CN116411122A - 番茄果实可溶性糖含量相关位点的caps分子标记及应用 - Google Patents
番茄果实可溶性糖含量相关位点的caps分子标记及应用 Download PDFInfo
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Abstract
本发明属于分子生物学技术领域,具体涉及番茄果实可溶性糖含量相关位点的CAPS分子标记及应用。本发明要解决的技术问题是为将加速番茄种质资源筛选提供一种新选择。本发明的技术方案是番茄果实可溶性糖含量相关位点的CAPS分子标记,其核苷酸序列如SEQ ID No.3和SEQ ID No.4所示。该分子标记是基于前期GWAS关联分析筛选得到的,是与番茄果实可溶性糖含量相关主效位点紧密连锁的共显性标记,可用于鉴定可溶性糖含量高低的番茄材料,分子标记与田间鉴定结果吻合率达90%。因此,该标记操作简洁、扩增结果精准度高、所需试剂成本较低,该标记的开发可用于番茄品质性状的分子标记辅助选择育种,进一步加速番茄常规育种进程。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及番茄果实可溶性糖含量相关位点的CAPS分子标记及应用。
背景技术
番茄是一种食用性果蔬,因其营养价值和丰富口感深受人们喜爱。随着人们生活水平逐渐提高,人们对番茄果实品质对需求也越来越多样化,不仅对其营养价值方面有较高要求,而且在果实口感和外观上也有一定要求,最直接影响番茄口感的因素即番茄果实的可溶性糖的含量。番茄中的糖主要由可溶性糖和非可溶性糖组成,而可溶性糖主要为葡萄糖、蔗糖、果糖等。在可溶性糖中,蔗糖所占的比例最大,番茄的品常甜味来源主要是蔗糖。此前,赵建涛等对174份不同品种的番茄进行了全基因组关联分析,找到与果糖相关的SSR标记9个,葡萄糖9个,蔗糖12个,分布在1、5、6、9、10号染色体上。Fridman等定位到了一个可溶性固形物总含量的位点,主要是影响了糖含量的积累QTL(Brix9-2-5)。Fulton等利用分子标记绘制了高密度的遗传连锁图谱,找到了一些与总糖、果糖、葡萄糖等相关的区域主要分布在2、3、4、9、10号染色体上。此外,之前的研究大都集中在番茄可溶性糖含量位点的定位方面,关于番茄可溶性糖含量相关位点的分子标记开发和应用的研究较少。
发明内容
本发明要解决的技术问题是为将加速番茄种质资源筛选提供一种新选择。
本发明的技术方案是番茄果实可溶性糖含量相关位点的CAPS分子标记,其核苷酸序列如SEQ ID No.3和SEQ ID No.4所示。
进一步的,所述CAPS分子标记位于番茄基因组的11号染色体上。
本发明还提供了扩增所述CAPS分子标记的引物对,其核苷酸序列如SEQ ID No.1和SEQ ID No.2所示。
本发明还提供了所述CAPS分子标记或引物对在番茄育种中的应用。
本发明还提供一种快速鉴定番茄材料的果实可溶性糖含量的方法,包括如下步骤:
a、提取番茄材料的基因组DNA;
b、以步骤a得到DNA为模板,采用SEQ ID No.1和SEQ ID No.2所示引物对进行扩增;
c、对步骤b扩增得到的片段进行测序或者酶切;若测序得到SEQ ID No.3所示序列则果实可溶性糖含量低,若测序得到SEQ ID No.4所示序列则果实可溶性糖含量高。
进一步的,步骤c中酶切是指用ClaI,若酶切产物出现2条长度为312bp和438p的片段,则果实可溶性糖含量低;若酶切产物出现1条长度为750p的片段,则果实可溶性糖含量高。
具体的,步骤b中扩增体系为:0.5μL正向引物,0.5μL反向引物,1μL模板DNA,5μL 2×Taq PCRMasterMix(Dye),3μL dd H2O。
其中,步骤b中扩增程序为:94℃预变性2min;其次94℃变性30s,57℃退火30s,72℃延伸40s,35个循环;72℃延伸5min;4℃保存。
优选的,步骤c中酶切体系为:ClaI 0.5μL,10×Cla I Buffer 1μL,PCR产物1μL,灭菌水7.5μL;酶切条件为37℃孵育15min。
本发明的有益效果:本发明公开了一种基于番茄果实可溶性糖含量相关位点的CAPS标记。该分子标记是基于前期GWAS关联分析筛选得到的,是与番茄果实可溶性糖含量相关主效位点紧密连锁的共显性标记。通过对番茄核心种质成熟果实进行可溶性糖含量分析,鉴定了一批可溶性糖含量显著差异的番茄材料,准确率可达95%以上。因此,该标记操作简洁、扩增结果精准度高、所需试剂成本较低,可用于分子标记辅助选择育种,该标记的开发应用将丰富番茄品质种质资源库。
附图说明
图1为CAPS标记SSC21在高糖番茄材料(High,H可溶性糖含量>5%)和低糖番茄材料(Low,L,可溶性糖含量<5%)中的部分DNA序列;L中存在ClaI酶切位点,H中无ClaI酶切位点。
图2为CAPS标记SSC21在番茄自然群体中的酶切结果;M:DL2000,H为高糖材料,L为低糖材料,1~24为不同番茄种质材料。
具体实施方式
下面结合实施例对本发明的过程进行进一步的说明。
实施例1CAPS分子标记的开发
基于番茄基因组SL3.0的数据,对100份番茄材料(由东北农业大学提供)的GWAS关联分析得到的番茄11号染色体的番茄果实可溶性糖含量位点信息,进行分子标记的开发。通过单倍型分析,进一步缩短关联区间,通过对目标区域进行CAPS标记开发,并对CAPS标记进行基因型和表型共分离情况进行统计,发现CAPS分子标记SSC21(高糖材料处为G;低糖材料处为A,ClaI酶切位点),在番茄高糖和低糖表型中,具有较高的共分离表现。SSC21标记:正向引物序列SEQ ID No.1:5’-GACCATCCAAAAGTTTTGCTGTATC-3’反向引物序列SEQ IDNo.2:5’-CATTTCAGGCTTGTCAGCATTAGTA-3’。
SEQ ID No.3:低可溶性糖含量位点SSC21-1,^表示酶切位置
SEQ ID No.4,高可溶性糖含量位点SSC21-2
实施例2CAPS标记在不同番茄种质材料中的验证
共选取了100份可溶性糖含量不同的番茄种质资源进行鉴定分析,表1为其中100份亲本自交系番茄材料(由东北农业大学提供,包含大果番茄34份、罗曼型番茄33份、樱桃番茄33份)。
待番茄材料长至4~5片真叶时,用CTAB法(Fulton et al.,1995)从幼苗叶片中提取基因组DNA。然后以番茄基因组DNA为模板与CAPS标记的引物进行PCR扩增,CAPS标记的PCR反应体系的总体积为10μL的PCR反应体系为:0.5μL正向引物,0.5μL反向引物,1μL模板DNA,5μL 2×Taq PCR StarMix,3μL dd H2O。总体积10μL的PCR扩增程序为:首先94℃预变性2min;其次94℃变性30s,57℃退火30s,72℃延伸40s,35个循环;最后72℃延伸5min;保存温度4℃。
上述方法中,总体积10μL的酶切体系为:ClaI 0.5μL,10×Taq I Buffer 1μL,PCR产物1μL,灭菌水7.5μL;酶切条件为37℃孵育15分钟。然后酶切产物在1.0%的琼脂糖凝胶上进行电泳,然后在凝胶成像仪上观察、记录实验结果。
部分材料酶切结果如图2所示。高糖与低糖番茄材料存在多态性,在低糖番茄材料中经过酶切可以酶切成312bp和438bp的两条特异性条带,而在高糖番茄材料中不能酶切目标条带,只可扩增出来750bp条带。
表1 100份番茄亲本自交系的标记鉴定
对上述100亲本自交系材料含糖量分析发现:可溶性糖含量最高的为10.4,最低的为3.0,番茄自然群体的可溶性糖含量的差异程度较大,相差三倍以上。樱桃番茄中可溶性糖含量偏高,普通番茄和罗曼型番茄可溶性糖含量偏低。统计结果表明SSC21标记在这些番茄材料的基因型和田间表型吻合率达90%以上。
上述的实施方案仅为了清楚地描述本发明而作出的一般性说明,在此基础上,可以对其做出一些修改或改进。在不违背本发明的原理的条件下所做的改动均属于本发明要求保护的范围。
Claims (9)
1.番茄果实可溶性糖含量相关位点的CAPS分子标记,其核苷酸序列如SEQ ID No.3和SEQ ID No.4所示。
2.如权利要求1所述CAPS分子标记,其特征在于,该CAPS分子标记位于番茄基因组的11号染色体上。
3.扩增权利要求1或2所述CAPS分子标记的引物对,其特征在于,其核苷酸序列如SEQID No.1和SEQ ID No.2所示。
4.权利要求1或2所述CAPS分子标记或权利要求3所述引物对在番茄育种中的应用。
5.一种快速鉴定番茄材料的果实可溶性糖含量的方法,其特征在于包括如下步骤:
a、提取番茄材料的基因组DNA;
b、以步骤a得到DNA为模板,采用SEQ ID No.1和SEQ ID No.2所示引物对进行扩增;
c、对步骤b扩增得到的片段进行测序或者酶切;若测序得到SEQ ID No.3所示序列则果实可溶性糖含量低,若测序得到SEQ ID No.4所示序列则果实可溶性糖含量高。
6.如权利要求5所述方法,其特征在于,步骤c中酶切是指用ClaI,若酶切产物出现2条长度为312bp和438p的片段,则果实可溶性糖含量低;若酶切产物出现1条长度为750p的片段,则果实可溶性糖含量高。
7.如权利要求5所述方法,其特征在于,步骤b中扩增体系为:0.5μL正向引物,0.5μL反向引物,1μL模板DNA,5μL 2×Taq PCRMasterMix(Dye),3μL dd H2O。
8.如权利要求5所述方法,其特征在于,步骤b中扩增程序为:94℃预变性2min;其次94℃变性30s,57℃退火30s,72℃延伸40s,35个循环;72℃延伸5min;4℃保存。
9.如权利要求5所述方法,其特征在于,步骤c中酶切体系为:ClaI 0.5μL,10×Cla IBuffer 1μL,PCR产物1μL,灭菌水7.5μL;酶切条件为37℃孵育15min。
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