CN114438249B

CN114438249B - Primer group, kit and identification method for identifying cultivation types of valium odoratum honey pomelos

Info

Publication number: CN114438249B
Application number: CN202210163279.XA
Authority: CN
Inventors: 王雨亭; 胡钟东; 万水林; 杨惠栋; 汤雨晴; 徐超; 刘心澄
Original assignee: Horticultural Research Institute Jiangxi Academy Of Agricultural Sciences
Current assignee: Horticultural Research Institute Jiangxi Academy Of Agricultural Sciences
Priority date: 2022-02-22
Filing date: 2022-02-22
Publication date: 2022-10-21
Anticipated expiration: 2042-02-22
Also published as: CN116287364B; CN114438249A; CN115927712B; CN115927713A; CN116287364A; CN115927713B; CN115927712A

Abstract

The invention discloses a primer group, a kit and an identification method for identifying cultivation types of jinggangmi pomelos, and relates to the technical field of molecular markers. Based on the whole genome re-sequencing result, 8 InDel labeled primers with good polymorphism are developed, and 2 main cultivars can be distinguished in the early stage by using any 1 pair of the 8 pairs of primers, the detection rate is 92.31-100%, the detection accuracy is 100%, a basis is provided for the early accurate identification of the Jinggang honey pomelo variety, and a positive effect is played on clarifying the name of the variety in production.

Description

Primer group, kit and identification method for identifying cultivation types of valium odoratum honey pomelos

Technical Field

The invention belongs to the technical field of molecular markers, and particularly relates to a primer group, a kit and an identification method for identifying a cultivation type of valium glaucum.

Background

The jinggang honey pomelo is a general name of sweet pomelo varieties which are produced and cultivated in Jian city of Jiangxi province by taking 'Jinggang mountain' as a brand, is a geographical landmark agricultural product of China, is one of six wealth people industries which are mainly supported and developed by Jian, and is also one of three golden signboards of fruit industry of Jiangxi province. Up to now, the planting area of Jinggang honey pomelos reaches more than 40 ten thousand mu, 3 main cultivars are golden Sha pomelos, golden orchid pomelos and Taoxi honey pomelos respectively, and according to statistics, the planting area of the first two cultivars reaches 71.27% of the total area of Jinggang honey pomelos as the leading cultivar by the end of 2020, and the planting area of the Taoxi honey pomelos accounts for 13.39%. The Taoxi honey pomelo is a variety of Shatian pomelo in the west of the river, has early mature period and obvious fruit characteristics, and is easy to be identified with other two Jinggang honey pomelo varieties. However, since the golden pomelo is a filial generation of the golden orchid pomelo and the shatian pomelo, the two jinggang honey pomelo varieties have close relativity and are difficult to distinguish by morphological characteristics. In the process of rapid development of Jinggang honey pomelos, 2 main cultivated varieties of golden orchid pomelos and golden Sha pomelos are mixed, so that seedling confusion in production and wrong name of the varieties are caused, and the healthy sustainable development of the industry is seriously influenced.

Disclosure of Invention

In view of the above, the invention aims to provide a primer group, a kit and an identification method for identifying the type of cultivation of jinggang honey pomelos, which can effectively distinguish 2 main cultivars, provide a basis for early accurate identification of the jinggang honey pomelo cultivars, and play a positive role in clarifying the cultivar names in production.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a primer for identifying the cultivation type of jinggangmei pomelo, wherein the primer group comprises 1 pair or a combination of more than 1 pair of PA1, PA2, PA3, PA4, PA5, PA6, PA7 and PA 8; the sequences of the primers are shown in table 1:

TABLE 1 sequences of primer sets

Based on the whole genome re-sequencing result, the invention develops the 8 InDel labeled primers with good polymorphism, and the 8 pairs of primers can distinguish 2 main cultivars by using any 1 pair, thereby providing a basis for the early accurate identification of the Jinggang honey pomelo varieties and playing an active role in clarifying the name of the varieties in production. The Jinggang honey pomelo cultivar preferably comprises golden orchid pomelos and golden Sha pomelos, wherein the golden Sha pomelos are filial generations of the golden orchid pomelos (female parent) and the Shatian pomelos (male parent), and the golden Sha pomelos are filial generations.

The method for obtaining the primer group is not specially limited, preferably, young leaves are put into liquid nitrogen to be ground, then, the total DNA of the genome is extracted, the obtained original data is subjected to quality evaluation through FastQC, a connector sequence is removed by utilizing Fastp software, data quality control is carried out, indexes are established by taking the late white pomelo (Citrus maxima) (wet: http:// Citrus hzau.edu.eu.cn/orange/download/index.php) as a reference genome, the fragments obtained by sequencing are compared with the reference genome sequence by utilizing BWA software, and InDel detection and filtration are carried out through GATK. InDel marker screening was performed using stringent parameters (Simona et al, 2017): (1) QUAL at the variant site is greater than 400; (2) the GQ value of the mutation site is 99; (3) the depth of coverage is greater than 20; (4) the length of the InDel insertion or deletion is between 5 and 8 bp. The information of the sequences flanking the variant positions was screened according to the reference genome annotation and InDel-labeled amplification primers were designed using Primer3.0 (http:// bioinfo. Ut. Ee/primer3 /). And then performing primer specificity detection by using e-PCR, and finally selecting 8 pairs of specific InDel labeled amplification primers.

The invention also provides a kit for identifying the cultivation type of valium glaucum maxim, which comprises the primer group.

The kit of the present invention preferably further comprises PCR reaction reagents such as Mix solution and/or ddH ₂ O。

The invention also provides a method for identifying the cultivation type of valium odoratum honey pomelo, which comprises the following steps: performing PCR amplification by using the genome DNA of the Jinggang honey pomelo cultivar as template DNA and the primer, wherein the amplification product is homozygous when only one band exists, and the variety is male parent Shatian pomelo or female parent Jinlan pomelo; when there are two or more bands, it is heterozygous, and the variety is offspring.

Specifically, the genotype of the female parent Jinlan pomelo, the male parent Shatian pomelo and the offspring Jinsha pomelo is taken as a reference, and the variety type is judged according to the genotype of the sample. Record 1 consistent with maternal genotype, this breed is golden orchid pomelo; record of the same genotype as the male parent is 2, and the variety is Shatian pomelo; record of genotype consistent with the progeny is 3, the variety is golden pomelo; the record of other genotypes is 4, other varieties; genotype deletions were recorded as 0.

The primer group or the kit can be applied to early identification, and only the young leaves of the young trees are utilized, so that the genome DNA is preferably extracted from the young leaves. The method for extracting the genomic DNA is not particularly limited in the present invention, and preferably a CTAB method or a kit method is used.

The PCR amplification system of the present invention is preferably 15. Mu.l, and preferably comprises: 7 μ L of Mix solution, 0.3 μ L of 10mmol/L forward primer, 0.3 μ L of 10mmol/L reverse primer, 1.5 μ L of template DNA and the balance ddH ₂ And O. The PCR amplification procedure of the present invention preferably comprises pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, and reaction for 35 cycles; further extension was carried out at 72 ℃ for 5min.

When the primer group or the kit is used for identification, at least two rounds of PCR identification are preferably carried out, then agarose gel electrophoresis is used, primers with clear gel pictures and completely consistent sizes with predictions are reserved, two bands can be seen in primer amplification, the fact that the site is heterozygous is shown, the size difference which can be distinguished by the gel pictures exists, and the mark with good effect can be determined to be used. The agarose gel electrophoresis of the invention preferably adopts 1.5 to 2 mass percent of agarose gel, the voltage is 120V, and the electrophoresis is carried out for 15 to 20min.

Has the advantages that: based on the whole genome re-sequencing result, 8 InDel labeled primers with good polymorphism are developed, and 2 main cultivars can be distinguished in the early stage by using any 1 pair of the 8 pairs of primers, so that a basis is provided for the early accurate identification of the Jinggang honey pomelo variety, and a positive effect is played on the clarification of the variety name in production.

Drawings

FIG. 1 is a diagram showing the verification of the specificity of PA1 and PA 2; in each figure, lane 1 and 2 are maternal Jinlan pomelos, lane 3 is offspring golden Sha pomelos, lane 4 and 5 are paternal Shatian pomelos, and the rest are cultivars of Jinggang honey pomelos in the producing area, the same applies below;

FIG. 2 is a diagram showing the verification of the specificity of PA3 and PA 4;

FIG. 3 is a diagram showing the verification of the specificity of PA5 and PA 6;

FIG. 4 is a diagram showing the verification of the specificity of PA7 and PA 8.

Detailed Description

The following will explain in detail the primer set, the kit and the method for identifying the type of cultivation of jinggangmi pomelo according to the present invention with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

The primers shown in Table 1 were synthesized by Biotech, inc., of Onychoma, beijing.

Materials: extracting the genomic DNA of the young and tender leaves of the honey pomelo tree.

The PCR reaction system was 15. Mu.l: 7 μ L of Mix solution, 0.3 μ L of 10mmol/L forward primer, 0.3 μ L of 10mmol/L reverse primer, 1.5 μ L of 100ng/μ L template DNA and 6 μ L of ddH2O.

The PCR amplification procedure was: pre-denaturation at 94 ℃ for 5min; carrying out denaturation at 94 ℃ for 30s, annealing at Tm 55 ℃ for 30s, extending at 72 ℃ for 30s, and carrying out reaction for 35 cycles; finally, the extension is carried out for 5min at 72 ℃.

The agarose gel electrophoresis adopts 1.5-2% agarose gel by mass percent, the voltage is 120V, and the electrophoresis is carried out for 15-20min. After at least two rounds of PCR identification, the primers with clear gel pictures and completely consistent sizes with the predictions are reserved, the results are shown in figures 1-4, two bands can be seen by primer amplification, the site is heterozygous, size differences distinguishable by the gel pictures exist, the usable marker with good effect can be determined, the detection rate is 92.31-100%, and the detection accuracy is 100% (table 2).

Table 2 statistics of InDel molecular marker identification results

Note: record consistent with the maternal genotype is 1, record consistent with the paternal genotype is 2, record consistent with the offspring genotype is 3, record of the other genotypes is 4, and the deletion record is 0.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Claims

1. A primer group for identifying cultivation types of jinggangmi pomelos is characterized by comprising PA1; the PA1 comprises PA1-F with a nucleotide sequence shown as SEQ ID NO.1 and PA1-R with a nucleotide sequence shown as SEQ ID NO. 2.

2. The primer set according to claim 1, wherein the Jinggang honey pomelo cultivars include golden orchid pomelos and golden shavings pomelos.

3. A kit for identifying a type of cultivation of valium glaucum fruit, comprising the primer set according to claim 1 or 2.

4. The kit of claim 3, wherein the kit further comprises PCR reaction reagents.

5. A method for identifying the cultivation type of Jinggang honey pomelos is characterized by comprising the following steps: performing PCR amplification by using the genomic DNA of a Jinggang honey pomelo cultivar as a template DNA and using the primer of claim 1 or 2, wherein the cultivar is homozygous when only one band exists in the amplification product, and the cultivar is male parent Shatian pomelo or female parent Jinlan pomelo; when two or more bands exist, the hybrid is obtained, and the variety is filial generation golden pomelo.

6. The method of claim 5, wherein the genomic DNA is extracted from young and young leaves of the body of honey pomelo.

7. The method of claim 5, wherein the PCR amplification system is 15 μ l, and comprises: 7 μ L of Mix solution, 0.3 μ L of 10mmol/L forward primer, 0.3 μ L of 10mmol/L reverse primer, 1.5 μ L of template DNA and the balance ddH ₂ O。

8. The method of claim 5 or 7, wherein the PCR amplification procedure comprises pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, and reaction for 35 cycles; further extension was carried out at 72 ℃ for 5min.