CN112063737B - 构建蚕豆指纹图谱的引物组及指纹图谱与应用 - Google Patents
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Abstract
本发明公开了构建蚕豆指纹图谱的引物组及指纹图谱与应用。属于分子生物技术领域。本发明筛选出了17对用于蚕豆指纹图谱构建的引物对,并在此基础上构建了蚕豆指纹图谱,将其应用于蚕豆杂交种纯度鉴定。具有快速、准确、操作方便等优势,并且鉴定结果不受环境影响。
Description
技术领域
本发明涉及分子生物技术领域,更具体的说是涉及构建蚕豆指纹图谱的引物组及指纹图谱与应用。
背景技术
蚕豆是世界上第三大重要的冬季食用豆作物。蚕豆营养价值较高,其蛋白质含量为25~35%。蚕豆还富含糖、矿物质、维他命、钙和铁,作为固氮作物,蚕豆可以将自然界中分子态氮转化为氮素化合物,增加土壤氮素含量。蚕豆隶属于小杂粮,在生活中有十分重要的价值。既可作为传统口粮,又是现代绿色食品和营养保健食品。是富含营养及蛋白质的粮食作物和动物饲料。
种子是主要的农业生产资料,种子纯度是种子质量的核心指标,直接影响农产品质量和优良品种的增产潜力,开展种子纯度检测对于保证种子质量具有重要意义。蚕豆是常异花授粉作物,自然异交率较高,容易导致种性退化,种子纯度、商品性和生产能力不断下降,严重影响品种的推广与应用。“三圃制”是繁育蚕豆新品种原种的传统方法。目前原种繁育过程中通常采用形态学对不同繁殖圃进行鉴定,但传统的表型鉴定对于质量性状是有效的,对数量性状的鉴定不尽准确,易受环境影响且鉴定时期多在成熟期,不利于规模化种子繁育。
综上,如何提供一种快速高效的蚕豆原种纯度鉴定方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了构建蚕豆指纹图谱的引物组及指纹图谱与应用。
为了实现上述目的,本发明采用如下技术方案:
构建蚕豆指纹图谱的引物组,包括如下17对引物组:
EST1117上游引物:TCTTTGTTGTTTTGCTTTTTCATC;SEQ ID NO.1;
EST1117下游引物:CAGACAAGGTTCTGGGTTTG;SEQ ID NO.2;
EST1106上游引物:CCATTGTTTACACTGGCTTTCA;SEQ ID NO.3;
EST1106下游引物:TTCCTGGAGGAACCTAAATAACA;SEQ ID NO.4;
EST1162上游引物:CCATTGTTTACACTGGCTTTCA;SEQ ID NO.5;
EST1162下游引物:TTCCTGGAGGAACCTAAATAACA;SEQ ID NO.6;
SSR13510上游引物:GCTGTCGGAAGCATACACAA;SEQ ID NO.7;
SSR13510下游引物:GGCCTGACTGTACCCATCTT;SEQ ID NO.8;
SSR14387上游引物:CCGATTGGAGTGTGGTAAGG;SEQ ID NO.9;
SSR14387下游引物:GCTCAAGTTTCTCTCAAAAAGCTC;SEQ ID NO.10;
SSR12479上游引物:ATTCTGCGCAGGTGTAGGTT;SEQ ID NO.11;
SSR12479下游引物:AGACTGCAGCGGATTCAGAT;SEQ ID NO.12;
SSR11989上游引物:GGTCCACCACAATGAAGAGG;SEQ ID NO.13;
SSR11989下游引物:TGGAAATGGGGGTGTAGAAA;SEQ ID NO.14;
SSR10910上游引物:ACGCTTTCCCTTCACATCAC;SEQ ID NO.15;
SSR10910下游引物:GTGAAGGCGATCCTGAGGTA;SEQ ID NO.16;
SSR13701上游引物:CACCTTCCTGTGGTTGTGTG;SEQ ID NO.17;
SSR13701下游引物:TGCTCGATCACCTGAGAAAA;SEQ ID NO.18;
SSR12868上游引物:AAATTGGTGGGAGACACCAG;SEQ ID NO.19;
SSR12868下游引物:AAGAACTACCGGAAGCAGACA;SEQ ID NO.20;
SSR12232上游引物:GACCATCTACGTAGCACCAACA;SEQ ID NO.21;
SSR12232下游引物:TGAAAACACAAACACTTACATTAACA;SEQ ID NO.22;
SSR12193上游引物:GGCGCTATGGCAAAGACTAC;SEQ ID NO.23;
SSR12193下游引物:CGATTGGTTCGATCCCTATG;SEQ ID NO.24;
SSR17631上游引物:AGAGCCAACGATATCCAAAAA;SEQ ID NO.25;
SSR17631下游引物:GGGAAAGGAACGACCATGTA;SEQ ID NO.26;
SSR17611上游引物:AGCAATTCACCCACAATCCT;SEQ ID NO.27;
SSR17611下游引物:TAACGGCCTCCAAAAACTTG;SEQ ID NO.28;
SSR14069上游引物:ATGAAGACATTGCCGGTTTC;SEQ ID NO.29;
SSR14069下游引物:AGATTTTGGGGCATTGACAG;SEQ ID NO.30;
SSR13598上游引物:ACTCGGACACGTTGAGTGTG;SEQ ID NO.31;
SSR13598下游引物:TACCATTGGGGAAACCAAAC;SEQ ID NO.32;
SSR12670上游引物:TCATGGCACTGAAAATCAACA;SEQ ID NO.33;
SSR12670下游引物:TGGTGGTGAAGATGGAGACA;SEQ ID NO.34。
利用权利要求1所述引物组构建的蚕豆指纹图谱,所述指纹图谱代码为A211B198C220D204E173F190G185H166I153_180J147K144L142M135N128O127_143_149P120Q123,其具体含义为经引物EST1117扩增出分子量为“211bp”的条带,经引物EST1106扩增出分子量为“198bp”的条带,经引物EST1162扩增出分子量为“220bp”的条带,经引物SSR13510扩增出分子量为“204bp”的条带,经引物SSR14387扩增出分子量为“173bp”的条带,经引物SSR12479扩增出分子量为“190bp”的条带,经引物SSR11989扩增出分子量为“185bp”的条带,经引物SSR10910扩增出分子量为“166bp”的条带,经引物SSR13701扩增出分子量为“153bp”和“180bp”的两条带,经引物SSR12868扩增出分子量为“147bp”的条带,经引物SSR12232扩增出分子量为“144bp”的条带,经引物SSR12193扩增出分子量为“142bp”的条带,经引物SSR17631扩增出分子量为“135bp”的条带,经引物SSR17611扩增出分子量为“128bp”的条带,经引物SSR14069扩增出分子量为“127bp”、“143bp”和“1149bp”的3条带,经引物SSR13598扩增出分子量为“120bp”的条带,经引物SSR12670扩增出分子量为“123bp”的条带。
构建的蚕豆指纹图谱在TF39杂交种纯度鉴定中的应用。
利用分子标记技术建立DNA指纹图谱是鉴定品种、品系的有力工具。目前用于绘制植物DNA指纹图谱的标记技术主要有RAPD、RFLP、AFLP、SSR等,其中SSR分子标记与其他分子标记相比,具有多态信息含量高、共显性遗传、技术简单、重复性好、特异性强等特点。
优选的,包括如下步骤:
(1)待测样抽样:随机选取100粒TF39蚕豆种子;
(2)催芽:用质量浓度1%的升汞对所述TF39蚕豆种子处理10min杀菌,再用清水浸泡12小时移入培养皿中进行催芽;
(3)DNA提取:催芽后采用DNA提取试剂盒提取DNA;
(4)分子标记检测:经过PCR扩增的产物,在6%聚丙烯凝胶电泳上扩增,读带。
经由上述的技术方案可知,与现有技术相比,本发明取得的有益效果为:本发明筛选出了17对用于蚕豆指纹图谱构建的引物对,并在此基础上构建了蚕豆指纹图谱,将其应用于蚕豆杂交种纯度鉴定。具有快速、准确、操作方便等优势,并且鉴定结果不受环境影响。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为实施例2中初选70个株行基于分子标记聚类图;
图2附图为实施例2中引物SSR12479在部分原始群体中的表现;
图3附图为图1附图的局部放大图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明所需药剂为常规实验药剂,采购自市售渠道;未提及的实验方法为常规实验方法,在此不再一一赘述。
实施例1
(一)SSR方法进行纯度鉴定
利用筛选出的适于蚕豆纯度鉴定的33对引物,对TF39(通过蚕鲜蚕豆和青蚕14号选育的蚕豆品系,其系谱图如表1所示)的原始群体的一致性进行分析,所用33对引物序列如表2所示:
表1系谱图
年份 | 选育世代 | 组合(代号) |
2002 | 配置组合 | 130×166(0220) |
2003 | F1 | 混收(0220) |
2004-2005 | F2、F3 | 混合选择(0220) |
2006 | F4 | 选择优良单株(0220-4) |
2007 | F5 | 选择优良单株(0220-4-3) |
2008-2011 | F6 | 混选,(0220-4-3) |
2012-2016 | 混繁,代号:TF39 | |
2017 | 产量鉴定比较;代号TF39 | |
2018 | 区域试验 |
表2 33对引物名称及序列
具体步骤如下:
(1)200份种植7代以上的群体进行SSR分子标记检测,记录每一对引物在每一份材料中的带型,有条带的记为“1”,无条带的记为“0”。
(2)利用NTSYS软件计算Dice参试200个单株间遗传相似系数,利用Powermarker软件基于Nei's1973计算参试材料间遗传距离。
结果:得到遗传相似系数为0.417-1,平均遗传相似系数为0.903(s=ns/(ns+nd),其中,ns代表两个种系都具有性状的特征数,nd代表一个种系性状的特征数。),表明该群体的遗传相似性较高,个体之间差异较小,群体的纯度较高。
(二)利用表型数据进行纯度鉴定
具体步骤如下:
(1)对株高、有效分枝、荚长、荚宽、荚质、荚数、粒数、脐色、子叶色、百粒重10个表型性状进行了调查分析。
(2)将株高、有效分枝、荚长、荚宽、荚数、粒数、百粒重的数量性状进行10级分类。1级<X-2δ,10级≥X+2δ,级差为0.5δ(X为调查性状的平均值,δ为性状的标准差)。
(3)用EXCEL计算各性状的变异系数,7个数量性状的变异系数均值为0.277,其中粒数的变异系数最大为0.492,荚宽的变异系数最小为0.098。有效分枝(0.445)、荚数(0.456)、粒数(0.492)、百粒重(0.168)4个数量性状的变异系数超过0.15,说明原始群体大多数表型性状的主要性状变异较大。
综上所述,SSR分子标记方法更能反映出种子纯度的准确性。
实施例2
(一)SSR进行株行圃鉴定
利用实施例1中的33对引物,对株行圃进行鉴定:根据生产中实际纯度要求95%以上的现实需求,以相似系数0.95为标准,根据表3基于SSR分子标记的相似系数进行株行内的筛选,共筛选出70个株行(表3中用“*”标注)。用于下一步株行间的鉴定。
表3中,遗传相似系数s=ns/(ns+nd),其中,ns代表两个种系都具有性状的特征数,nd代表一个种系性状的特征数。形态学通过性状调查,以有和无量化成0或1,分子标记根据读取的条带。
表3株行内个体之间的遗传相似系数比较
利用PowerMark V3.25软件对初选出的70个株行的分子数据进行分析,计算70个株行间的Nei's1972遗传距离,非加权分组平均法(UPGMA)进行聚类分析,结果如图1所示。
由图1可知,在遗传距离为0.008时70个株行被分成六大类,第Ⅰ大类包括1个株行,第Ⅱ大类包括1个株行,第Ⅲ大类包括1个株行,第Ⅳ大类包括1个株行,第Ⅴ大类包括49个株行,第Ⅵ大类包括17个株行。结果说明应淘汰除第Ⅴ大类外的21个株行。
根据图1结果,最终筛选出株行内多样性低、相似性高、株行间表现均一致的49个株行。当选株行分别收获、保存,第二年将每株行种子种为一个株系。
选择遗传距离较近的49个株行进行分析[根据聚类分析(图1)选择分类中较多的株行进行分析],发现与原始群体相比,10个表型性状的变异系数大幅度下降,株高、百粒重、荚长、荚宽等的变异系数均降为0.1以下。基于表型性状计算得到的相似系数明显低于SSR分子标记得到的相似系数,说明利用SSR分子标记对原种繁育过程中的株系铺进行鉴定,快速、有效。
(二)SSR进行株系圃鉴定
利用实施例1中的33对引物鉴定49个株系圃:基于SSR分子标记的相似系数进行株系内的鉴定,筛选出株系内平均相似系数大于0.95的共计20个株系(表4中用“*”标注)。
表4中,遗传相似系数s=ns/(ns+nd),其中,ns代表两个种系都具有性状的特征数,nd代表一个种系性状的特征数。形态学通过性状调查,以有和无量化成0或1,分子标记根据读取的条带。
表4株系内个体之间的遗传相似系数比较
(三)TF39原种指纹图谱构建:
构建指纹图谱通常选择较少数量的引物形成引物组合,经济、高效地鉴定品种纯度。综合考虑引物在株行及株系中的表现,从33对引物中筛选出17对在不同繁殖圃间扩增稳定、在原原种间带型一致、条带清晰的核心引物(SEQ ID NO.1~SEQ ID NO.34),这17对引物条带清楚、稳定、容易判断(引物SSR12479在部分原始群体中的表现如图2所示)。构建TF39原种的指纹图谱。
每对引物可以扩增出分子量不同的多态性条带(单位为bp,此处省去单位),记录每对引物在TF39中扩增出的多态性条带,若同一个引物在TF39中出现多个条带,按分子量从小到大的顺序用“_”连接不同分子量的条带(每个引物代码顺序为引物编号在前,分子量在后)。TF39的DNA经17对SSR引物扩增形成的由阿拉伯数字和字母组成的一系列带型编码,即为TF39的指纹图谱。
按照此方法构建的TF39的SSR指纹图谱代码为A211B198C220D204E173F190G185H166I153_180J147K144L142M135N128O127_143_149P120Q123(具体含义为TF39经引物EST1117扩增出分子量为“211bp”的条带,经引物EST1106扩增出分子量为“198”的条带,经引物EST1162扩增出分子量为“220bp”的条带,经引物SSR13510扩增出分子量为“204bp”的条带,经引物SSR14387扩增出分子量为“173bp”的条带,经引物SSR12479扩增出分子量为“190bp”的条带,经引物SSR11989扩增出分子量为“185bp”的条带,经引物SSR10910扩增出分子量为“166bp”的条带,经引物SSR13701扩增出分子量为“153bp”和“180”的两条带,经引物SSR12868扩增出分子量为“147bp”的条带,经引物SSR12232扩增出分子量为“144bp”的条带,经引物SSR12193扩增出分子量为“142bp”的条带,经引物SSR17631扩增出分子量为“135bp”的条带,经引物SSR17611扩增出分子量为“128bp”的条带,经引物SSR14069扩增出分子量为“127bp”、“143bp”和“1149bp”的3条带,经引物SSR13598扩增出分子量为“120bp”的条带,经引物SSR12670扩增出分子量为“123bp”的条带)。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 青海省农林科学院
<120> 构建蚕豆指纹图谱的引物组及指纹图谱与应用
<160> 66
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tctttgttgt tttgcttttt catc 24
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cagacaaggt tctgggtttg 20
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccattgttta cactggcttt ca 22
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ttcctggagg aacctaaata aca 23
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccattgttta cactggcttt ca 22
<210> 6
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ttcctggagg aacctaaata aca 23
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gctgtcggaa gcatacacaa 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggcctgactg tacccatctt 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccgattggag tgtggtaagg 20
<210> 10
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gctcaagttt ctctcaaaaa gctc 24
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
attctgcgca ggtgtaggtt 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
agactgcagc ggattcagat 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggtccaccac aatgaagagg 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tggaaatggg ggtgtagaaa 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
acgctttccc ttcacatcac 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gtgaaggcga tcctgaggta 20
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
caccttcctg tggttgtgtg 20
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tgctcgatca cctgagaaaa 20
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aaattggtgg gagacaccag 20
<210> 20
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aagaactacc ggaagcagac a 21
<210> 21
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gaccatctac gtagcaccaa ca 22
<210> 22
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tgaaaacaca aacacttaca ttaaca 26
<210> 23
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ggcgctatgg caaagactac 20
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
cgattggttc gatccctatg 20
<210> 25
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
agagccaacg atatccaaaa a 21
<210> 26
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gggaaaggaa cgaccatgta 20
<210> 27
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
agcaattcac ccacaatcct 20
<210> 28
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
taacggcctc caaaaacttg 20
<210> 29
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
atgaagacat tgccggtttc 20
<210> 30
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
agattttggg gcattgacag 20
<210> 31
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
actcggacac gttgagtgtg 20
<210> 32
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
taccattggg gaaaccaaac 20
<210> 33
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
tcatggcact gaaaatcaac a 21
<210> 34
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tggtggtgaa gatggagaca 20
<210> 35
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gtcatggtac acttcctaag aaca 24
<210> 36
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
agcccgtaga taaccacgaa 20
<210> 37
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
ccgatttcag caacctgttt 20
<210> 38
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gtgaccccat ttgcagactc 20
<210> 39
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
catggaactc gaagccgtat 20
<210> 40
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
aaagttccgt gggtcaagtg 20
<210> 41
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
gagagaggtg aagaaagtgg tga 23
<210> 42
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
cgccttccat gcttacaaat 20
<210> 43
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
aataaggggc tgcagcaata 20
<210> 44
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
cacaccataa gcgatttcgt a 21
<210> 45
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
aaaccactca ccctttcacc 20
<210> 46
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
tcagcacaca caacaacaaa a 21
<210> 47
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
ctctctagtg gcctgggtgt 20
<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
cgatggggtg tttctctctc 20
<210> 49
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
ctccgcgagc atcactttat 20
<210> 50
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
ttgcacgatc tcaactcacc 20
<210> 51
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
agcaggtctc acccttctca 20
<210> 52
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
cgaaagcttc ttcggacttg 20
<210> 53
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
caaaggcaac agaaacacac a 21
<210> 54
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
agggagagca tgattgtcca 20
<210> 55
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
ttttggggca tcaaagaaag 20
<210> 56
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
ttttgtgcac tgacccactc 20
<210> 57
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
aaaaccccct caaaatctca a 21
<210> 58
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
ttgtgggaaa tgtggagtga 20
<210> 59
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
tcctccaggt ccaaaaacac 20
<210> 60
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
aacccgatcc gtttcatct 19
<210> 61
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
cctcgtcttc tcccttcctc 20
<210> 62
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
gcctaaagtt gccgtttctg 20
<210> 63
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
agaagggggt tccattcact 20
<210> 64
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
gcggttaaag tttggttctt g 21
<210> 65
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
aaggccgcat ttatgtcaag 20
<210> 66
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
ttgctaacat tactgaaaca aaatca 26
Claims (2)
1.一种构建的蚕豆指纹图谱在TF39杂交种纯度鉴定中的应用,其特征在于,所述构建蚕豆指纹图谱的引物组包括:
EST1117上游引物:TCTTTGTTGTTTTGCTTTTTCATC;SEQ ID NO.1;
EST1117下游引物:CAGACAAGGTTCTGGGTTTG;SEQ ID NO.2;
EST1106上游引物:CCATTGTTTACACTGGCTTTCA;SEQ ID NO.3;
EST1106下游引物:TTCCTGGAGGAACCTAAATAACA;SEQ ID NO.4;
EST1162上游引物:CCATTGTTTACACTGGCTTTCA;SEQ ID NO.5;
EST1162下游引物:TTCCTGGAGGAACCTAAATAACA;SEQ ID NO.6;
SSR13510上游引物:GCTGTCGGAAGCATACACAA;SEQ ID NO.7;
SSR13510下游引物:GGCCTGACTGTACCCATCTT;SEQ ID NO.8;
SSR14387上游引物:CCGATTGGAGTGTGGTAAGG;SEQ ID NO.9;
SSR14387下游引物:GCTCAAGTTTCTCTCAAAAAGCTC;SEQ ID NO.10;
SSR12479上游引物:ATTCTGCGCAGGTGTAGGTT;SEQ ID NO.11;
SSR12479下游引物:AGACTGCAGCGGATTCAGAT;SEQ ID NO.12;
SSR11989上游引物:GGTCCACCACAATGAAGAGG;SEQ ID NO.13;
SSR11989下游引物:TGGAAATGGGGGTGTAGAAA;SEQ ID NO.14;
SSR10910上游引物:ACGCTTTCCCTTCACATCAC;SEQ ID NO.15;
SSR10910下游引物:GTGAAGGCGATCCTGAGGTA;SEQ ID NO.16;
SSR13701上游引物:CACCTTCCTGTGGTTGTGTG;SEQ ID NO.17;
SSR13701下游引物:TGCTCGATCACCTGAGAAAA;SEQ ID NO.18;
SSR12868上游引物:AAATTGGTGGGAGACACCAG;SEQ ID NO.19;
SSR12868下游引物:AAGAACTACCGGAAGCAGACA;SEQ ID NO.20;
SSR12232上游引物:GACCATCTACGTAGCACCAACA;SEQ ID NO.21;
SSR12232下游引物:TGAAAACACAAACACTTACATTAACA;SEQ ID NO.22;
SSR12193上游引物:GGCGCTATGGCAAAGACTAC;SEQ ID NO.23;
SSR12193下游引物:CGATTGGTTCGATCCCTATG;SEQ ID NO.24;
SSR17631上游引物:AGAGCCAACGATATCCAAAAA;SEQ ID NO.25;
SSR17631下游引物:GGGAAAGGAACGACCATGTA;SEQ ID NO.26;
SSR17611上游引物:AGCAATTCACCCACAATCCT;SEQ ID NO.27;
SSR17611下游引物:TAACGGCCTCCAAAAACTTG;SEQ ID NO.28;
SSR14069上游引物:ATGAAGACATTGCCGGTTTC;SEQ ID NO.29;
SSR14069下游引物:AGATTTTGGGGCATTGACAG;SEQ ID NO.30;
SSR13598上游引物:ACTCGGACACGTTGAGTGTG;SEQ ID NO.31;
SSR13598下游引物:TACCATTGGGGAAACCAAAC;SEQ ID NO.32;
SSR12670上游引物:TCATGGCACTGAAAATCAACA;SEQ ID NO.33;
SSR12670下游引物:TGGTGGTGAAGATGGAGACA;SEQ ID NO.34。。
2.如权利要求1所述的应用,其特征在于,包括如下步骤:
(1)待测样抽样:随机选取100粒TF39蚕豆种子;
(2)催芽:用质量浓度1%的升汞对所述TF39蚕豆种子处理10min杀菌,再用清水浸泡12小时移入培养皿中进行催芽;
(3)DNA提取:催芽后采用DNA提取试剂盒提取DNA;
(4)分子标记检测:经过PCR扩增的产物,在6%聚丙烯凝胶电泳上扩增,读带。
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利用SSR标记分析蚕豆品种(品系)与优异种质的遗传多样性;张红岩等;《中国蔬菜》;20181231(第2期);34-41 * |
基于SSR标记的蚕豆DNA指纹图谱构建及品种纯度鉴定;张红岩;《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》;20190115(第12期);D048-55 * |
蚕豆SSR-PCR体系建立及优化;沙伟超等;《青海大学学报》;20170430;第35卷(第2期);13-18,27 * |
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