Development and application of a SNP marker for identifying ethylene-dependent / independent types of sand pear

Abstract

The present invention discloses a SNP molecular marker for identifying the ethylene-dependent / independent type of sand pear fruit and its application. The SNP site is located at the nucleotide 20023924 of chromosome 15 of the sand pear genome, and the nucleotide is C or T. The nucleotide polymorphism marker site obtained by the present invention can be used to identify the ethylene-dependent / independent type of sand pear fruit. The SNP marker of the present invention is a SNP molecular marker that is closely linked to the ethylene-dependent / independent type of sand pear fruit and is developed based on whole genome association analysis. The present invention does not need to wait until the pear seedlings have passed the juvenile stage and entered the fruiting stage before harvesting mature fruits for measurement. The ethylene-dependent / independent type of sand pear fruit can be accurately and quickly identified in the seedling stage, which can improve breeding efficiency, shorten identification time, and is suitable for sand pear molecular design breeding.

Description

Technical Field

[0001] The invention belongs to the field of sand pear molecular breeding and relates to the development and application of a SNP marker for identifying the ethylene-dependent / independent type of sand pear. Background Art

[0002] Pear (Pyrus L.) is a plant of the tribe Maleae, subfamily Amygdaloideae, family Rosaceae. Based on their evolutionary origins, the genus Pyrus can be divided into Asian and European pear populations. The sand pear (P. pyrifolia Nakai) is the primary cultivated species of the Asian pear.

[0003] Ethylene is a key hormone involved in fruit ripening, softening, and aging. The level of ethylene released after fruit ripens and is harvested determines its storage and shelf life. Fruits can be divided into ethylene-dependent and ethylene-independent types based on whether there is a peak in ethylene release after ripening. Sand pears are generally considered ethylene-dependent and not storage-resistant. However, a growing number of studies have revealed significant differences in the ability of different sand pear varieties to produce ethylene after ripening and harvest. Most varieties, especially early- and mid-ripening varieties, exhibit significant ethylene and respiration peaks after fruit ripening, resulting in a short shelf life and poor storage tolerance, indicating ethylene dependence. In contrast, some sand pear varieties produce very low levels of ethylene during ripening, indicating ethylene independence and good storage. Measuring changes in ethylene release after ripening in different pear varieties can clarify their ethylene dependence or independence, but these experiments are complex and time-consuming, and the results are easily affected by factors such as fruit maturity and storage conditions. The development of molecular markers that can be used for early identification of the ethylene-dependent / independent types of sand pear fruits has important practical significance for accelerating the discovery and breeding of ethylene-independent and storage-resistant sand pear varieties. Summary of the Invention

[0004] The present invention aims to provide a SNP marker development and application for identifying the ethylene-dependent / independent type of sand pear fruit.

[0005] In order to achieve the above object, the present invention adopts the following technical measures:

[0006] A SNP marker for identifying ethylene dependence and independence in sand pear fruit is located at nucleotide 20023924 on chromosome 15 of the pear genome. This nucleotide is either C or T. A T / T or C / T genotype indicates ethylene dependence, while a C / C genotype indicates ethylene independence. Ethylene dependence is dominant over ethylene independence.

[0007] A primer pair for detecting the above-mentioned SNP marker,

[0008] The forward primer is SEQ ID NO. 1, 5'-ACTGAAACCGCTCTGGAAGA-3', and the reverse primer is SEQ ID NO. 2, 5'-ATCGCTAATGAGGTGGATGC -3'.

[0009] A method for identifying the ethylene-dependent / independent type of sand pear fruit comprises the following steps:

[0010] Step 1: extracting genomic DNA from the pear to be tested;

[0011] Step 2: Using the pear genomic DNA to be tested as a template, PCR amplification is performed using the above primer pair to obtain a PCR amplification product;

[0012] Step 3: Direct sequencing and analysis of the PCR amplification products. The ethylene-dependent / independent type of the fruit can be determined based on the SNP genotype. A T / T or C / T genotype indicates ethylene-dependence, while a C / C genotype indicates ethylene-independence.

[0013] Furthermore, the total volume of the PCR amplification system in step 2 was 20 μl, including 2 μl of 10 ng genomic template DNA, 10 μl of 2×Es Taq MasterMix, 2 μl each of 10 mM forward and reverse primers, and 4 μl of ddH2O.

[0014] Furthermore, the PCR amplification program in step 2 was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 150 s, 35 cycles; and finally extension at 72°C for 10 min.

[0015] The beneficial effects of the present invention are:

[0016] 1. The present invention utilizes genome resequencing and genome-wide association analysis (GWAS) to enrich for gene intervals related to the ethylene-dependent / independent types of sand pear fruit, and develops SNP molecular markers tightly linked to the target traits. These markers can be effectively used in sand pear molecular marker-assisted selection breeding, thereby improving the accuracy of selection for the ethylene-dependent / independent types of sand pear fruit and accelerating the molecular breeding process for storability-resistant sand pear.

[0017] 2. The present invention can accurately identify the ethylene-dependent / independent type of sand pear fruit based on the single nucleotide polymorphism difference at position 20023924 of chromosome 15 of the sand pear genome.

[0018] 3. The SNP molecular markers of the present invention were used to identify 20 registered sand pear varieties. The results showed that the accuracy of the SNP molecular markers in identifying the ethylene-dependent / independent types of sand pear fruits reached 100%. This shows that using the SNP molecular markers of the present invention to identify the ethylene-dependent / independent types of sand pear fruits has the advantages of being simple, rapid, and highly accurate, and can be used on a large scale in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Manhattan plot for GWAS analysis of ethylene-dependent / independent types of sand pear fruit. DETAILED DESCRIPTION

[0020] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional in the art.

[0021] Example 1: Development of a SNP marker for identifying the ethylene-dependent / independent type of sand pear fruit.

[0022] 1. Phenotypic identification of ethylene-dependent / independent types of sand pear fruits of different varieties.

[0023] By sampling 100 sand pear varieties during the fruit ripening period for two consecutive years, the changes in their ethylene release were measured to clarify their ethylene-dependent / independent types.

[0024] 2. SNP sites were obtained by genome-wide association analysis.

[0025] Genome resequencing of 450 sand pear cultivars was performed using next-generation sequencing technology and compared with the '20th Century' sand pear reference genome. 32,453,152 single-nucleotide polymorphisms (SNPs) and 4,079,097 indels were identified. A genome-wide association analysis of fruit ethylene dependence and independence using a mixed linear model (MLM) revealed that the ethylene dependence and independence patterns were significantly enriched in the gene interval 19,875,007 to 2,069,394 on chromosome 15 (see ). Figure 1 Association analysis of SNPs in this interval with different ethylene-dependent / independent sand pear varieties revealed that SNP 20023924 was completely consistent with the ethylene-dependent / independent phenotype. Specifically, sand pear fruits with the C / C genotype at this SNP were ethylene-independent, while those with the T / T or T / C genotypes were ethylene-dependent. Brown is dominant over green. This confirms that this SNP is tightly linked to the ethylene-dependent / independent phenotype of the fruit.

[0026] Example 2: Using the above SNP markers to identify the ethylene-dependent / independent type of sand pear fruit.

[0027] 1. DNA extraction.

[0028] The genomic DNA of sand pear leaves was extracted by conventional CTAB method. The OD values ​​of genomic DNA samples at 260 nm and 280 nm were measured by ultraviolet spectrophotometer. The DNA content and OD 260 / 280 ratio were calculated. Genomic DNA samples with OD 260 / 280 values ​​between 1.8 and 2.0 were selected and diluted to 100 ng / μL for later use.

[0029] 2. SNP marker primer design and PCR amplification.

[0030] Based on the 150 bp upstream and downstream sequences of the SNP site obtained in Example 1, primers were designed using Primer software to develop a SNP marker. The forward primer was 5'-ACTGAAACCGCTCTGGAAGA-3' (SEQ ID NO. 1), and the downstream reverse primer was 5'-ATCGCTAATGAGGTGGATGC -3' (SEQ ID NO. 2). The amplified product was 120 bp in size. The total volume of the PCR amplification system was 20 μL, including 2 μL of 10 ng genomic template DNA, 10 μL of 2× Es Taq MasterMix, 2 μL each of 10 mM forward and reverse primers, and 4 μL of ddH2O. The PCR amplification program was an initial denaturation at 94°C for 5 min, followed by denaturation at 94°C for 10 s, 35 cycles of annealing at 60°C for 30 s, and extension at 72°C for 150 s, followed by a final extension at 72°C for 10 min. The PCR products were detected by 1% agarose gel electrophoresis. If a bright single electrophoresis band was found at about 120 bp, it indicated that the amplification was successful. The PCR products were directly sent for sequencing.

[0031] 3. SNP site detection and identification of ethylene-dependent / independent types of sand pear fruits.

[0032] Sequencing results were compared with the reference genome for sequence analysis, and the ethylene-dependent / independent type of the sand pear fruit was determined based on the bases at the SNP site. Sand pear fruits with a SNP site genotype of C / C were ethylene-independent, while sand pear fruits with a SNP site genotype of T / T or T / C were ethylene-dependent.

[0033] Example 3: Identification of ethylene-dependent / independent types of 20 sand pear fruits using SNP molecular markers.

[0034] Twenty registered sand pear varieties from non-major crop varieties preserved at the National Sand Pear Germplasm Resource Center (Wuhan) were used as materials. Ethylene release was measured after fruit ripening and numbered to determine ethylene dependence / independence. The ethylene dependence / independence characteristics of the fruit were then identified using the SNP molecular markers of the present invention. The specific identification method was similar to that described in Example 2. The identification results showed that the phenotypes and genotypes of the 20 sand pear varieties were completely consistent, with an accuracy rate of 100%, demonstrating that the SNP molecular markers of the present invention can accurately identify the ethylene dependence / independence of sand pear fruit. The specific varieties, phenotypes, and genotypes are shown in Table 1 below.

[0035] Table 1 Ethylene-dependent / independent types and genotypes of fruits of 20 sand pear varieties

[0036] Variety name origin Ethylene-dependent / independent SNP genotype

[0037] Fresh fragrance Zhejiang Academy of Agricultural Sciences ethylene-dependent C / T

[0038] E-Pear No. 2 Hubei Academy of Agricultural Sciences Ethylene-independent C / C

[0039] Jinxin Hubei Academy of Agricultural Sciences ethylene-dependent T / T

[0040] Jinjing Hubei Academy of Agricultural Sciences Ethylene-dependent C / T

[0041] Jinfeng Pear Hubei Academy of Agricultural Sciences Ethylene-independent C / C

[0042] Jinxiu Hubei Academy of Agricultural Sciences Ethylene-dependent C / T

[0043] Baojing Yangdong Pear Hunan Academy of Agricultural Sciences Ethylene-independent C / C

[0044] Cuiyu Zhejiang Academy of Agricultural Sciences Ethylene-independent C / C

[0045] Jin Yuli Hubei Academy of Agricultural Sciences Ethylene-dependent C / T

[0046] Jinmi Hubei Academy of Agricultural Sciences ethylene-dependent C / T

[0047] Longhua Hubei Zhijiang City Ethylene non-dependent C / C

[0048] Jinbi Hubei Academy of Agricultural Sciences Ethylene-dependent C / T

[0049] Jintong Hubei Academy of Agricultural Sciences Ethylene-dependent C / T

[0050] Jincuisu Hubei Academy of Agricultural Sciences Ethylene-dependent C / T

[0051] Jin Zaocui Hubei Academy of Agricultural Sciences Ethylene-independent C / C

[0052] Su Cui No. 1 Jiangsu Academy of Agricultural Sciences ethylene-independent C / C

[0053] Early Red Jade Ethylene-dependent C / T from Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences

[0054] Zhongli No. 4 Ethylene-dependent C / T from Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences

[0055] Su Cui No. 2 Jiangsu Academy of Agricultural Sciences ethylene-independent C / C

[0056] Xinyu Zhejiang Academy of Agricultural Sciences ethylene-independent C / C

[0057] In summary, the SNP molecular markers provided by the present invention can accurately and rapidly identify the ethylene-dependent / independent type of sand pear fruit, thereby improving breeding efficiency and shortening identification time, making them suitable for molecular-assisted breeding of sand pear. The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A method for identifying the ethylene-dependent / independent type of sand pear fruit, characterized in that: The following steps are involved: Step 1: extracting genomic DNA from the pear to be tested; Step 2: Using the pear genomic DNA to be tested as a template, PCR amplification is performed using a primer pair to obtain a PCR amplification product; Step 3: Direct sequencing and analysis of the PCR amplification product; when the SNP site genotype is T / T or C / T, it is ethylene-dependent; When the SNP site genotype is C / C, it is ethylene-independent; The forward primer of the primer pair is SEQ ID NO.1, 5'-ACTGAAACCGCTCTGGAAGA-3', and the reverse primer of the primer pair is SEQ ID NO.2, 5'-ATCGCTAATGAGGTGGATGC -3'; The SNP site is located at position 20023924 of chromosome 15 of the pear genome, the base is C or T, and the pear genome is the '20th Century' sand pear reference genome.

2. The method for identifying the ethylene-dependent / independent type of sand pear fruit according to claim 1, wherein: The total volume of the PCR amplification system in step 2 is 20 μl, including 2 μl of 10 ng genomic template DNA, 10 μl of 2×EsTaq MasterMix, 2 μl each of 10 mM forward and reverse primers, and 4 μl of ddH2O.

3. The method for identifying the ethylene-dependent / independent type of sand pear fruit according to claim 1, wherein: The PCR amplification program in step 2 was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 150 s, 35 cycles; and final extension at 72°C for 10 min.

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