CN113652497A - 与辣椒果实朝向紧密相关SNP37458393的应用、特异性dCAPS引物 - Google Patents

与辣椒果实朝向紧密相关SNP37458393的应用、特异性dCAPS引物 Download PDF

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CN113652497A
CN113652497A CN202111018429.XA CN202111018429A CN113652497A CN 113652497 A CN113652497 A CN 113652497A CN 202111018429 A CN202111018429 A CN 202111018429A CN 113652497 A CN113652497 A CN 113652497A
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曹亚从
于海龙
王立浩
程锋
张宝玺
张正海
张亢
陈姝敏
张伟丽
景雅欣
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Abstract

本发明涉及分子标记领域，具体涉及与辣椒果实朝向紧密相关SNP37458393的应用、特异性dCAPS引物。所述SNP位点的位置为Chr12：37458393，与果实朝向极显著关联，Chr12：37458393位点处碱基为C时，果实朝下；碱基为T时，不能被酶切开，果实朝上。此标记的开发利用使得苗期就可以鉴定出果实朝向，并且本发明设计的分子标记位于基因内部，具有高效鉴定效果。

Description

与辣椒果实朝向紧密相关SNP37458393的应用、特异性dCAPS 引物
技术领域
本发明涉及分子标记领域，具体涉及与辣椒果实朝向紧密相关SNP37458393的应用、特异性dCAPS引物。
背景技术
辣椒(Capsicum spp.)属茄科(solanaceae)辣椒属(Capsicum)，起源于美洲的热带和亚热带地区。辣椒具有重要的经济价值，在世界范围内被广泛种植。在过去的几十年里，随着辣椒分子标记的发展，已将其应用于评估辣椒的多样性、构建遗传图谱和进行标记辅助选择(MAS)。随着测序技术的快速发展，第三代标记系统如单核苷酸多态性(SNPs)和插入/缺失多态性(InDels)在辣椒中大量发现，用于遗传定位、多样性分析等。
在茄科作物中，果实朝向是辣椒的一个独特性状。果尖方向从野生型直立到栽培下垂的转变特别重要。野生辣椒的果实朝向是向上，这可能更容易让鸟类取食，从而使其种子传播更远。在选择驯化过程中，出现了果实朝下的辣椒材料，这种变化可能与果实大小的增加或更好地抵御生物和非生物因素的威胁有关，果实朝下能减少鸟类取食，减轻阳光直射造成伤害。辣椒果实朝向为简单遗传，受到up位点的控制，该位点定位于P12染色体，已报道开发了与其相关的分子标记，包括A2C7469(与果实朝向的遗传距离为1.7cM)和upCAPS(与果实朝向的遗传距离为4.3cM)，在开发这些分子标记进行遗传定位研究时尚缺乏辣椒基因组序列信息，并未确定准确的物理候选区域和基因。2014年辣椒基因组序列公布(Kimet al.,2014；Qin et al.,2014)，这为定位性状物理位置的确定提供了基础。2016年，Han等(2016)对重组自交系群体和双亲进行重测序，构建超高密度图谱，将果实朝向的QTLs分别定位到第1、4、12号染色体。Cheng等(2016)以Zunla-1为参考基因组，利用重测序数据构建芯片进行遗传定位，将辣椒果实朝向定位到12号染色体36.54Mb至41.06Mb区间，根据Zunla-1基因组的注释，预测总共有65个蛋白质编码基因。
发明内容
本发明的目的是提供与辣椒果实朝向紧密相关SNP的应用。
本发明的目的是提供与辣椒果实朝向紧密相关SNP的特异性dCAPS引物。
本发明的再一目的是提供上述与辣椒果实朝向紧密相关SNP的特异性dCAPS引物的应用。
本发明的再一目的是提供鉴定辣椒材料的果实朝向的方法。
本发明提供了与辣椒果实朝向紧密相关SNP的应用，其中，所述SNP位点的位置为Chr12：37458393，与果实朝向极显著关联，Chr12：37458393位点处碱基为C时，果实朝下；碱基为T时，不能被酶切开，果实朝上。
根据本发明的与辣椒果实朝向紧密相关SNP的应用，其中，基于所述SNP位点设计特异性引物，并将PCR产物采用进行酶切，Chr12：37458393位点处碱基为C时，能够被酶切开，果实朝下；碱基为T时，不能被酶切开，果实朝上。
与辣椒果实朝向紧密相关SNP的特异性CAPS引物，其核苷酸序列如下所示：
F-5’TATCTACTTGAGACATCTTATAAGTCT3’；
R-5’GACGGTTCAATAACGGAGCA3’。
本发明提供了上述与辣椒果实朝向紧密相关SNP的特异性dCAPS引物的应用。
根据本发明的应用，所述特异性dCAPS引物用于鉴定辣椒果实的朝向。
根据本发明的鉴定辣椒果实朝向的方法，包括使用上述与辣椒果实朝向紧密相关SNP的特异性CAPS引物对待测材料进行PCR扩增的步骤。
根据本发明的鉴定辣椒果实朝向的方法，其中，PCR产物的条带长度均为244bp,将PCR产物采用Hpy188I进行酶切，在Chr12：37458393位点处碱基为C时，能够被酶切开，果实朝下；碱基为T时，不能被酶切开，果实朝上。
本发明利用重测序SNP数据进行全基因组关联分析，将控制果实朝向的基因定位到12号染色体，并通过进一步查找结构变异，确定了决定果实朝向的基因及其关键变异位点，并根据变异位点设计分子标记引物，扩大群体验证了此引物确定果实朝向的准确率。辣椒的果实朝向在现代育种工作中仍是一个重要的选育性状，如朝天椒的果实朝上。此标记的开发利用使得苗期就可以鉴定出果实朝向，并且本发明设计的分子标记位于基因内部，具有高效鉴定效果。
附图说明
图1显示果实朝向表型示例，其中，左图果实朝上，右图果实朝下；
图2显示.PCR扩增结果以及酶切结果，其中，a.PCR扩增琼脂糖电泳结果，b.PCR扩增产物酶切后琼脂糖电泳结果，1-6为果实朝上材料，7-12为果实朝下材料。M，markerD2000。
具体实施方式
实施例1可用于辅助选择辣椒果实朝向的分子标记。
对311份一年生辣椒(Capsicum annuum L.)重测序，测序平台为IlluminaSolexa，采用Trimmomatic v0.33处理原始数据，去除接头、poly-N和低质量片段，得到clean data，利用BWA0.75将clean data比对到Zunla-1基因组数据(Qin et al.,2014)，设置参数为aln–o 1–e 10–t 4–l 32–I 15–q 10；利用Samtools 0.1.19(Li et al.,2009)，采用Bayesian算法，设置参数-q 1–C 50–S–D–m 2–F 0.002–u，运行命令mpileup，查找SNP，过滤SNP，设置最小基因等位频率大于0.01，缺失率小于0.1。
对311份一年生辣椒材料的果实朝向进行表型鉴定，果实朝向表型性状分为朝上、朝下(图1)，对上述过滤得到的SNP与果实朝向的表型数据进行全基因组关联分析，全基因组关联分析采用Gemma(Zhou and Stephens 2012)中的线型混合模型，将果实朝向的显著信号位点定位到12号染色体。
其中SNP位点Chr12：37458393与果实朝向极显著关联，参考基因组碱基为T，突变碱基为C，此位点的GWAS的显著性指数p值为1.204258E-134，对此位点设计dCAPS引物，引物序列为：F-TATCTACTTGAGACATCTTATAAGTCT；R-GACGGTTCAATAACGGAGCA。对果实朝上和果实朝下材料分别进行PCR扩增，将PCR产物进行琼脂糖电泳，电泳结果如图2所示，条带长度均为244bp，将PCR产物采用Hpy188I进行酶切，在Chr12：37458393位点处碱基为C时，能够被酶切开，果实朝下；碱基为T时，不能被酶切开，果实朝上。
利用果实朝向的分子标记验证一自然群体。取嫩叶5-8片用锡箔纸包好液氮预冷，-80℃冻存用于提取DNA。
SNP标记PCR扩增体系(20ul)：
Figure BDA0003240793200000031
取5μL PCR产物或酶切产物点样，用D2000 bp DNA Ladder作为标准指示分子量大小，电泳缓冲液为0.5×TBE，180V恒压30min。电泳结束后在BIO-RAD紫外凝胶成像系统下显影，照相统计多态性条带。
该群体包含283份辣椒材料，其中田间表现果实朝上的材料有33份，在Chr12：37458393位点处碱基为T，PCR产物均未被Hpy188I酶切开；果实朝下的材料有250份，其中248份材料的Chr12：37458393位点处碱基为C，PCR产物能够被Hpy188I酶切开。利用此dCAPS标记验证这283份辣椒材料群体的果实朝向，鉴定准确率为99.29％。
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<110> 中国农业科学院蔬菜花卉研究所
<120> 与辣椒果实朝向紧密相关SNP37458393的应用、特异性dCAPS引物
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tatctacttg agacatctta taagtct 27
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gacggttcaa taacggagca 20

Claims (8)

1.与辣椒果实朝向紧密相关SNP用于鉴定辣椒果实朝向的应用，其特征在于，所述SNP位点的位置为Chr12：37458393，与果实朝向极显著关联，Chr12：37458393位点处碱基为C时，果实朝下；碱基为T时，不能被酶切开，果实朝上。
2.根据权利要求1所述的与辣椒果实朝向紧密相关SNP的应用，其特征在于，基于所述SNP位点设计特异性引物，并将PCR产物采用进行酶切，Chr12：37458393位点处碱基为C时，能够被酶切开，果实朝下；碱基为T时，不能被酶切开，果实朝上。
3.与辣椒果实朝向紧密相关SNP的特异性CAPS引物，其特征在于，所述引物的核苷酸序列如下所示：
F-5’TATCTACTTGAGACATCTTATAAGTCT3’；
R-5’GACGGTTCAATAACGGAGCA3’。
4.权利要求3所述的与辣椒果实朝向紧密相关SNP的特异性dCAPS引物的应用。
5.根据权利要求3所述的应用，其特征在于，所述特异性dCAPS引物用于鉴定辣椒果实的朝向。
6.根据权利要求5所述的应用，其特征在于，将PCR产物采用MspI进行酶切，在Chr12：37458043位点处碱基为C时，能够被酶切开，则辣椒果实朝上，碱基为T时，不能被酶切开，则辣椒果实朝下。
7.鉴定辣椒果实朝向的方法，其特征在于，所述方法包括使用与辣椒果实朝向紧密相关SNP的特异性CAPS引物对待测材料进行PCR扩增的步骤，所述引物的核苷酸序列如下所示：
F-5’AGCTTATCACAACTCCGCACT3’；R-5’CCTGCACATCTGATCGCACTA3’。
8.根据权利要求7所述的鉴定辣椒果实朝向的方法，其特征在于，PCR产物的条带长度均为398bp，将PCR产物采用MspI进行酶切，在Chr12：37458043位点处碱基为C时，能够被酶切开，则辣椒果实朝上，碱基为T时，不能被酶切开，则辣椒果实朝下。
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