阅读说明：本技术 一组芥蓝InDel分子标记及其开发方法与应用 (Mustard blue InDel molecular markers and development method and application thereof ) 是由 张艳 洪晓如 陈汉才 黎庭耀 沈卓 杨易 于 2019-11-29 设计创作，主要内容包括：本发明提供了一组芥蓝InDel分子标记及其开发方法与应用,涉及分子标记技术领域。本发明提供的一组芥蓝InDel分子标记包括60个位点所对应的引物对,其核苷酸序列如SEQ ID NO.1～SEQ ID NO.120所示。本发明开发的标记具有操作简单、稳定性好、多态性高的特点；且上述引物对稳定性好,均匀分布于9个连锁群,可用于芥蓝重要农艺性状基因的定位、遗传多样性分析、指纹图谱构建、全基因组关联分析与遗传连锁图谱的构建或分子标记辅助选择育种,可以提高工作效率。(The invention provides a group of cabbage mustard InDel molecular markers and a development method and application thereof, and relates to the technical field of molecular markers. The group of mustard blue InDel molecular markers provided by the invention comprises primer pairs corresponding to 60 sites, and the nucleotide sequences of the primer pairs are shown as SEQ ID No. 1-SEQ ID No. 120. The marker developed by the invention has the characteristics of simple operation, good stability and high polymorphism; the primer pair has good stability, is uniformly distributed in 9 linkage groups, can be used for positioning important agronomic trait genes of the cabbage mustard, analyzing genetic diversity, constructing a fingerprint map, performing whole genome association analysis and constructing a genetic linkage map or performing molecular marker-assisted selective breeding, and can improve the working efficiency.)

1. A group of mustard blue InDel molecular markers is characterized in that: the molecular marker of the cabbage mustard InDel comprises primer pairs corresponding to 60 sites, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1-SEQ ID NO. 120.

2. The method for developing the molecular marker of the mustard blue InDel of claim 1, which is characterized in that: the method comprises the following steps:

(1) extracting genome DNA of purple flower cabbage mustard "HJL" and green flower cabbage mustard "612F";

(2) performing re-sequencing on the obtained genome DNA by utilizing the illumina HiSeqTM, performing data analysis by utilizing bioinformatics software, and finding out an InDel site between parents by taking a reference sequence as a bridge;

(3) selecting InDel sites uniformly distributed on 9 chromosomes of cabbage mustard, extracting sequences of 400bp before and after the InDel sites, and designing corresponding InDel primers; the length of the InDel primer is 100-300 bp, and the Tm value is 55-60 ℃;

(4) identifying amplification polymorphism of the InDel sites in the step (3) in multiple materials through a plurality of representative cabbage mustard materials, and screening to obtain a group of InDel sites on the basis of the principle that at least 2 materials present amplification polymorphism;

(5) and (4) further selecting InDel sites with clear amplified bands from the InDel sites obtained in the step (4), wherein the number of the InDel sites on each chromosome is not less than 5, and thus obtaining the Chinese kale InDel molecular marker.

3. The method for developing the molecular marker of the mustard blue InDel according to claim 2, wherein the method comprises the following steps:

the plurality of representative kale materials in step (4) are 8 representative kale materials, specifically selected from: brassica juncea "HJL", Tao mountain Brassica juncea, big leaf red foot Brassica juncea, Chaoyang red foot Brassica juncea, Brassica juncea "612F", 07M Brassica juncea, 612M Brassica juncea, and Dongqiang M Brassica juncea.

4. Use of the mustard blue InDel molecular marker of claim 1 in the construction of a genetic map of mustard blue.

5. Use of the mustard blue InDel molecular marker of claim 1 in the QTL mapping of mustard blue.

6. Use of the mustard blue InDel molecular marker of claim 1 in mustard blue whole genome association analysis.

7. The use of the mustard blue InDel molecular marker of claim 1 in molecular assisted breeding of mustard blue.

8. Use of the mustard blue InDel molecular marker of claim 1 in the analysis of genetic diversity in mustard blue.

9. The use of the method for developing a molecular marker of mustard blue InDel as claimed in claim 2 or 3 in developing a molecular marker of Brassicaceae vegetable InDel.

10. Use according to claim 9, characterized in that:

the cruciferous vegetables include cabbage, and Chinese cabbage.

Technical Field

The invention relates to the technical field of molecular markers, in particular to a group of cabbage mustard InDel molecular markers and a development method and application thereof.

Background

Cabbage mustard (Brassica alboglabra) is a subspecies of Brassica species in brassicaceae, is mainly cultivated in places such as Guangdong, Guangxi, Fujian and the like, is a special vegetable in south China, and takes the flower shoots and leaves as food. The traditional breeding method of the cabbage mustard has long period and low efficiency. The molecular marker auxiliary selection is combined with the traditional breeding, so that the new variety breeding and genetic improvement process can be accelerated. The development of the molecular marker lays a foundation for molecular marker-assisted breeding.

Molecular Markers (Molecular Markers) are genetic Markers based on nucleotide sequence variations in the genetic material between individuals and are a direct reflection of genetic polymorphisms at the DNA level. The method is widely applied to the aspects of genetic breeding, gene localization, species genetic relationship identification and the like. With the continuous development of biotechnology, more than 20 markers have been established, such as rflp (restriction fragment length polymorphism), rapd (random amplified polymorphism dnas), ssr (simple sequence repeat), srap (sequence delayed amplified polymorphism), snp (single nucleotide polymorphism), InDel (induction-deletion), etc., wherein InDel is widely used for gene localization and genetic diversity analysis (Li and mao, 2018; Shu et al, 2018; Wu et al, 2014). InDel markers refer to molecular markers in which a certain nucleotide fragment is inserted or deleted at the allelic site of homologous sequences between closely related species or different individuals of the same species, and the occurrence of InDel is mainly related to the base type of genomic sequences and DNA replication errors (Yangjie et al, 2016; Jander et al, 2002). InDel is widely distributed in a genome, has large density and is numerous. InDel is second only to SNP in terms of distribution density, but much higher than SSR. The average density in rice was one InDel per 953bp and in eggplant was one InDel per 1.4M. The InDel marker has high accuracy and stable variation, avoids fuzzy follow-up analysis caused by specificity and complexity, and is gradually applied to crops such as rice, cucumber, hot pepper, wheat, cabbage, Chinese cabbage and the like. However, mustard has fewer InDel markers than other crops and is of limited use.

Disclosure of Invention

The invention aims to solve the technical problem of establishing a technical system for developing the InDel marker of the kalium mustard, providing more new InDel markers for the gene positioning of important characters of the kalium mustard, the construction of a genetic map, the analysis of genetic diversity and the auxiliary selection breeding of molecular markers, and making up for the problem of insufficient InDel markers of the kalium mustard at present. One object of the present invention is to provide a set of molecular markers of mustard blue InDel.

The invention also aims to provide a development method of the mustard blue InDel molecular marker.

The invention further aims to provide application of the above mentioned molecular marker of the mustard blue InDel.

The purpose of the invention is realized by the following technical scheme:

a group of kale InDel molecular markers comprises primer pairs corresponding to 60 sites, wherein the name and nucleotide sequence of each primer pair are shown in table 1 (also shown in SEQ ID NO. 1-SEQ ID NO.120 in a sequence table):

TABLE 160 primer pair information for InDel molecular markers

The development method of the mustard blue InDel molecular marker comprises the following steps:

(1) extracting genome DNA of purple flower cabbage mustard "HJL" and green flower cabbage mustard "612F";

(2) performing re-sequencing on the obtained genome DNA by utilizing the illumina HiSeqTM, performing data analysis by utilizing bioinformatics software, and finding out an InDel site between parents by taking a reference sequence as a bridge;

(3) selecting InDel sites uniformly distributed on 9 chromosomes of cabbage mustard, extracting sequences of 400bp before and after the InDel sites, and designing corresponding InDel primers; the conditions for primer design were: the length is 100-300 bp, and the Tm value is 55-60 ℃;

(4) identifying amplification polymorphism of the InDel sites in the step (3) in multiple materials through a plurality of representative cabbage mustard materials, and screening to obtain a group of InDel sites on the basis of the principle that at least 2 materials present amplification polymorphism;

(5) and (4) further selecting InDel sites with clear amplified bands from the InDel sites obtained in the step (4), wherein the number of the InDel sites on each chromosome is not less than 5, and thus obtaining the Chinese kale InDel molecular marker.

The plurality of representative kale materials in step (4) is preferably 8 representative kale materials, specifically selected from: brassica juncea "HJL", Tao mountain Brassica juncea, big leaf red foot Brassica juncea, Chaoyang red foot Brassica juncea, Brassica juncea "612F", 07M Brassica juncea, 612M Brassica juncea, and Dongqiang M Brassica juncea.

Wherein, the peach mountain kale: GD II 4E00078 of Guangdong province crop germplasm resource protection library; big leaf red foot kale: GD II 4E00068 of Guangdong province crop germplasm resource protection library; chaoyang red-foot cabbage mustard: GD II 4E00077 of Guangdong province crop germplasm resource protection library; 07M cabbage mustard: GD II 4E00039 of Guangdong province crop germplasm resource protection library; 612M cabbage mustard: GD II 4E00042 of Guangdong province crop germplasm resource protection library; wintergreen M cabbage: and GD II 4E00046 of Guangdong province crop germplasm resource protection library. The 8 representative cabbage mustard materials involved were all available from vegetable research institute of agricultural sciences, Guangdong province.

The method can also be applied to the development of InDel molecular markers of all cruciferous vegetables such as cabbage mustard, cabbage, Chinese cabbage and the like.

The molecular marker of the Chinese kale InDel is applied to the construction of a Chinese kale genetic map, QTL positioning, whole genome association analysis, molecular assisted breeding and genetic diversity analysis.

Compared with the prior art, the invention has the following advantages and effects:

(1) according to the invention, 2 cabbage mustard inbred lines are subjected to whole genome re-sequencing and InDel locus difference analysis, and an InDel primer is designed by taking insertion/deletion 5-20 bp as a standard, so that an InDel marker covering a whole genome is obtained. The InDel polymorphic marker developed aiming at the genome difference of two representative varieties of cabbage mustard has high success rate, has higher efficiency than the primer designed according to SSR locus re-screening polymorphism, and has distribution density far higher than SSR. Has the characteristics of simple operation, good stability and high polymorphism.

(2) The 60 pairs of InDel molecular markers have good stability and are uniformly distributed on 9 linkage groups. The method is used for positioning important agronomic trait genes of the cabbage mustard, analyzing genetic diversity, constructing a fingerprint map, performing whole genome association analysis and constructing a genetic linkage map or performing molecular marker-assisted selective breeding, and can improve the working efficiency.

Drawings

FIG. 1 is a diagram showing the results of gel electrophoresis of PCR amplification products of 8 parts of cabbage mustard material with 5 InDel molecular markers; wherein, Lane M is Marker, Lane A1-A8 is InDel Marker J113, Lane B1-B8 is InDel Marker J115, Lane C1-C8 is InDel Marker J117, Lane D1-D8 is InDel Marker J125, and Lane E1-E8 is InDel Marker J128; the 8 parts of cabbage mustard material are purple cabbage mustard "HJL", peach mountain cabbage mustard, big leaf red-foot cabbage mustard, Chaoyang red-foot cabbage mustard, green cabbage mustard "612F", 07M cabbage mustard, 612M cabbage mustard and Dongqiang M cabbage mustard in sequence.

FIG. 2 is a diagram showing the results of gel electrophoresis of PCR amplification products of InDel marker J252 in 33 portions of cabbage mustard material; wherein, Lane M is Marker, and the numbers of the other lanes correspond to the DNA sequence numbers of 33 kinds of cabbage mustard materials.

FIG. 3 is a diagram showing the results of gel electrophoresis of PCR amplification products of InDel marker J117 in 33 portions of cabbage mustard material; wherein, Lane M is Marker, and the numbers of the other lanes correspond to the DNA sequence numbers of 33 kinds of cabbage mustard materials.

FIG. 4 is a diagram showing the results of gel electrophoresis of PCR amplification products of InDel marker J813 in 33 portions of cabbage mustard material; wherein, Lane M is Marker, and the numbers of the other lanes correspond to the DNA sequence numbers of 33 kinds of cabbage mustard materials.

FIG. 5 is a graph of the cluster analysis results of a set of Chinese kale InDel molecular markers of the present invention on 33 parts of Chinese kale materials.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.