CN108384877A - The InDel molecular labeling primers of BcGGP genes and application - Google Patents

Abstract

The invention discloses a kind of InDel molecular labelings of assisted Selection Chinese cabbage ascorbic acid content related gene B cGGP, the sense primer of the InDel molecule labelled series is BcGGPindel F：AGAACAACATCCCTTTCAAC；Downstream primer is BcGGPindel R：TAATCCTCTTTCCTCTTCAG.BcGGPindel can be used for the auxiliary identification of Chinese cabbage ascorbic acid content detection, and have the advantages that easy to operate, of low cost, the period is short, and the stability of the label is good, it is not influenced by other gene effects and environmental factor, it can be in early generation selection, shorten the breeding time limit, improves breeding efficiency.The breed improvement that ascorbic acid content is improved to Chinese cabbage is of great significance, and is suitable for the assisted selection of gene.

Description

InDel Molecular Marker Primer of BcGGP Gene and Its Application

technical field

The invention belongs to the field of molecular assisted breeding of non-heading Chinese cabbage, and relates to an InDel molecular marker primer for BcGGP gene and its application; in particular, it relates to an InDel molecular marker primer for assisting selection of ascorbic acid content of non-heading Chinese cabbage BcGGP gene and its application.

Background technique

Ascorbic acid (L-Ascorbic acid, AsA), also known as vitamin C, is a multifunctional metabolite in animals and plants, and is also an essential nutrient for maintaining the life activities of animals and plants. Since humans and some primates cannot synthesize ascorbic acid by themselves, they can only be obtained from vegetables and fruits. Non-heading cabbage is one of the most important vegetable crops in my country. It originated in the middle and lower reaches of the Yangtze River and has a long history of cultivation. Because of its strong adaptability, short growth cycle and rich nutritional quality, it is widely used in South Asia, Japan , the United States, Europe and other places are widely planted and become an important world vegetable. The ASA content of different varieties of non-heading Chinese cabbage is significantly different, and improving the ASA content of non-heading Chinese cabbage is one of the important breeding goals. The molecular marker-assisted breeding can speed up the breeding process, so the development of molecular markers related to ascorbic acid content is of great significance for improving the quality of non-heading Chinese cabbage.

The L-galactose pathway is the main pathway of plant ascorbic acid synthesis, T-DNA insertion or knockout of genes on this pathway can reduce the content of ASA in leaves and overexpress most genes in the L-galactose pathway, which has a significant effect on ASA content However, it has a lower impact, but overexpression of the GDP-L-galactose phosphorylase (GGP) gene can increase the ASA content of tobacco leaves by 2 times, the ASA content of Arabidopsis leaves by 3 times, and increase the ASA content in potato, tomato , and strawberry have been shown that the overexpression of GGP can increase the content of ASA. The analysis of ASA-related QTL in apple showed that the allelic variation of GGP is the main determinant of regulating ascorbic acid content. It indicated that the GGP gene plays an important role in the synthesis of ascorbic acid. Based on the resequencing of different varieties of non-heading Chinese cabbage, the present invention detects the allelic variation of the BcGGP gene, and develops the InDel molecular marker of BcGGP; and selects Wutaicai varieties with high ascorbic acid content and Erqing with low ascorbic acid content. As the parent, the reliability of the marker was verified on 100 recombinant inbred lines. The development of this marker has the advantages of simple operation, low cost, and short cycle. Moreover, the marker has good stability and is not affected by other gene effects and environmental factors. It can be selected in the early generation, shorten the breeding period, and improve the breeding efficiency. It is of great significance to improve the ascorbic acid content of non-heading Chinese cabbage varieties, and it is suitable for gene-assisted selection breeding.

Contents of the invention

Purpose of the invention: The purpose of the present invention is to develop the molecular marker and primers of BcGGP gene of non-heading Chinese cabbage with high ascorbic acid content, which can be used for molecular marker-assisted selection breeding of non-heading Chinese cabbage ascorbic acid content.

The purpose of the present invention can be achieved through the following technical solutions:

The method for BcGGPindel molecular labeling of non-heading Chinese cabbage comprises:

The ascorbic acid synthesis gene GDP-L-galactose phosphorylase (GGP) carries allelic variation in non-heading Chinese cabbage, and the upstream and downstream primers of the molecular markers of BcGGPindel developed according to the allelic variation of the gene are as follows:

BcGGPindel-F: AGAACAACATCCCTTTCAAC (SEQ ID NO. 1);

BcGGPindel-R: TAATCCTCTTTCCTCTTCAG (SEQ ID NO. 2)

Using this pair of primers to amplify non-heading cabbage Wutaicai variety and Erqing variety as well as individual plants in the isolated population obtained, the amplification results are three types of fragments, the long fragment represents the insertion of InDel, denoted as In, and The amplification result of Wutaicai is consistent, generally it is a plant with high ascorbic acid content; the short fragment indicates the absence of InDel, denoted as Del, which is consistent with the amplification result of Erqing, generally it is a plant with low ascorbic acid content; both fragments have The fragments are heterozygous, denoted as H, and are generally plants with high ascorbic acid content. The BcGGPInDel molecular marker was significantly correlated with the ascorbic acid content of non-heading Chinese cabbage, so it could be applied to molecular marker-assisted selection of ascorbic acid content in non-heading Chinese cabbage.

Indel molecular marker primers for non-heading Chinese cabbage BcGGP, which consist of the following primers: BcGGPindel-F: SEQ ID NO.1; BcGGPindel-R: SEQ ID NO.2.

Application of the molecular marker of the present invention in molecular marker-assisted selection of ascorbic acid content in non-heading Chinese cabbage.

Application of the Indel molecular marker primer of non-heading Chinese cabbage BcGGP in the molecular marker-assisted selection of ascorbic acid content in non-heading Chinese cabbage.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The InDel marker can be analyzed using the same separation and detection technology as the SSR marker, which is easy to apply, and is more convenient to detect and analyze than the SNP marker, and has stability.

(2) Auxiliary selection is convenient and cost saving. The conventional breeding methods of non-heading Chinese cabbage with high Vc content generally have a long cycle, high cost, time-consuming and laborious. The BcGGPindel molecular marker obtained by the invention is used for auxiliary selection, which can realize seedling selection, reduce workload, and greatly improve the selection efficiency of non-heading Chinese cabbage lines with high Vc content, thereby speeding up the breeding process.

Description of drawings

Fig. 1 Genotyping of the InDel molecular marker of the BcGGP gene

Figure 2 The ascorbic acid content corresponding to the RIL population of different genotypes

Detailed ways

Implementation procedure of the present invention is:

Step 1. Based on the site provided by resequencing and the sequencing results of the full-length DNA clone of the BcGGP gene, it was determined that there was an InDeL insertion site of GAAAATAAAT at the fifth intron of the BcGGP gene.

Step 2: Design specific primers at a distance of 100 bp from the InDel site. The upstream primer of the BcGGPindel sequence is BcGGPindel-F: AGAACAACATCCCTTTCAAC; the downstream primer is BcGGPindel-R: TAATCCTCTTTCCTCTTCAG. The fragment length is about 200 bp, and the PCR amplification product is subjected to polypropylene gel electrophoresis Perform genotyping. The difference in electrophoresis bands can be used to distinguish genotypes. Long fragments are marked as In, short fragments are marked as Del, and heterozygous bands are marked as H (Figure 1)

Step 3, genotyping and association analysis of the InDel molecular marker of the BcGGP gene in the F6 recombinant inbred line population obtained from Wutaicai and Erqing as parents. The results verified that among the 100 recombinant inbred lines populations, 41 were detected as the In genotype and had the same band type as the parent Wutaicai, and the average ASA content was 67.32 mg/100g; it was the Del genotype and had the same band type as the parent Erqing had 39 samples with the same band type, with an average ASA content of 39.79 mg/100g; 5 samples with H genotype with two band types, with an average ascorbic acid content of 63.70 mg/100g (Figure 2). Significant difference analysis (P=0.0018, P<0.01) was extremely significant difference. The person correlation analysis between the marker genotype and the ASA content phenotype showed that the material ASA content was significantly correlated with the marker genotype at the 0.1 level.

sequence listing

<110> Nanjing Agricultural University

<120> InDel molecular marker primer and application of BcGGP gene

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 1

agaacaacat ccctttcaac 20

<210> 2

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 2

taatcctctt tcctcttcag 20

Claims (4)

1. the method for Chinese cabbage BcGGP Indel molecular labelings it is characterized in that：

Ascorbic acid synthetic gene GDP-L- gala saccharophosphorylases carry allelic variation in Chinese cabbage, according to the gene The molecular labeling upstream and downstream primer of BcGGPindel of allelic variation exploitation be respectively：

BcGGPindel-F：SEQ ID NO.1；

BcGGPindel-R：SEQ ID NO.2.

Using this to the single plant in primer amplification Chinese cabbage Wuta-tsai kind and two green kinds and the segregating population of acquisition, Amplification is the segment of three kinds of forms, and long segment indicates the insertion of InDel, is denoted as In, consistent with the amplification of Wuta-tsai, For the plant of high ascorbic acid content；Short-movie section indicates the missing of InDel, is denoted as Del, consistent with the amplification of two blueness, is The plant of low ascorbic acid content；Two kinds of segment some are hybrid fragments, are denoted as H, are the plant of high ascorbic acid content.

2. the Indel molecular labeling primers of Chinese cabbage BcGGP, it is characterised in that be made of following primer：BcGGPindel- F：SEQ ID NO.1；BcGGPindel-R：SEQ ID NO.2.

3. the molecular labeling described in claim 1 is in the molecular marker assisted selection of Chinese cabbage ascorbic acid content Using.

4. the Indel molecular labeling primers of the Chinese cabbage BcGGP described in claim 2 contain in Chinese cabbage ascorbic acid Application in the molecular marker assisted selection of amount.