双蒸水(ddH2O)补齐到14μL;扩增程序为DNA 95℃预变性5m in;95℃变性30s,55℃退火30s,72℃复性45s,35个循环;72℃延伸10min;4℃结束保存;DNA扩增产物与6×上样液混合,经8%非变性聚丙烯酰胺凝胶电泳,进行电泳分离,电泳结束后进行银染检测分析
与现有技术相比,本发明具有如下有益效果:
(1)本发明方法首次使用EST-SSR标记对香葱进行遗传多样性进行分析,操作简单,能对香葱种质进行有效区分,填补香葱SSR分子标记开发的空白。通过这些EST-SSR标记的开发不仅丰富了香葱的分子标记数量,而且为其种质资源的遗传分析提供了理论依据。
(2)本发明提供的分子标记组合、引用组合可用于香葱品种分子身份证构建、品种鉴定及遗传多样性分析等,应用前景十分广阔。
附图说明
图1是部分SSR引物在6份香葱种质的初筛多样性条带图;
图2是SSR引物Cluster-10188.0在48份香葱种质的多样性条带;
图3是SSR引物Cluster-19764.0在48份香葱种质的多样性条带;
图4是SSR引物Cluster-19959.3在48份香葱种质的多样性条带;
图5是供试香葱材料的UPGMA聚类图。
具体实施方式
下面结合附图,对本发明的具体实施方式进行详细描述,但应当理解本发明的保护范围并不受具体实施方式的限制。
除非另有其它明确表示,否则在整个说明书和权利要求书中,术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分,而并未排除其它元件或其它组成部分。
材料与方法
一;材料与DNA提取
56份香葱种质取至广西壮族自治区农业科学院蔬菜研究所科研展示基地。香葱叶片中DNA的提取参照传统的CTAB法。
1;香葱EST数据的获得与处理
从GenBank公共数据库(https://www.ncbi.nlm.nih.gov/sra/?term= SRR3144570)中,下载该物种所有的转录组测序结果,共5.9G。分析中采用MISA1.0版对拼接后的结果进行其重复序列位点的搜索,对应的各个unit size的最少重复次数分别为:1-10,2-6,3-5,4-5,5-5,6-5(如:1-10,以单核苷酸为重复单位时,其重复数至少为10才可被检测到;2-6,以双核甘酸为重复单位时,其最少重复数为6)对数据进行SSR检测。
2;香葱EST-SSR引物设计
利用Primer3.0(2.3.5版)软件,对含有SSR重复位点的EST序列进行EST-SSR分子标记设计。EST序列的设定标准为EST序列长度大于100bp,且SSR位点距离左右两末端大于50bp。引物的设置标准为:GC含量在40%~60%,预期扩增片段大小为100~300bp,Tm值在40℃~65℃之间,上下游引物Tm值差异不超过5℃,引物长度为18~22bp。
3;香葱EST-SSR引物多态性筛选
PCR反应体系为14μL:Mix(含DNA聚合酶)5μL;上下游引物10μmol/L各1μmol,模板DNA 60ng(2μL),即模板DNA为2μL中含有60ng,加双蒸水补齐到14μL。PCR反应条件设置为:DNA 95℃预变性5min;95℃变性30s,55℃退火30s,72℃复性45s,35个循环;72℃延伸10min;4℃结束保存。DNA扩增产物与6×上样液混合,经8%非变性聚丙烯酰胺凝胶电泳,在北京六一仪器厂生产的G20型垂直板电泳仪,进行电泳分离,电泳结束后进行银染检测分析。
二;结果与分析
1;香葱SSRs的基本统计
SSRs即简单重复序列(Simple Sequence Repeat,SSRs),也即通常所说的微卫星DNA,又被称作短串连重复(Short Tandem Repeats,STRs)。是由2-4个碱基对不断重复而成的DNA片断。
从NCBI的SRA数据库下载获得了葱转录组数据(SRR3144570),软件分析检测的序列碱基数共计94030015bp,序列总数为94805个,得到总SSR序列9511条,占10.03%(表1;2),说明香葱EST序列中SSR数量和类型较为丰富。其中,869条EST序列含有1个以上的SSR位点序列,352条SSR以复合形式存在。其中,在重复数类型中,一核苷酸占频率最大,为55.08%;其次是三核苷酸和二核苷酸,分别为为21.51%和21.32%;最少的为五核苷酸,仅为0.03%。
表1.NCBI的SRA数据库葱转录组数据SSR分析的基本统计
表2.SSR在数据库中出现的比例及频率
备注:SSR的类型,p1,单碱基重复;p2,两个碱基重复;p3三个碱基重复;p4四个碱基重复;p5五个碱基重复;p6六个碱基重复;c,复杂重复类型;类型比例=SSR数目/确定的ssr总数*100%;频率=SSR数目/检查的序列总数*100%。
2;香葱SSRs的主要重复基元及其频率
在所有的SSR类型中,主要包括有41种重复基元(表3)。其中以一核苷酸重复类型A/T;二核苷酸重复类型AC/GT和AT/AT含量频率最高,分别占总SSR数量的55.01%;8.11%和7.95%。其它类型的重复基元类型比例在5.47%~0.0011%之间。
其中以二核苷酸重复类型ag/ct;三核苷酸重复类型cag和六核苷酸重复类型aaccct含量相对较高,分别占总SSR的0.90%;0.26%和0.11%,其它类型的重复基元类型频率在0.04%~0.57%之间,其值含量相对较低。根据SSR重复基元类型是否存在间断,可将香葱的SSR类型分为429个完全重复基元和54个不完全重复基元EST-SSR,所占已有EST-SSR序列的88.8%和11.2%,表明完全重复基元类型占绝对优势(表3)。
表3.香葱SSR模序重复类型的频数及频率
备注:所占比例=重复基元数目/重复类型总数;b发生频率:重复基元数目/总EST数目类型。
3;香葱SSR标记的开发
利用Primer3软件设计引物9492对,占中EST的99.8%。如表4在已收集的香葱EST序列中,其SSR的平均长度为53.23bp,并且不同的SSR其长度范围变化差异幅度大,最短的为10bp,最长的为158bp。其中,复杂重复类型(c)变化范围最大,在20~158bp之间,而五核苷酸变化范围最小,在25~25bp之间。
表4:香葱EST中SSR重复的长度和性质
备注:SSR的类型,p1,单碱基重复;p2,两个碱基重复;p3三个碱基重复;p4四个碱基重复;p5五个碱基重复;p6六个碱基重复;c,复杂重复类型。
4;香葱SSR分子标记的筛选
对已设计出的引物对,随机挑选200对进行合成,并在不同香葱品种基因组DNA中进行PCR随机扩增验证。PCR扩增结果发现,200对引物中有197对(占98.5%)有扩增产物,其中173对(占86.5%)有清晰的目标条带。在200对SSR引物中,共有61对引物扩物扩增的条带超过10条,61对引物扩物扩增的条带10条在5条之间,51对扩物扩增的条带5条在1条之间。10条以下引物的扩增平均条带数为5.71条(图1)。其中有37对引物具有较好的多态性(表5;图2-4)。说明本实验设计的引物,可用于香葱种质资源的SSR遗传分析。
5;基于SSR标记的香葱种质亲缘关系聚类分析
基于UPGMA法,用NTSYS-PcVer.2.10e软件进行聚类树状图绘制。在相似系数为0.68时,可将56个香葱种质分为4个类群。如图5所示。
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式,并且很显然,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
序列表
<110> 广西壮族自治区农业科学院
<120> 香葱EST-SSR分子标记及其应用
<160> 74
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
accggaaatc agtccacgtc 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gagagggtga agtgccactc 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
agaagcacaa tcccagcagg 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tctccggaca ccaactacct 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttctgggact cgtacctgct 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
acctccaccc actccagaaa 20
<210> 7
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
acagctatga gatgtttcag aca 23
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
agggtgtttg tgttggacga 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tcgaacaggc gacgaaatga 20
<210> 10
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cgtaattggc tgaatgtggc a 21
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atagcgcctc agcaaacctt 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
taaaacacac gcgcacacac 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cgcgtgtccc agttgttttt 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gtctcagctc cgctactgac 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gcgacaccat tcatttggga 20
<210> 16
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tgcattacgg atatgctccc c 21
<210> 17
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ccatgtgtcg ggactcaatc a 21
<210> 18
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tccctaccct atacgcacac a 21
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aggttggcat atctgggtca 20
<210> 20
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tgccatttca tgcatgtcca c 21
<210> 21
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tgatgttcct tccctcacaa tca 23
<210> 22
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
agaaacagtc acaaaacgga ct 22
<210> 23
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
accaattgtg tcacacctcc a 21
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gcattgctgg ccaatctgag 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tgccccgtga tcacaatcaa 20
<210> 26
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ccgaagcaaa ccacacagtg 20
<210> 27
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
agcaggtagt ccaaactggc 20
<210> 28
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
cctgcaaatc attccagacg c 21
<210> 29
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
acattgtgag agttgccaca 20
<210> 30
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
aggactggtt cttcataggc a 21
<210> 31
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
ccgacaaggt ggacaaactc t 21
<210> 32
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
ccgtcatcta caatgttttt ggct 24
<210> 33
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
aagccggtat tcccaaaggg 20
<210> 34
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ccgcttcaga tgtagcaggt 20
<210> 35
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
aaaggtgtag gcagacgtgg 20
<210> 36
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gccagtacag catcttcggt 20
<210> 37
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gcctatggcg gctcctaatt 20
<210> 38
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
atggggataa tacggctgcg 20
<210> 39
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
cgttacaaat cactcatttc ctgt 24
<210> 40
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
acgttgaaga gacagagcgg 20
<210> 41
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
tggatttcag actttatgga agca 24
<210> 42
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
agagcctaag ttcacttccc a 21
<210> 43
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
agcaacagcg actagtaggc 20
<210> 44
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
gctgcccggt tttatatggc 20
<210> 45
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
gacggttcgg gttgtgtttg 20
<210> 46
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
ccaaatcaaa acatctttca aaaaggg 27
<210> 47
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
ccgaaccctg cttctcctac 20
<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
caatagcgac ggtggagaca 20
<210> 49
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
acgtgaacgc aacctaatgg 20
<210> 50
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
tgtgacgttt tgcgcaagtt 20
<210> 51
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
gctgaaatca ggctcctcca 20
<210> 52
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
tgaggcgtgt gcatgttact 20
<210> 53
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
gcttggtggg actggctaat 20
<210> 54
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
aagcgaggag atgttgcaga 20
<210> 55
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
ttgggccttt ggtggttctt 20
<210> 56
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
cccccgctgc taaacctaaa 20
<210> 57
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
tgcgcacatc gatcttctct 20
<210> 58
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
atggaggtga ccagagacca 20
<210> 59
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
tgtgcacttg aagaggcact 20
<210> 60
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
agcaactact ggatcaaggt gg 22
<210> 61
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
cctcggaccc aattgcaaac 20
<210> 62
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
tcgggagtgc tctaatgcag 20
<210> 63
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
aggagggggt ggatagcaat 20
<210> 64
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
cccttttcta atcggccccc 20
<210> 65
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
accgtatttc atgagaggga gc 22
<210> 66
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
agaattcacc aagcggtcaa 20
<210> 67
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
tccagttcgc atgacagtga 20
<210> 68
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
tgtaggaaag ctgaggcaca 20
<210> 69
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
ggaaagagtg gggtccatgg 20
<210> 70
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
tgccacatca ccaccaaact 20
<210> 71
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
tttccgtgaa catgcacaca 20
<210> 72
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
tggaccatgg tctgaaagtt tg 22
<210> 73
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
tcgttgtcgg ttcttcctgg 20
<210> 74
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
tgctggccta atgaggtgtg 20
Claims (6)
1.一种香葱EST-SSR分子标记,其特征在于,所述香葱EST-SSR分子标记具有如编号为Cluster-10188.0;Cluster-11228.0;Cluster-11382.0;Cluster-11719.0;Cluster-12571.0;Cluster-13488.0;Cluster-13953.0;Cluster-14907.0;Cluster-1547.0;Cluster-1574.0;Cluster-168.0;Cluster-17176.0;Cluster-18261.0;Cluster-18284.1;Cluster-18905.0;Cluster-19764.0;Cluster-19959.3;Cluster-22324.0;Cluster-22847.0;Cluster-24908.0;Cluster-2567.0;Cluster-25807.1;Cluster-2608.10612;Cluster-2608.1143;Cluster-2608.11633;Cluster-2608.12824;Cluster-2608.13377;Cluster-2608.18176;Cluster-2608.19044;Cluster-2608.19078;Cluster-2608.19327;Cluster-2608.19412;Cluster-2608.19628;Cluster-2608.19820;Cluster-2608.20126;Cluster-2608.2015;Cluster-2608.22133所示的序列;各编号对应的香葱EST-SSR分子标记的引物对序列按顺序如SEQ ID NO.1-SEQ ID NO.74所示。
2.香葱的引物组合物,其特征在于,包括SEQ ID NO.1-SEQ ID NO.74所示的引物组合对。
3.包含权利要求2所述引物组合物的试剂盒。
4.根据权利要求1所述的香葱EST-SSR分子标记或权利要求2所述引物组合物或权利要求3所述的试剂盒的如下任一应用:
(1)在香葱品种鉴定中的应用;
(2)在香葱遗传图谱或分子身份证构建中的应用;
(3)在香葱遗传育种中的应用;
(4)在香葱亲缘关系或遗传多样性分析中的应用。
5.香葱种质资源遗传多样性分析的方法,其特征在于,包括以下步骤:
(1)提取香葱的基因组DNA;
(2)以所述基因组DNA为模板采用权利要求2所述引物组合物进行PCR扩增;
(3)数据分析:对电泳结果进行读带和进行聚类分析。
6.根据权利要求5所述香葱种质资源遗传多样性分析的方法,其特征在于,步骤(2)中引物组合物进行PCR扩增包括:
PCR扩增总体积为14μL:含DNA聚合酶Mix 5μL、上下游引物10μmol/L各1μmol,模板DNA为2μL中含有60ng,加双蒸水补齐到14μL;
扩增程序为DNA 95℃预变性5min;95℃变性30s,55℃退火30s,72℃复性45s,35个循环;72℃延伸10min;4℃结束保存;
DNA扩增产物与6×上样液混合,经8%非变性聚丙烯酰胺凝胶电泳,进行电泳分离,电泳结束后进行银染检测分析。
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