技术领域Technical field
本发明涉及生物技术领域，尤其是一种基于小麦单核苷酸多态性位点的小麦表型筛选和鉴定方法，可应用于小麦育种科研和实践。The invention relates to the field of biotechnology, in particular to a wheat phenotypic screening and identification method based on wheat single nucleotide polymorphism sites, which can be applied to scientific research and practice of wheat breeding.
背景技术Background technique
小麦(Triticum aestivum L.)是我国最主要的粮食作物之一。提升小麦单产是满足粮食需求不断提高的重要方式，同时也是保证粮食安全的战略目标。植物株型对其产量、品质和光能利用率起着举足轻重的作用，株高、穗型、分蘖数和分蘖角度对株型构成有着重要作用。其中穗型的改良是超高产育种的重要途径，穗型是亲本选择、后代筛选的重要指标之一，穗部的结构决定了小麦的产量。因此对小麦穗型的农艺性状加以改良，对提高产量有重要作用。Wheat (Triticum aestivum L.) is one of the most important food crops in my country. Increasing wheat yield is an important way to meet the ever-increasing demand for food, and it is also a strategic goal to ensure food security. Plant type plays a vital role in its yield, quality and light energy utilization. Plant height, ear type, number of tillers and tiller angle play an important role in plant type composition. Among them, the improvement of ear type is an important way to breed super-high-yield varieties. Ear type is one of the important indicators for parent selection and offspring screening. The structure of the ear determines the yield of wheat. Therefore, improving the agronomic traits of wheat ear type plays an important role in increasing yield.
在目前的研究中，小麦中关于穗发育的研究主要集中于遗传定位和标记开发。Hu等使用中国小麦品种Yanzhan 1作为共同亲本构建RIL群体进行QTL分析，发现并验证了8个稳定的QTL，千粒重QTL(QSPS-2A.4)也在自然群体中得到了验证。Li等构建了Kechengmai1/Chuanmai42的DH群体并通过小麦55K SNP芯片构建了高密度遗传图谱进行穗部QTL检测。检测到27个与每穗总小穗数和每穗可育小穗数相关的QTL。其中QTsn/Fsn.cib-3D为总小穗数和可育小穗数的共定位QTL，解释表型变异为5.97-23.28％，并利用开发的KASP标记KASP_AX-110914105在两个不同遗传背景的群体中进一步验证了该QTL的作用。并且利用55K芯片在小麦2B和2D染色体上鉴定到3个新的控制小麦小穗粒数的QTL，解释19.59％-26.57％的表型变异。但是，由于绝大多数QTL的表型贡献率较小，且在不同年际及环境间重复性差，所以QTL难以应用于小麦千粒重的遗传改良。In the current study, research on panicle development in wheat has mainly focused on genetic mapping and marker development. Hu et al. used the Chinese wheat variety Yanzhan 1 as a common parent to construct a RIL population for QTL analysis and found and verified 8 stable QTL. The thousand-grain weight QTL (QSPS-2A.4) was also verified in the natural population. Li et al. constructed a DH population of Kechengmai1/Chuanmai42 and constructed a high-density genetic map through wheat 55K SNP chip for ear QTL detection. 27 QTL related to the total number of spikelets per panicle and the number of fertile spikelets per panicle were detected. Among them, QTsn/Fsn.cib-3D is a co-localized QTL for total spikelet number and fertile spikelet number, explaining 5.97-23.28% of phenotypic variation, and using the developed KASP marker KASP_AX-110914105 in two different genetic backgrounds The effect of this QTL was further verified in the population. And three new QTLs controlling wheat spikelet grain number were identified on wheat chromosomes 2B and 2D using the 55K chip, explaining 19.59%-26.57% of the phenotypic variation. However, because the phenotypic contribution rate of most QTL is small and the repeatability between different years and environments is poor, it is difficult to apply QTL to the genetic improvement of thousand-kernel weight in wheat.
CAPS标记又称为PCR-RFLP(限制性片段长度多态性聚合酶链反应)，是一类以PCR为基础的共显性分子标记，揭示的是特异PCR片段的限制性长度变异信息，其基本原理是用PCR扩增目的DNA，扩增产物再用特异性内切酶消化切割成不同大小片段，直接在凝胶电泳上分辨。不同等位基因的限制性酶切位点分布不同，产生不同长度的DNA片段条带。优点是避免了RFLP分析中繁琐的转移、杂交步骤，又能保持RFLP分析的精确度。然而，SNP恰好位于限制性酶切位点的情况比较少，因此，提出了dCAPS标记，即在CAPS标记的基础上通过在扩增引物中引入错配碱基，结合SNP位点引入新的限制性内切酶作用位点，产生和CAPS标记类似的多态性。CAPS markers, also known as PCR-RFLP (restriction fragment length polymorphism polymerase chain reaction), are a type of PCR-based co-dominant molecular markers that reveal the restriction length variation information of specific PCR fragments. The basic principle is to use PCR to amplify the target DNA, and then use specific endonucleases to digest and cut the amplified products into fragments of different sizes, which are directly resolved on gel electrophoresis. Different alleles have different distributions of restriction enzyme sites, producing DNA fragment bands of different lengths. The advantage is that it avoids the tedious transfer and hybridization steps in RFLP analysis while maintaining the accuracy of RFLP analysis. However, it is rare that a SNP happens to be located at a restriction enzyme site. Therefore, the dCAPS marker is proposed, that is, based on the CAPS marker, mismatched bases are introduced in the amplification primers and new restrictions are introduced based on the SNP site. Sexual endonuclease action site, resulting in polymorphisms similar to CAPS markers.
目前，与小麦表型尤其是小麦千粒重表型相关的单核苷酸多态性位点的开发是亟需的，具有重要的科研和应用价值。At present, the development of single nucleotide polymorphism sites related to wheat phenotypes, especially wheat thousand-kernel weight phenotypes, is urgently needed and has important scientific research and application value.
发明内容Contents of the invention
本发明要解决的技术问题是提供一种基于A583G SNP位点的开发而鉴定、筛选或控制小麦千粒重的方法。The technical problem to be solved by the present invention is to provide a method for identifying, screening or controlling the thousand-kernel weight of wheat based on the development of the A583G SNP site.
为解决上述技术问题，本发明所采取的技术方案如下。In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.
控制小麦千粒重的育种方法，基于小麦基因组中单核苷酸多态性位点的特定基因型控制小麦的千粒重。The breeding method for controlling the thousand-grain weight of wheat controls the thousand-grain weight of wheat based on specific genotypes of single nucleotide polymorphism sites in the wheat genome.
作为本发明的一种优选技术方案，所述单核苷酸多态性位点对应于SEQ ID NO.1所示序列自5’末端第583位碱基。As a preferred technical solution of the present invention, the single nucleotide polymorphism site corresponds to the 583rd base from the 5' end of the sequence shown in SEQ ID NO.1.
作为本发明的一种优选技术方案，所述单核苷酸多态性位点处的核苷酸为G/G纯合时，对应基因型I小麦；所述单核苷酸多态性位点处的核苷酸为A/A纯合时，对应基因型II小麦；基因型I小麦的千粒重大于基因型II小麦的千粒重。As a preferred technical solution of the present invention, when the nucleotide at the single nucleotide polymorphism site is G/G homozygous, it corresponds to genotype I wheat; the single nucleotide polymorphism site When the nucleotide at the point is A/A homozygous, it corresponds to genotype II wheat; the 1,000-kernel weight of genotype I wheat is greater than the 1,000-kernel weight of genotype II wheat.
用于鉴定、筛选或控制小麦千粒重的试剂盒，用于检测小麦基因组中如下SNP位点的单核苷酸多态性，所述SNP位点对应于SEQ ID NO.1所示序列自5’末端第583位碱基；所述SNP位点处的核苷酸为G或A；所述SNP位点处的核苷酸为G/G纯合时，相对应的基因型是I；所述SNP位点处的核苷酸为A/A纯合时，相对应的基因型是II。A kit for identifying, screening or controlling the thousand-kernel weight of wheat, for detecting the single nucleotide polymorphism of the following SNP site in the wheat genome, the SNP site corresponding to the sequence shown in SEQ ID NO.1 starting from 5' The 583rd base at the end; the nucleotide at the SNP site is G or A; when the nucleotide at the SNP site is G/G homozygous, the corresponding genotype is I; the When the nucleotide at the SNP site is homozygous for A/A, the corresponding genotype is II.
作为本发明的一种优选技术方案，所述试剂盒包含SEQ ID NO.2和SEQ ID NO.3组成的引物对1F和1R，和SEQ ID NO.4和SEQ ID NO.5组成的引物对2F和2R。As a preferred technical solution of the present invention, the kit comprises a primer pair 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3, and a primer pair 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO.5.
基于SNP位点鉴定小麦千粒重的方法，该方法包括如下步骤：对待测小麦基因组DNA中任意一段包含如下SNP位点的DNA片段进行PCR扩增和鉴定；所述SNP位点对应于SEQID NO.1所示序列自5’末端第583位碱基；然后基于步骤A中的扩增产物确定待测小麦的基因型；最后根据待测小麦的基因型鉴定待测小麦的千粒重性状。A method for identifying thousand-kernel weight of wheat based on SNP sites, the method includes the following steps: PCR amplification and identification of any DNA fragment containing the following SNP site in the wheat genomic DNA to be tested; the SNP site corresponds to SEQ ID NO.1 The sequence shown starts from the 583rd base at the 5' end; then the genotype of the wheat to be tested is determined based on the amplification product in step A; and finally the thousand-kernel weight trait of the wheat to be tested is identified based on the genotype of the wheat to be tested.
作为本发明的一种优选技术方案，该方法包括如下步骤：As a preferred technical solution of the present invention, the method comprises the following steps:
A、对待测小麦基因组DNA中任意一段包含如下SNP位点的DNA片段进行PCR扩增，并将该PCR扩增产物进行酶切；所述SNP位点对应于SEQ ID NO.1所示序列自5’末端第583位碱基；A. Perform PCR amplification of any DNA fragment containing the following SNP site in the wheat genomic DNA to be tested, and perform enzyme digestion on the PCR amplification product; the SNP site corresponds to the sequence shown in SEQ ID NO.1. 5' end base 583;
B、确定待测小麦的基因型，所述SNP位点处的核苷酸为G/G纯合时，相对应的基因型是I；所述SNP位点处的核苷酸为A/A纯合时，相对应的基因型是II；根据待测小麦基因型按照如下标准确定待测小麦的千粒重性状：基因型I纯合小麦的千粒重大于/候选大于基因型II纯合小麦的千粒重。B. Determine the genotype of the wheat to be tested. When the nucleotide at the SNP site is homozygous for G/G, the corresponding genotype is I; when the nucleotide at the SNP site is homozygous for A/A, the corresponding genotype is II. Determine the thousand-grain weight trait of the wheat to be tested according to the genotype of the wheat to be tested according to the following standard: the thousand-grain weight of the genotype I homozygous wheat is greater than/candidate greater than the thousand-grain weight of the genotype II homozygous wheat.
作为本发明的一种优选技术方案，步骤A中，所述PCR扩增的特异性引物对为SEQID NO.2、SEQ ID NO.3组成的引物对1F和1R，和/或SEQ ID NO.4、SEQ ID NO.5组成的引物对2F和2R。As a preferred technical solution of the present invention, in step A, the specific primer pair for PCR amplification is primer pair 1F and 1R composed of SEQ ID NO.2 and SEQ ID NO.3, and/or primer pair 2F and 2R composed of SEQ ID NO.4 and SEQ ID NO.5.
作为本发明的一种优选技术方案，步骤A中，所述扩增和酶切包括以下步骤：以小麦基因组DNA为模板，以引物1F和1R为引物对扩增得到PCR产物；将此PCR产物作为模板，以引物2F和2R为引物对扩增得到PCR产物；用限制性内切酶PstI进行酶切，得到酶切产物。As a preferred technical solution of the present invention, in step A, the amplification and enzyme digestion include the following steps: using wheat genomic DNA as a template and using primers 1F and 1R as a primer pair to amplify the PCR product; As a template, primers 2F and 2R were used as a primer pair to amplify the PCR product; the restriction endonuclease PstI was used to digest the product to obtain the digested product.
作为本发明的一种优选技术方案，步骤B中，如果所述酶切产物为一条DNA片段，则所述SNP位点处的核苷酸为A/A纯合，待测小麦为基因型II；如果所述酶切产物为两条DNA片段，则所述SNP位点处的核苷酸为G/G纯合，待测小麦为基因型I。As a preferred technical solution of the present invention, in step B, if the enzyme cleavage product is a DNA fragment, the nucleotide at the SNP site is homozygous A/A, and the wheat to be tested is genotype II; if the enzyme cleavage product is two DNA fragments, the nucleotide at the SNP site is homozygous G/G, and the wheat to be tested is genotype I.
采用上述技术方案所产生的有益效果在于：基于本发明的技术，通过检测所述SNP即可找到千粒重相对较高的小麦。本发明为小麦的分子标记辅助选择育种提供了一个新方法，在培育高产稳产小麦品种和研究中具有重要意义。The beneficial effect of adopting the above technical solution is that based on the technology of the present invention, wheat with a relatively high thousand-kernel weight can be found by detecting the SNP. The present invention provides a new method for molecular marker-assisted selection breeding of wheat, and is of great significance in cultivating and researching high- and stable-yielding wheat varieties.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为利用F1、R1对自然群体中24个品种小麦TaJMJ12-5A基因进行扩增结果。图中展示的是对照和24个品种小麦的检测结果，1-24的品种依次为Drysdale、冀麦30、冀麦32、冀麦38、冀麦41、冀麦6号、冀麦9号、冀麦一号、冀审5099、鉴26、金光、晋2148-7、晋麦13、晋麦17、晋麦33、晋麦39、晋麦44、晋麦47、晋麦50、晋麦51、晋麦53、晋麦54、晋麦57、晋麦63。Figure 1 shows the amplification results of TaJMJ12-5A genes of 24 wheat varieties in natural populations using F1 and R1. The picture shows the test results of the control and 24 varieties of wheat. The varieties 1-24 are Drysdale, Jimai 30, Jimai 32, Jimai 38, Jimai 41, Jimai No. 6, Jimai No. 9, Jimai No. 1, Jishen 5099, Jian 26, Jinguang, Jin 2148-7, Jinmai 13, Jinmai 17, Jinmai 33, Jinmai 39, Jinmai 44, Jinmai 47, Jinmai 50, Jinmai 51 , Jinmai 53, Jinmai 54, Jinmai 57, Jinmai 63.
图2为自然群体中部分小麦TaJMJ12-5A基因的基因检测结果。图中展示的是对照和24个品种小麦的检测结果，1-24的品种依次为Drysdale、冀麦30、冀麦32、冀麦38、冀麦41、冀麦6号、冀麦9号、冀麦一号、冀审5099、鉴26、金光、晋2148-7、晋麦13、晋麦17、晋麦33、晋麦39、晋麦44、晋麦47、晋麦50、晋麦51、晋麦53、晋麦54、晋麦57、晋麦63。Figure 2 shows the genetic detection results of TaJMJ12-5A gene in some wheat in natural populations. The figure shows the detection results of the control and 24 varieties of wheat. The varieties 1-24 are Drysdale, Jimai 30, Jimai 32, Jimai 38, Jimai 41, Jimai 6, Jimai 9, Jimai 1, Jishen 5099, Jian 26, Jinguang, Jin 2148-7, Jinmai 13, Jinmai 17, Jinmai 33, Jinmai 39, Jinmai 44, Jinmai 47, Jinmai 50, Jinmai 51, Jinmai 53, Jinmai 54, Jinmai 57, Jinmai 63.
具体实施方式Detailed ways
以下实施例详细说明了本发明。本发明所使用的各种原料及各项设备均为常规市售产品，均能够通过市场购买直接获得。应当理解，当在本申请说明书和所附权利要求书中使用时，术语“包括”指示所描述特征、整体、步骤、操作、元素和/或组件的存在，但并不排除一个或多个其它特征、整体、步骤、操作、元素、组件和/或其集合的存在或添加。还应当理解，在本申请说明书和所附权利要求书中使用的术语“和/或”是指相关联列出的项中的一个或多个的任何组合以及所有可能组合，并且包括这些组合。如在本申请说明书和所附权利要求书中所使用的那样，术语“如果”可以依据上下文被解释为“当...时”或“一旦”或“响应于确定”或“响应于检测到”。类似地，短语“如果确定”或“如果检测到[所描述条件或事件]”可以依据上下文被解释为意指“一旦确定”或“响应于确定”或“一旦检测到[所描述条件或事件]”或“响应于检测到[所描述条件或事件]”。另外，在本申请说明书和所附权利要求书的描述中，术语“第一”、“第二”、“第三”等仅用于区分描述，而不能理解为指示或暗示相对重要性。在本申请说明书中描述的参考“一个实施例”或“一些实施例”等意味着在本申请的一个或多个实施例中包括结合该实施例描述的特定特征、结构或特点。由此，在本说明书中的不同之处出现的语句“在一个实施例中”、“在一些实施例中”、“在其他一些实施例中”、“在另外一些实施例中”等不是必然都参考相同的实施例，而是意味着“一个或多个但不是所有的实施例”，除非是以其他方式另外特别强调。术语“包括”、“包含”、“具有”及它们的变形都意味着“包括但不限于”，除非是以其他方式另外特别强调。The following examples illustrate the invention in detail. Various raw materials and various equipment used in the present invention are conventional commercially available products and can be directly obtained through market purchase. It will be understood that, when used in this specification and the appended claims, the term "comprising" indicates the presence of the described features, integers, steps, operations, elements and/or components but does not exclude one or more other The presence or addition of features, integers, steps, operations, elements, components and/or collections thereof. It will also be understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items. As used in this specification and the appended claims, the term "if" may be interpreted as "when" or "once" or "in response to determining" or "in response to detecting" depending on the context. ". Similarly, the phrase "if determined" or "if [the described condition or event] is detected" may be interpreted, depending on the context, to mean "once determined" or "in response to a determination" or "once the [described condition or event] is detected ]" or "in response to detection of [the described condition or event]". In addition, in the description of this application and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the description, and cannot be understood as indicating or implying relative importance. Reference in this specification to "one embodiment" or "some embodiments" or the like means that a particular feature, structure or characteristic described in connection with the embodiment is included in one or more embodiments of the application. Therefore, the phrases "in one embodiment", "in some embodiments", "in other embodiments", "in other embodiments", etc. appearing in different places in this specification are not necessarily References are made to the same embodiment, but rather to "one or more but not all embodiments" unless specifically stated otherwise. The terms “including,” “includes,” “having,” and variations thereof all mean “including but not limited to,” unless otherwise specifically emphasized.
下述实施例中所用的小麦材料均来自国家作物种质库(网址为：http://icscaas.com.cn/jiguoku/zhongzhiku.htm)，材料信息见中国作物种质信息网(网址为：http://icgr.caas.net.cn)。由于小麦材料均为栽培种，因此通常被默认为是高度纯合的植物材料，基因型均为纯合型。The wheat materials used in the following examples are all from the National Crop Germplasm Bank (website: http://icscaas.com.cn/jiguoku/zhongzhiku.htm), and the material information can be found in the China Crop Germplasm Information Network (website: http://icgr.caas.net.cn). Since the wheat materials are all cultivated varieties, they are usually assumed to be highly homozygous plant materials, and the genotypes are all homozygous.
实施例1、小麦TaJMJ12-5A基因中A583G SNP的发现及小麦基于TaJMJ12-5A基因的基因型分型方法的建立Example 1. Discovery of A583G SNP in wheat TaJMJ12-5A gene and establishment of wheat genotyping method based on TaJMJ12-5A gene
一、小麦TaJMJ12-5A基因中A583G SNP的发现1. Discovery of A583G SNP in wheat TaJMJ12-5A gene
本发明的发明人经过大量实验，在小麦TaJMJ12-5A基因(核苷酸序列如SEQ IDNO:1所示)上发现了一个SNP位点，命名为A583G SNP。A583G SNP位于SEQ ID NO:1自5’末端起的第583位，基因型为AA纯合型和GG纯合型。由于基因组DNA是由反向互补的两条单链DNA分子组成双链DNA分子，因此一般将编码蛋白质的DNA分子命名为正义DNA分子；将与正义DNA分子的反向互补的DNA分子命名为反义DNA分子。A583G SNP的基因型均为正义DNA的基因型。After extensive experiments, the inventor of the present invention discovered a SNP site on the wheat TaJMJ12-5A gene (the nucleotide sequence is shown as SEQ IDNO: 1), named A583G SNP. A583G SNP is located at position 583 from the 5' end of SEQ ID NO:1, and the genotypes are AA homozygous and GG homozygous. Since genomic DNA is composed of two reverse-complementary single-stranded DNA molecules to form a double-stranded DNA molecule, the DNA molecule encoding the protein is generally named a sense DNA molecule; the DNA molecule that is reverse complementary to the sense DNA molecule is named reverse sense DNA molecule. The genotypes of A583G SNP are all positive-sense DNA genotypes.
根据小麦TaJMJ12-5A基因的不同将小麦分为两种基因型：TaJMJ12-5A-G(以下简称基因型I)和TaJMJ12-5A-A(以下简称基因型II)。According to the differences in the wheat TaJMJ12-5A gene, wheat is divided into two genotypes: TaJMJ12-5A-G (hereinafter referred to as genotype I) and TaJMJ12-5A-A (hereinafter referred to as genotype II).
二、合成用于扩增包括A583G SNP的靶序列的引物对甲和引物对乙2. Synthesis of Primer Pair A and Primer Pair B for Amplification of Target Sequences Including A583G SNP
设计并合成用于扩增包括A583G SNP的靶序列的引物对甲和引物对乙。引物对甲由引物F1和引物R1组成。引物对乙由引物F2和引物R2组成。Primer pair A and primer pair B for amplifying target sequences including the A583G SNP were designed and synthesized. Primer pair A consists of primer F1 and primer R1. Primer pair B consists of primer F2 and primer R2.
各个引物的核苷酸序列具体如下：The nucleotide sequences of each primer are as follows:
引物F1：5′-GGAGGGGGACTGTCCGG-3′(SEQ ID NO:2)Primer F1: 5′-GGAGGGGGACTGTCCGG-3′ (SEQ ID NO: 2)
引物R1：5′-CGGTGCGGTGTAGTCATTCGCG-3′(SEQ ID NO:3)Primer R1: 5′-CGGTGCGGTGTAGTCATTCGCG-3′ (SEQ ID NO: 3)
引物F2：5′-CAGGACACAACGGTTG-3′(SEQ ID NO:4)Primer F2: 5′-CAGGACACAACGGTTG-3′ (SEQ ID NO: 4)
引物R2：5′-ACGTCACTTCTCCTCTG-3′(SEQ ID NO:5)Primer R2: 5′-ACGTCACTTCTCCTCTG-3′ (SEQ ID NO: 5)
引物对甲扩增的靶序列为TaJMJ12-5A基因自5’末端起第14016-14032位(13981CAATAGGTGC ACTATAGCCA CGCCCGCCCG TCGTAGGAGG GGGACTGTCC GGTCGTCTGG14040)。The target sequence amplified by primer pair A is positions 14016-14032 from the 5' end of the TaJMJ12-5A gene (13981CAATAGGTGC ACTATAGCCA CGCCCGCCCG TCGTAGGAGG GGGACTGTCC GGTCGTCTGG14040).
三、小麦基于TaJMJ12-5A基因的基因型分型方法的建立3. Establishment of wheat genotyping method based on TaJMJ12-5A gene
1、提取待测小麦(科农199)的基因组DNA。1. Extract the genomic DNA of the wheat to be tested (Konong 199).
2、以步骤1的待测小麦的基因组DNA为模板，采用引物F1和引物R1组成的引物对甲进行PCR扩增，得到PCR扩增产物P1。2. Using the genomic DNA of the wheat to be tested in step 1 as a template, use the primer pair A composed of primer F1 and primer R1 to perform PCR amplification to obtain the PCR amplification product P1.
反应体系为10μL，由3.6μL ddH2O、5μL 2×Taq酶Mix、0.2μL引物F1水溶液(浓度为10μmol/L)、0.2μL引物R1水溶液(浓度为10μmol/L)、和1μL待测小麦的基因组DNA(浓度为20ng/μL)组成。The reaction system was 10 μL, consisting of 3.6 μL ddH2 O, 5 μL 2×Taq enzyme Mix, 0.2 μL primer F1 aqueous solution (concentration 10 μmol/L), 0.2 μL primer R1 aqueous solution (concentration 10 μmol/L), and 1 μL genomic DNA of wheat to be tested (concentration 20 ng/μL).
2×Taq酶Mix为南京诺唯赞公司的产品，产品目录号为P131。2×Taq enzyme Mix is a product of Nanjing Novozymes Co., Ltd., with the product catalog number of P131.
反应条件为：95℃3min；95℃15s，68℃15s，72℃1min，35次循环；72℃10min；16℃保存。The reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 68°C for 15 s, 72°C for 1 min, 35 cycles; 72°C for 10 min; and storage at 16°C.
3、完成步骤2后，以PCR扩增产物P1的稀释液(1体积份PCR扩增产物P1和9体积份水混合而成)为模板，采用引物F2和引物R2组成的引物对乙进行PCR扩增，得到PCR扩增产物P2。3. After completing step 2, using the diluted solution of PCR amplification product P1 (a mixture of 1 volume of PCR amplification product P1 and 9 volumes of water) as a template, PCR amplification is performed on primer pair B using primer F2 and primer R2 to obtain PCR amplification product P2.
反应体系为10μL，由3.6μL ddH2O、5μL 2×Taq酶Mix、0.2μL引物F2水溶液(浓度为10μmol/L)、0.2μL引物R2水溶液(浓度为10μmol/L)、和1μL PCR扩增产物P1的稀释液组成。The reaction system is 10 μL, consisting of 3.6 μL ddH2 O, 5 μL 2×Taq enzyme mix, 0.2 μL primer F2 aqueous solution (concentration: 10 μmol/L), 0.2 μL primer R2 aqueous solution (concentration: 10 μmol/L), and 1 μL PCR amplification Dilution composition of product P1.
反应条件为：95℃3min；95℃15s，56℃15s，72℃15s，35次循环；72℃10min；16℃保存。The reaction conditions were: 95°C for 3 min; 95°C for 15 s, 56°C for 15 s, 72°C for 15 s, 35 cycles; 72°C for 10 min; and stored at 16°C.
4、将步骤3得到的PCR扩增产物P2用限制性内切酶PstI进行酶切消化，得到酶切产物；将酶切产物进行4％琼脂糖凝胶电泳检测，进行如下判断：如果酶切产物为带型A(显示两条带，分别为94bp和15bp)，则待测小麦A583G SNP为GG纯合型，即待测小麦基于TaJMJ12-5A基因的基因型为基因型I；如果酶切产物为带型B(显示一条带，为109bp)，则待测小麦A583G SNP为AA纯合型，即待测小麦基于TaJMJ12-5A基因的基因型为基因型II。4. Digest the PCR amplification product P2 obtained in step 3 with the restriction endonuclease PstI to obtain the digested product; conduct 4% agarose gel electrophoresis detection of the digested product, and make the following judgment: If the enzyme is digested The product is band type A (showing two bands, 94bp and 15bp respectively), then the A583G SNP of the wheat to be tested is GG homozygous type, that is, the genotype of the wheat to be tested based on the TaJMJ12-5A gene is genotype I; if the enzyme is digested If the product is band type B (showing one band, 109 bp), then the A583G SNP of the wheat to be tested is AA homozygous type, that is, the genotype of the wheat to be tested based on the TaJMJ12-5A gene is genotype II.
实施例2、小麦TaJMJ12-5A基因的基因型与小麦千粒重的关联分析及验证Example 2: Association analysis and verification between the genotype of wheat TaJMJ12-5A gene and wheat thousand-grain weight
一、自然群体中各个小麦TaJMJ12-5A基因的基因分型1. Genotyping of various wheat TaJMJ12-5A genes in natural populations
采用实施例1中步骤三的方法对自然群体中的各个小麦品种进行基因分型。自然群体由296个小麦品种(均为六倍体)组成。小麦品种名称详见表1。The method of step 3 in Example 1 was used to perform genotyping on each wheat variety in the natural population. The natural population consisted of 296 wheat varieties (all hexaploid). The names of the wheat varieties are detailed in Table 1.
部分检测结果见图1(M为DNA Marker，其它泳道为不同小麦品种；其中G为基因型I的小麦品种，A为基因型II的小麦品种)。Some of the test results are shown in Figure 1 (M is a DNA Marker, and the other lanes are different wheat varieties; G is a wheat variety of genotype I, and A is a wheat variety of genotype II).
基于TaJMJ12-5A基因的基因型见表1：212个小麦品种基于TaJMJ12-5A基因的基因型为基因型I，85个小麦品种基于TaJMJ12-5A基因的基因型为基因型II。The genotypes based on the TaJMJ12-5A gene are shown in Table 1: the genotypes of 212 wheat varieties based on the TaJMJ12-5A gene are genotype I, and the genotypes of 85 wheat varieties based on the TaJMJ12-5A gene are genotype II.
表1Table 1
注：I为基因型I，II为基因型II。Note: I is genotype I, II is genotype II.
2、千粒重性状检测2. Thousand-grain weight trait detection
将小麦品种分别种植于不同环境，收获后分别统计两种基因型的小麦的平均千粒重。统计结果见表2。Wheat varieties were planted in different environments, and the average thousand-kernel weight of the two genotypes of wheat was calculated after harvest. The statistical results are shown in Table 2.
表2Table 2
注：P value为关联分析的显著性水平，“*”表示P<0.05,“**”表示P<0.01,“****”表示P<0.0001。Note: P value is the significance level of correlation analysis. “*” means P<0.05, “**” means P<0.01, and “****” means P<0.0001.
利用Tassel2.1软件选择单线性模型+群体结构(GLM+Q)方法将自然群体中小麦TaJMJ12-5A基因的基因型与千粒重进行关联分析。结果表明，296个小麦品种组成的自然群体中，“基因型I的小麦”的千粒重>“基因型II的小麦”的千粒重；其余4个环境虽然均值数据同样为I型＞II型，但是统计检验大于0.05，因此不计入。所述“>”为在统计学上的>。对自然群体的研究表明，基因型I是提高小麦千粒重的优异基因型。Tassel2.1 software was used to select the single linear model + population structure (GLM + Q) method to conduct correlation analysis between the genotype of the wheat TaJMJ12-5A gene and the thousand-kernel weight in the natural population. The results show that in a natural population composed of 296 wheat varieties, the thousand-kernel weight of "genotype I wheat" is > the thousand-kernel weight of "genotype II wheat"; although the mean data of the remaining four environments are also type I > type II, the statistics The test is greater than 0.05, so it is not counted. The ">" means > in statistics. Studies on natural populations have shown that genotype I is an excellent genotype for increasing thousand-grain weight of wheat.
基于上述实施例可见，通过检测待测小麦基于TaJMJ12-5A基因的基因型可以筛选或辅助筛选小麦千粒重性状，在小麦分子标记辅助育种过程中具有重要的应用价值。Based on the above examples, it can be seen that detecting the genotype of the wheat to be tested based on the TaJMJ12-5A gene can screen or assist in screening the thousand-grain weight trait of wheat, which has important application value in the process of molecular marker-assisted breeding of wheat.
在上述实施例中，对各个实施例的描述都各有侧重，某个实施例中没有详述或记载的部分，可以参见其它实施例的相关描述。In the above embodiments, each embodiment is described with its own emphasis. For parts that are not detailed or documented in a certain embodiment, please refer to the relevant descriptions of other embodiments.
以上所述实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围，均应包含在本发明的保护范围之内。The above-described embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still implement the above-mentioned implementations. The technical solutions described in the examples are modified, or some of the technical features are equivalently replaced; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of each embodiment of the present invention, and should be included in within the protection scope of the present invention.
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