SNP (Single Nucleotide Polymorphism) molecular marker related to size of sweet cherry fruit and application

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) molecular marker related to the size of a sweet cherry fruit and application, and belongs to the technical field of genetic breeding of sweet cherries. The molecular marker disclosed by the invention is derived from an exon of FUN001609 and is positioned at a position 16366288 of a first chromosome of Prunus avium Tieton Genome v2.0, and nucleotide at the position is G or A. The molecular marker disclosed by the invention has the advantages that the molecular marker disclosed by the invention can be used for detecting the molecular marker; the SNP site is obviously related to the size of the sweet cherry fruit, and the genotype of the size of the sweet cherry fruit is AAgt; gGgt; gA. Meanwhile, a primer is designed according to the SNP site to carry out PCR amplification on the sweet cherries to be detected, and a PCR product is subjected to sequencing inspection. The SNP molecular marker disclosed by the invention can be used as an auxiliary selection marker of fruit size characters in a sweet cherry breeding process, so that the screening efficiency of large-fruit sweet cherry varieties is improved, the breeding period is shortened, and the breeding time and workload of the large-fruit varieties are greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit tree genetic breeding molecular marker preparation, and in particular relates to a SNP molecular marker related to sweet cherry fruit size and an application thereof. Background Art

[0002] Sweet cherry (Prunus avium L.) fruit is rich in nutrition and has high economic value. Fruit size is an important agronomic trait of sweet cherry and is the main indicator for fruit price classification. Sweet cherry fruit size is affected by multiple factors such as genetic basis, environmental factors and selection intensity, among which genetic basis plays a major role. Sweet cherry trees are tall, have a long juvenile period, and have low breeding efficiency. Molecular marker-assisted breeding can shorten the breeding cycle and save breeding costs. The screening of molecular markers for fruit size and their application in production practice is of great significance for improving breeding efficiency, reducing breeding costs, and breeding new varieties of large-fruited sweet cherries.

[0003] Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. Compared with other molecular markers, SNP molecular markers have the advantages of large number, wide distribution, rich polymorphism and high density. At present, SNP molecular markers are used in genetic diversity and kinship analysis, assisted breeding and variety identification. In sweet cherries, SNP molecular markers have been used to screen 19 main sweet cherry varieties and 5 sweet cherry rootstocks, but SNP molecular markers used to distinguish fruit size traits have not been reported. In view of the unclear genetic regulation mechanism of sweet cherry fruit size traits, the low efficiency and long cycle of traditional breeding methods. The development of new molecular markers and breeding techniques is of great significance for the cultivation of large-fruited sweet cherry varieties. Summary of the invention

[0004] The present invention aims to provide a SNP molecular marker significantly associated with sweet cherry fruit size, with the aim of improving breeding efficiency and cultivating new large-fruit sweet cherry varieties.

[0005] The invention also provides application of the molecular marker.

[0006] The present invention is specifically implemented through the following technical solutions:

[0007] A specific primer for amplifying a SNP molecular marker related to the fruit size of sweet cherry, wherein the nucleotide sequences of the primer and the reverse primer are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0008] Forward primer: 5'-CGCCATTCTTACTACGCTAT-3' (SEQ ID NO.1)

[0009] Reverse primer: 5'-TGAGGCAAACAATGAGGTAG-3' (SEQ ID NO. 2)

[0010] The SNP molecular marker related to the fruit size of sweet cherry was amplified using the genomic DNA of sweet cherry as a template using SEQID NO.1 and SEQ ID NO.2 to obtain an amplified product fragment of 433bp, which is the SNP molecular marker related to the fruit size of sweet cherry; among them, the base type at the 275bp position is AA, corresponding to the sweet cherry variety with larger fruit; the base type at the 275bp position is GA or AG, corresponding to the sweet cherry variety with smaller fruit; the base type at the 275bp position is GG, corresponding to the sweet cherry variety with medium fruit size.

[0011] A kit for detecting SNP molecular markers related to sweet cherry fruit size, comprising the above-mentioned specific primers.

[0012] The use of the above-mentioned SNP molecular marker or the above-mentioned kit in genetic breeding.

[0013] Application of the above SNP molecular marker or the above kit in detecting or identifying the size of sweet cherry fruit.

[0014] The application includes:

[0015] (1) extracting genomic DNA of the sweet cherry sample to be tested;

[0016] (2) using the genomic DNA of the sweet cherry sample to be tested as a template, performing a PCR amplification reaction using the above-mentioned specific primers to obtain an amplified product fragment;

[0017] (3) Detect the base type of the PCR amplification product fragment to determine the fruit size of the sample. When the genotype of the SNP site at the 275bp position is AA, the fruit size of the sweet cherry variety is large. When the genotype is GA or GA, the sweet cherry fruit is the smallest. The genotype is GG, and the corresponding sweet cherry variety has a medium fruit size. The specific primers are:

[0018] Forward primer: 5'-CGCCATTCTTACTACGCTAT-3',

[0019] Reverse primer: 5′-TGAGGCAAACAATGAGGTAG-3′.

[0020] The PCR reaction conditions are as follows: pre-denaturation at 95°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 10 s, extension at 72°C for 30 s, 34 cycles; final extension at 72°C for 5 min; and storage at 4°C.

[0021] The genomic DNA of the sweet cherry sample to be tested comes from sweet cherry seedling leaves.

[0022] The invention provides a SNP molecular marker related to the fruit size of sweet cherry. The SNP molecular marker comes from the third exon of the FUN_001609 gene, is located at 16366288 of the first chromosome of Prunus avium Tieton Genome v2.0, and has a polymorphism of G or A.

[0023] The genotypes of the sweet cherry fruit size molecular markers include GG, AA and GA / AG; wherein, when the genotype is AA, the sweet cherry fruit is a large fruit, when the genotype is GG, the sweet cherry fruit is a medium fruit, and when the genotype is GA / AG, the sweet cherry fruit is a small fruit.

[0024] The advantages of adopting the above technical solution are as follows:

[0025] The SNP molecular marker provided by the present invention is significantly correlated with the fruit size of sweet cherry. According to the significant association between the genome sequence polymorphism of FUN_001609 and the traits related to the fruit size of sweet cherry, a SNP molecular marker related to the fruit size of sweet cherry is obtained. Using the SNP molecular marker provided by the present invention and its specific primers, breeders can quickly screen out sweet cherry varieties with target traits at the seedling stage, thereby improving the breeding of new sweet cherry varieties. Through further in-depth research on the SNP molecular marker, the genetic mechanism and regulatory network in the development process of sweet cherry fruit can be revealed, providing a new theoretical basis for the genetic transformation and molecular biology research of sweet cherry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Significance analysis between genotypes

[0027] Figure 2 : Electrophoresis of PCR amplification products (partial)

[0028] Figure 3 :Sequencing results of different genotypes of PCR amplification products (partial) DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0030] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products.

[0031] The present invention is achieved through the following technical solutions:

[0032] A gene FUN_001609 related to the fruit size of sweet cherry, the related gene is located on the chromosome 1 of sweet cherry. In the preliminary study of the present invention, a high-density genetic map was constructed using 100 hybrid offspring of 'Labins' and 'Late Red Pearl', and one QTL related to the fruit size of sweet cherry was found on chromosomes 2 and 6, respectively, and two QTLs related to the fruit size of sweet cherry were found on chromosome 1. The extreme fruit size of the hybrid offspring was used to measure the transcriptome, and the QTL and transcriptome were used to screen candidate differential genes. The expression of the candidate gene in the population and the phenotype correlation analysis determined that the gene FUN_001609 had a significant correlation with the fruit size of sweet cherry.

[0033] A SNP molecular marker related to the fruit size of sweet cherry, wherein the SNP site of the related gene is located at 16366288 of chromosome 1, and the polymorphism is G or A. The invention analyzes the SNP site variation of FUN_001609 in sweet cherry germplasm by genome resequencing, uses the phenotypic data of the fruit size of sweet cherry for many consecutive years and the genotype of the SNP site to perform correlation analysis, and then develops a molecular marker.

[0034] SNP molecular markers are amplified in sweet cherry germplasm resources, and a primer pair for amplifying the molecular markers is used, wherein the forward primer sequence is SEQ ID NO.1, and the reverse primer sequence is SEQ ID NO.2. When the genotype is AA, the sweet cherry fruit is judged to be larger, and when the genotype is GA, the sweet cherry fruit is judged to be smaller.

[0035] The biological materials involved in the following examples are all commercially available products.

[0036] Example 1: Mining of SNP molecular markers associated with sweet cherry fruit size

[0037] 1.1 Test materials

[0038] A total of 101 sweet cherry varieties were selected and planted in the Forestry and Fruit Research Institute of Beijing Academy of Agricultural and Forestry Sciences.

[0039] 1.2 Statistics of sweet cherry fruit size traits

[0040] After the sweet cherry fruits matured, 30 fruits of each variety were randomly picked to measure the average single fruit weight and the maximum single fruit weight, and 10 fruits were randomly picked to measure the average horizontal diameter, average vertical diameter and average thickness of the fruits. There was a significant correlation between the average horizontal diameter, average vertical diameter, average thickness, average single fruit weight and maximum single fruit weight of the five sweet cherry fruit size traits (see Figure 1 , Table 1).

[0041] Table 1: Correlation analysis of five fruit size traits of sweet cherry

[0042]

[0043] Note: **. The correlation is significant at the 0.01 level (two-tailed)

[0044] 1.3 Sweet cherry genomic DNA extraction and sequencing

[0045] Take the young leaves of each variety, fully cool them in liquid nitrogen, put them into a 2ml centrifuge tube with built-in steel balls, grind them into powder using a grinder, and extract DNA using a commercial DNA extraction kit. After extracting the FUN_001609 sequence and all polymorphic sites within 5kb upstream and downstream using genome resequencing data, the genotype of the SNP site was correlated with the five traits of sweet cherry fruit size.

[0046] Correlation analysis between SNP loci and sweet cherry fruit size

[0047] The correlation between the five traits of sweet cherry fruit size and the three genotypes of SNP loci was analyzed using the single-factor ANOVA test of SPSS software. When the variance homogeneity test value was greater than 0.05, the significance of the ANOVA test was used, and when the variance homogeneity test value was less than 0.05, the significance of the Welch test was used. As shown in Table 2, the correlation between the sweet cherry varieties with the AA genotype of the SNP locus and the fruit size was significantly greater than that of other genotypes, among which the fruit of the heterozygous genotype GA was the smallest.

[0048] Table 2: Correlation analysis between sweet cherry fruit size and genotype

[0049]

[0050]

[0051] The significantly associated SNP loci were verified by PCR amplification in sweet cherry germplasm resources.

[0052] Example 2: Validation and application of molecular markers

[0053] First, the sequence of FUN_001609 was obtained using Prunus avium Tieton Genome v2.0 on the GDR website as the reference genome, and PCR amplification primers were designed upstream and downstream of the above SNP sites. The specificity of the primers was further tested using the NCBI website. PCR amplification was able to amplify a 433bp product. The specific primer sequences are as follows:

[0054] Forward primer: 5'-CGCCATTCTTACTACGCTAT-3' (SEQ ID NO.1)

[0055] Reverse primer: 5'-TGAGGCAAACAATGAGGTAG-3' (SEQ ID NO. 2)

[0056] PCR amplification: PCR reaction system composition

[0057]

[0058] PCR reaction program: pre-denaturation at 94°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 10 s, extension at 72°C for 30 s, 34 cycles; final extension at 72°C for 5 min; storage at 4°C.

[0059] The amplified product was 433 bp and was sent to Bioengineering (Shanghai) Co., Ltd. for sequencing. The base peak diagram of the sequencing results showed that there were three types of sequencing results, namely, the genotypes at 275 bp, i.e., chromosome 1, 16366288, were GG, AA, and GA. (See Figure 2 , Figure 3 )

[0060] The detection results of the above genes correspond to the fruit size.

[0061] Among them, when the genotype is AA, the sweet cherry fruit is a large fruit, when the genotype is GG, the sweet cherry fruit is a medium fruit, and when the genotype is GA / AG, the sweet cherry fruit is a small fruit.

Claims

1. A specific primer for amplifying a SNP molecular marker associated with sweet cherry fruit size, wherein the nucleotide sequences of the forward primer and the reverse primer are as shown in SEQ ID NO.1 and SEQ ID NO.2: Forward primer: 5'-CGCCATTCTTACTACGCTAT-3', Reverse primer: 5′-TGAGGCAAACAATGAGGTAG-3′.

2. The SNP molecular marker associated with the fruit size of sweet cherry was amplified using SEQ ID NO.1 and SEQ ID NO.2 with the genomic DNA of sweet cherry as a template to obtain a 433 bp amplified product fragment, i.e., the SNP molecular marker associated with the fruit size of sweet cherry; wherein the base type at the 275 bp position was AA, corresponding to a sweet cherry variety with larger fruit; the base type at the 275 bp position was GA or AG, corresponding to a sweet cherry variety with smaller fruit; the base type at the 275 bp position was GG, corresponding to a sweet cherry variety with medium fruit size. The forward primer and reverse primer shown in SEQ ID NO.1 and SEQ ID NO.2 are: Forward primer: 5'-CGCCATTCTTACTACGCTAT-3', Reverse primer: 5′-TGAGGCAAACAATGAGGTAG-3′.

3. A kit for detecting SNP molecular markers associated with sweet cherry fruit size, comprising the specific primers according to claim 1, wherein: Forward primer: 5'-CGCCATTCTTACTACGCTAT-3', Reverse primer: 5′-TGAGGCAAACAATGAGGTAG-3′.

4. Use of the SNP molecular marker according to claim 2 in detecting or identifying sweet cherry fruit size or genetic breeding.

5. Use of the kit according to claim 3 in detecting or identifying the fruit size of sweet cherry or in genetic breeding.

6. The application according to claim 4 or 5, comprising: (1) extracting genomic DNA of the sweet cherry sample to be tested; (2) Using the genomic DNA of the sweet cherry sample to be tested as a template, a PCR amplification reaction was performed using specific primers to obtain an amplified product fragment of 433 bp; (3) Detect the base type of the PCR amplification product fragment to determine the fruit size of the sample. When the genotype of the SNP site at the 275bp position is AA, the fruit size of the sweet cherry variety is large. When the genotype is GA or GA, the sweet cherry fruit is the smallest. The genotype is GG, and the corresponding sweet cherry variety has a medium fruit size. The specific primers are: Forward primer: 5'-cgccattcttactacgctat-3', Reverse primer: 5'-TGAGGCAAAACAATGAGGTAG-3'.

7. The use according to claim 6, wherein the PCR reaction conditions are: pre-denaturation at 95°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 10 s, extension at 72°C for 30 s, 34 cycles; final extension at 72°C for 5 min; and storage at 4°C.

8. The use according to claim 6, wherein the genomic DNA of the sweet cherry sample to be tested is derived from sweet cherry seedling leaves.

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