A SNP molecular marker related to fruit size of sweet cherry and application thereof

Abstract

The present application relates to a kind of SNP molecular marker related to sweet cherry fruit size and application, belong to sweet cherry genetic breeding technical field.The molecular marker of the present application is from the exon of FUN_001609, located at Prunus avium Tieton Genome v2.0 16366288 of the first chromosome, the nucleotide of this site is G or A.The SNP site is significantly related to sweet cherry fruit size, and the genotype of sweet cherry variety fruit size is AA>GG>GA in turn.According to the SNP site, the primer pair for the to-be-tested sweet cherry is designed to carry out PCR amplification, and the PCR product is sequenced to test.The SNP molecular marker of the present application can be used as the auxiliary selection marker of fruit size trait in the process of sweet cherry breeding, improve the screening efficiency of large fruit sweet cherry variety, be conducive to shortening breeding period, greatly reduce the breeding time and workload of large fruit variety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular marker preparation for fruit tree genetic breeding, and particularly relates to a SNP molecular marker related to fruit size of sweet cherry and application thereof. BACKGROUND

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

[0003] Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by single nucleotide variation at the genome 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 applied to genetic diversity and kinship analysis, assisted breeding and variety identification. In sweet cherry, SNP molecular markers are applied to screening of 19 main sweet cherry varieties and 5 sweet cherry rootstocks, but there is no report on SNP molecular markers applied to distinguishing fruit size traits. The genetic regulation mechanism of fruit size traits of sweet cherry is not clear, and traditional breeding methods are low in efficiency and long in cycle. Development of new molecular markers and breeding techniques is of great significance for breeding large-fruited sweet cherry varieties. SUMMARY

[0004] The application aims to provide a SNP molecular marker significantly related to fruit size of sweet cherry, so as to improve breeding efficiency and breed new varieties of large-fruited sweet cherry.

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

[0006] The application is implemented by the following technical solutions.

[0007] A specific primer for amplifying a SNP molecular marker related to fruit size of sweet cherry, wherein the nucleotide sequences of the forward primer and the reverse primer are shown in 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 is obtained by amplifying the genomic DNA of sweet cherry as a template using SEQ ID NO. 1 and SEQ ID NO. 2, and the fragment of the amplification product is 433 bp, that is, the SNP molecular marker related to the fruit size of sweet cherry; the base type at the 275th bp is AA, which corresponds to a larger fruit size of the sweet cherry variety; the base type at the 275th bp is GA or AG, which corresponds to a smaller fruit size of the sweet cherry variety; and the base type at the 275th bp is GG, which corresponds to a medium fruit size of the sweet cherry variety.

[0011] A kit for detecting the SNP molecular marker related to the fruit size of sweet cherry, comprising the specific primers described above.

[0012] The SNP molecular marker described above or the kit described above is applied in genetic breeding.

[0013] The SNP molecular marker described above or the kit described above is applied in detecting or identifying the fruit size of sweet cherry.

[0014] The application comprises:

[0015] (1) extracting the 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 PCR amplification reaction by using the specific primers described above, and obtaining the fragment of the amplification product;

[0017] (3) detecting the base type of the fragment of the PCR amplification product, and judging the fruit size of the sample; when the genotype of the SNP site at the 275th bp is AA, the fruit size of the sweet cherry variety is larger; when the genotype is GA or GA, the fruit size of the sweet cherry is the smallest; and when the genotype is GG, the fruit size of the sweet cherry variety is medium, and 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℃ for 3 min; denaturation at 98℃ for 10 s, annealing at 55℃ for 10 s, extension at 72℃ for 30 s, 34 cycles; final extension at 72℃ for 5 min; and preservation at 4℃.

[0021] The genomic DNA of the to-be-tested sweet cherry sample is derived from a sweet cherry seedling leaf.

[0022] The application provides a SNP molecular marker related to the size of a sweet cherry fruit, which is from a third exon of a 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 genotype of the sweet cherry fruit size molecular marker includes GG, AA and GA / AG; when the genotype is AA, the sweet cherry fruit is large, when the genotype is GG, the sweet cherry fruit is medium, and when the genotype is GA / AG, the sweet cherry fruit is small.

[0024] The above technical solution has the following advantages:

[0025] The SNP molecular marker provided by the application is significantly related to the size of a sweet cherry fruit, and the genomic sequence polymorphism of FUN_001609 is significantly associated with the size of a sweet cherry fruit, so that the SNP molecular marker related to the size of a sweet cherry fruit is obtained. Using the SNP molecular marker and the specific primer thereof provided by the application, a breeder can quickly screen a sweet cherry variety with a target trait at a seedling stage, thereby improving the breeding of a new sweet cherry variety. Through further in-depth research on the SNP molecular marker, the genetic mechanism and the regulation network in the development process of a sweet cherry fruit can be revealed, thereby providing a new theoretical basis for the genetic modification and molecular biology research of a sweet cherry. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 : Significant analysis between genotypes

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

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

[0029] In order for those skilled in the art to better understand the application scheme, the technical solutions in the embodiments of the application will be described clearly and completely in combination with the drawings in the embodiments of the application. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor should belong to the protection scope of the application.

[0030] The specific experimental steps or conditions not indicated in the examples can be carried out according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not indicated by the manufacturer, and are conventional reagent products.

[0031] The present application is obtained by the following technical solutions:

[0032] A gene FUN_001609 related to the fruit size of sweet cherry, wherein the related gene is located on the first chromosome of sweet cherry. In the previous study of the present application, a high-density genetic map was constructed using 100 hybrid offspring of 'Rabbith' and 'Late Red Pearl', and one QTL related to the fruit size of sweet cherry was found on the second and sixth chromosomes, respectively, and two QTLs related to the fruit size of sweet cherry were found on the first chromosome. The transcriptome was measured by mixing pools of extreme fruit size of hybrid offspring, and candidate differential genes were screened by QTL and transcriptome. The expression of candidate genes in the population and the correlation analysis of phenotypes determined that the gene FUN_001609 had 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 on the first chromosome, and the polymorphism is G or A. The present application analyzes the SNP site variation of FUN_001609 in sweet cherry germplasm by genome resequencing, and develops a molecular marker by correlation analysis of the phenotypic data of sweet cherry fruit size in consecutive years and the genotype of the SNP site.

[0034] The SNP molecular marker is amplified in sweet cherry germplasm resources, and the primer pair for amplifying the molecular marker is as follows: the sequence of the forward primer is SEQ ID NO. 1, and the sequence of the reverse primer is SEQ ID NO. 2. When the genotype is AA, it is judged that the fruit of sweet cherry is larger, and when the genotype is GA, it is judged that the fruit of sweet cherry is smaller.

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

[0036] Example 1: Mining of SNP molecular marker related to fruit size of sweet cherry

[0037] 1.1 Test materials

[0038] 101 sweet cherry varieties were selected and planted in the Beijing Academy of Forestry and Pomology Institute.

[0039] 1.2 Statistics of fruit size traits of sweet cherry

[0040] After the sweet cherry fruits were mature, 30 fruits were randomly picked for each variety to measure the average single fruit weight and the maximum single fruit weight. 10 fruits were randomly picked to measure the average transverse diameter, average longitudinal diameter and average thickness. The five traits of the sweet cherry fruit size, average transverse diameter, average longitudinal diameter, average thickness, average single fruit weight and maximum single fruit weight, had significant correlation (see Table 1). Figure 1

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

[0042]

[0043] Note: **. Significant correlation at 0.01 level (two-tailed)

[0044] 1.3 Extraction of sweet cherry genomic DNA and sequencing

[0045] The tender leaves of each variety were cooled in liquid nitrogen, put into 2ml centrifuge tubes with steel balls, and ground into powder using a grinder. The DNA was extracted using a commercial DNA extraction kit. The sequence of FUN_001609 and all polymorphic sites within the range of 5kb upstream and downstream were extracted using genomic resequencing data, and the genotypes of the SNP sites were correlated with the five traits of sweet cherry fruit size.

[0046] Correlation analysis of SNP sites and sweet cherry fruit size

[0047] The correlation of the five traits of sweet cherry fruit size and the three genotypes of SNP sites was analyzed using one-way ANOVA test of SPSS software. When the variance homogeneity test value was greater than 0.05, the significance was tested by ANOVA, and when the variance homogeneity test value was less than 0.05, the significance was tested by Welch test. As shown in Table 2, the correlation of the sweet cherry varieties with SNP site genotype AA and fruit size was significantly greater than that of other genotypes, and the fruit of the heterozygous genotype GA was the smallest.

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

[0049]

[0050]

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

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

[0053] ​First, the sequence of FUN_001609 was obtained by taking Prunus avium Tieton Genome v2.0 on the GDR website as the reference genome, and PCR amplification primers were designed on the upstream of the above SNP site, and the specificity of the primers was further tested by using the NCBI website. PCR amplification can amplify a 433bp product, and 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: 94℃ pre-denaturation for 3min; 98℃ denaturation for 10s, 55℃ annealing for 10s, 72℃ extension for 30s, 34 cycles; finally 72℃ extension for 5min; 4℃ preservation.

[0059] The amplification product is 433bp, and is sent to Bioengineering (Shanghai) Co., Ltd. for sequencing. The base peak chart of the sequencing result is observed, and the sequencing result has three types, that is, the genotype is GG, AA and GA at the 275bp site, that is, the 16366288 site of chromosome 1. (See Figure 2 , Figure 3 )

[0060] The detection result of the above-mentioned gene corresponds to the fruit size.

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

Claims

1. SNP molecular markers related to sweet cherry fruit size: Using sweet cherry genomic DNA as a template, amplification was performed using SEQ ID NO.1 and SEQ ID NO.2, yielding a 433bp amplification product fragment, which represents the SNP molecular markers related to sweet cherry fruit size. Specifically, the base type at 275bp is AA, corresponding to a larger sweet cherry variety; GA or AG at 275bp, corresponding to a smaller sweet cherry variety; and GG at 275bp, corresponding to a medium-sized sweet cherry variety. The forward and reverse primers shown in SEQ ID NO.1 and SEQ ID NO.2: Forward primer: 5'-CGCCATTCTTACTACGCTAT-3', Reverse primer: 5'-TGAGGCAAACAATGAGGTAG-3'.

2. The application of the SNP molecular marker as described in claim 1 in detecting or identifying the size of sweet cherry fruits or in the genetic breeding selection of sweet cherry fruit size.

3. The application of a kit in detecting or identifying the size of sweet cherry fruits or in the genetic breeding selection of sweet cherry fruit size, said kit comprising specific primers, wherein: Forward primer: 5'-CGCCATTCTTACTACGCTAT-3', Reverse primer: 5'-TGAGGCAAACAATGAGGTAG-3'.

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

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

6. The application according to claim 4, wherein the genomic DNA of the sweet cherry sample to be tested is derived from the leaves of a sweet cherry seedling.

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