CN115505649A - SNP molecular marker for identifying watermelon peel thickness and application thereof - Google Patents

Abstract

The invention relates to an SNP molecular marker for identifying the thickness of watermelon peel and application thereof, and relates to the technical field of biology, wherein the SNP molecular marker is positioned at 32344170 th base of No. 2 chromosome of watermelon genome, the base is C or T, if the genotype is CC, the thickness of the corresponding watermelon peel is less than or equal to 0.6cm, and if the genotype is GG, the thickness of the corresponding watermelon peel is more than 0.6cm. Effectively quickens the breeding process, improves the breeding efficiency and has important significance for watermelon breeding.

Description

SNP molecular marker for identifying watermelon peel thickness and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an SNP molecular marker for identifying watermelon peel thickness and application thereof.

Background

Watermelon is an important cucurbitaceae crop, is an important fruit for people to relieve summer heat and quench thirst in summer, and plays an important role in horticultural crops in the world. China is the first major country in the world for watermelon production and consumption. The thickness of the peel is one of the important agronomic traits of the watermelon, directly influences the edibility and commodity rate of the watermelon, and is an important breeding index of the watermelon. At present, researches on the properties of watermelon peel mainly focus on gene positioning and marker development of peel color, grain coating, wax powder, hardness and the like, but researches on the peel thickness of watermelon are not reported.

In recent years, a multigene marker screening method is developed rapidly, genome-Wide Association Study (GWAS) has become one of the mainstream methods, which can screen related variation of a certain trait in the whole Genome range, and identification of a genetic marker with a low heritability trait by a whole Genome Association analysis method has become a common means of modern breeding work. SNP molecular markers (single nucleotide polymorphisms) refer to changes in the DNA sequence at the genomic level due to mutations of individual nucleotides. The molecular marker is widely existed on a genome, has the characteristics of high throughput, simplicity, stability, high sensitivity and the like, and is a molecular marker with great development prospect in the current molecular marker-assisted breeding work.

With the advent of the watermelon genome era, the functions of important agronomic trait genes of watermelons are defined, key genes for controlling the traits are searched, corresponding molecular markers are developed, and the application of the molecular markers to breeding is an important content of the current watermelon breeding work. Therefore, the invention provides an SNP molecular marker for identifying the thickness of watermelon peel and application thereof.

Disclosure of Invention

The invention aims to solve the technical problem of providing an SNP molecular marker for identifying the thickness of watermelon peel and application thereof. The method aims to develop a molecular marker related to the thickness of watermelon peel and apply the molecular marker to the breeding work of watermelon so as to accelerate the breeding process of watermelon.

In order to solve the technical problems, the first object of the present invention is to provide a SNP molecular marker for identifying watermelon peel thickness, wherein the SNP molecular marker is located at 32344170 th base of chromosome 2 of watermelon genome, and the base is C or T.

The invention has the beneficial effects that: the method utilizes whole genome association analysis (GWAS) to identify and obtain 5 significant SNP sites related to the watermelon peel, then combines transcriptome sequencing to analyze candidate genes near 100kb upstream and downstream of the significant SNP sites, determines the candidate genes related to the thickness of the watermelon peel, and finally determines that the 32344170 th SNP site of No. 2 chromosome of the watermelon genome is tightly linked with the thickness of the watermelon peel based on the candidate genes. Therefore, the SNP marker related to the watermelon peel thickness can promote the molecular marker-assisted selection of the watermelon peel thickness, effectively accelerate the breeding process, improve the breeding efficiency and have important significance for watermelon breeding.

On the basis of the technical scheme, the invention can be further improved as follows.

Furthermore, if the genotype is CC, the thickness of the corresponding watermelon peel is less than or equal to 0.6cm, and if the genotype is GG, the thickness of the corresponding watermelon peel is more than 0.6cm.

Further, the watermelon is 97103.

The second purpose is to provide the application of the SNP molecular marker for identifying the thickness of the watermelon peel, and the application of the SNP molecular marker in identifying the thickness of the watermelon peel; or the application of the SNP molecular marker in watermelon breeding.

In the above application, the substance of the polymorphism of the SNP molecular marker is a reagent, a kit or an instrument for detecting the SNP molecular marker by whole genome sequencing, and can be a primer pair, a probe or a chip for detecting the SNP marker, and the like.

The third purpose is to provide a breeding method of the watermelon, which comprises the following steps:

and (3) taking the sample watermelon with the SNP molecular marker CC in the genome obtained by detection as a parent to breed the watermelon.

The invention also protects a gene chip containing the SNP molecular marker or the SNP molecular marker combination.

Primer pairs, probes or chips for detecting the SNP molecular markers and the corresponding gene chips or SNP molecular marker combinations and the corresponding gene chips are also within the protection scope of the invention.

The application of the SNP molecular marker or the SNP molecular marker combination and a primer pair, a probe or a chip for detecting the SNP molecular marker or the SNP molecular marker combination in the watermelon whole genome selection are also within the protection scope of the invention.

The application of the SNP molecular marker or the SNP molecular marker combination and the primer pair, the probe or the chip for detecting the SNP molecular marker or the SNP molecular marker combination in watermelon breeding are also within the protection scope of the invention.

Drawings

Figure 1 is a GWAS-based manhattan plot of watermelon peel thickness.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Example 1: mining SNP (Single nucleotide polymorphism) marker related to watermelon peel thickness based on whole genome association analysis (GWAS)

1. Materials and methods

1.1 obtaining, quality control and type judgment of watermelon whole genome re-sequencing data

112 parts of watermelon natural population materials (see table 1 in detail) are selected from a watermelon middle-term bank, planted in a greenhouse, and young leaves at the top of the plant are collected in the plant elongation period, and complete genome DNA is extracted by adopting a traditional CTAB method. The quality of the extracted genome DNA is detected by a NanodropND1000 spectrophotometer, and the quality standard is reached when the ratio of A260/280 is 1.8-2.0 and the ratio of A260/230 is about 1.7-1.9.

The concentration of the DNA samples meeting the standard was diluted to 50 ng/. Mu.l and each DNA sample was re-sequenced using the sequencing platform from Illumina (average sequencing depth 9.24X). Sequencing reads for each variety were aligned to the watermelon reference genome using SOAP2 (http:// cucurbitangenomics. Org/organissm/V1). The parameters are set as-m 100, -x888, -s35, -132, -v3, and filtering to remove the weight. All reads on the double-ended and single-ended alignments were SNP calling with the SOAPsnp software with the parameters-L100-u-F156. Filtering the SNPs, and reserving the data with the SNP quality more than or equal to 40 and the base quality more than or equal to 40.

Determination of 1.2, 112 portions of peel thickness of natural population material of watermelon

And continuously planting 112 natural groups for 3 years, selecting 3 melons with consistent watermelon node positions and fruit types in the fruit ripening period, measuring the thickness of the watermelon peel by using a ruler, and taking the average value as the final peel thickness of each material by using data of different field phenotypes in 3 years. The peel thickness of 112 watermelon natural population materials shown in Table 1 is measured, and when the peel thickness is less than or equal to 0.6cm, the watermelon natural population materials are taken as thin peels, and when the peel thickness is more than 0.6cm, the watermelon natural population materials are not taken as thin peels.

1.3 Whole genome Association analysis

Using a mixed linear model in GEMMA 0.98.1 software, taking a Q matrix and an affinity matrix as covariates, using tasselv5.2.43 software to perform genome-wide association analysis on 112 watermelon natural population materials, making manhattan charts by R software, calculating significance thresholds through Bonferroni correction, and setting-log 10 (P) 0.1/Ne (Ne = number of significant SNPs) and-log 10 (P) 0.01/Ne as two threshold lines for screening significant SNPs. The result is shown in figure 1, the watermelon peel thickness gene is positioned at the end of chromosome 2, and the region has 5 significant SNPs, namely S2:32344170, S2:32321308, S2:32304738, S2: 328501 and S2:32311192.

2. Results

Obtaining target SNP: expression quantity and gene function analysis are carried out on 21 candidate genes near 100kb of the upstream and downstream of the significant SNP locus, and the MADS family gene Cla97C02G044160 in the watermelon is found to be a key candidate gene for regulating and controlling the thickness of the watermelon fruit peel and to be closely linked with the significant SNP S2:32344170 of the upstream of the gene and the thickness of the watermelon peel. Therefore, the SNP marker related to the watermelon peel thickness is positioned on No. 2 chromosome of the watermelon genome (http:// cucurbitangenomics. Org/, V2), and is T or C at the 32344170 th nucleotide. The homozygous genotypes of the SNP markers are TT genotype and CC genotype respectively, the CC genotype is a watermelon with thin skin in a watermelon variety, the TT genotype is a non-watermelon with thin skin in the watermelon variety, and the detection efficiency is 91% (see tables 1 and 2 for details).

TABLE 1 pericarp thickness and genotype of 112 natural watermelon populations

TABLE 2 pericarp thickness and genotype statistics of 112 natural watermelon populations

Example 2: application evaluation

1.1 verification of watermelon peel thickness identification method

The genotype of 30 natural population materials at the 32344170 th position on the No. 2 chromosome is detected by a genome re-sequencing method, and the accuracy of detecting the watermelon material by the SNP marker is 97%. As shown in tables 3 and 4.

TABLE 3 pericarp thickness and genotype of 30 watermelon natural populations

TABLE 4 statistics of pericarp thickness and genotype of 30 watermelon natural populations

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (5)

1. The SNP molecular marker for identifying the thickness of the watermelon peel is characterized by being located at 32344170 th base of No. 2 chromosome of a watermelon genome, wherein the base is C or T.

2. The SNP molecular marker for identifying the thickness of watermelon peel according to claim 1, wherein if the genotype is CC, the thickness of the corresponding watermelon peel is less than or equal to 0.6cm, and if the genotype is GG, the thickness of the corresponding watermelon peel is more than 0.6cm.

3. The SNP molecular marker for identifying the thickness of the watermelon peel according to claim 1, wherein the watermelon is a watermelon 97103.

4. The application of the SNP molecular marker for identifying the thickness of watermelon peel is characterized in that the SNP molecular marker of claim 1 is applied to the identification of the thickness of watermelon peel; or the application of the SNP molecular marker of claim 1 in watermelon breeding.

5. A breeding method of watermelon is characterized by comprising the following steps:

and (3) taking the sample watermelon with the SNP molecular marker CC in the genome obtained by detection as a parent to breed the watermelon.

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