Development and application of SNP (Single Nucleotide Polymorphism) marker for identifying ethylene-dependent / non-dependent type of Chinese pears

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for identifying ethylene-dependent / non-dependent types of Chinese pear fruits and application of the SNP molecular marker. The SNP site is located at the 20023924th nucleotide of the No.15 chromosome of a Chinese pear genome, and the nucleotide is C or T. The nucleotide polymorphism marker site obtained by the invention can be used for identifying the ethylene dependence / non-dependence type of the Chinese pear fruit. The SNP marker disclosed by the invention is an SNP molecular marker which is developed on the basis of whole genome association analysis and is closely linked with the ethylene-dependent / non-dependent type of the Chinese pear fruit. The method does not need to harvest mature fruits for determination after pear seedlings pass through the juvenile phase and fruit, can accurately and rapidly identify the ethylene dependence / non-dependence type of the Chinese pear fruits in the seedling stage, can improve the breeding efficiency and shorten the identification time, and is suitable for molecular design breeding of Chinese pears.

Description

Technical Field

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

[0002] Pear (Pyrus L.) is a plant of the Maleae family, subfamily Amygdaloideae, of the Rosaceae family. It originated in my country and is cultivated and distributed in all provinces, municipalities and autonomous regions except Hainan Province, Hong Kong, Macao and Taiwan. It is the fruit tree with the widest distribution in my country. my country is also the world's largest producer of pears, and its pear production accounts for more than 2 / 3 of the world's pear production. According to the evolutionary path of the origin of the genus Pyrus, it can be divided into Asian pear and European pear populations. Sand pear (P.pyrifoliaNakai) is the main cultivated species of Asian pear, accounting for more than two-thirds of my country's pear cultivation area. Sand pear originated in the Yangtze River Basin and the areas to the south of it, and is the main cultivated species of pear in my country, Japan and South Korea.

[0003] Ethylene is an important hormone involved in fruit ripening, softening and aging. The level of ethylene release after the fruit is ripe and harvested determines the storage and shelf life of the fruit. According to whether there is a transition peak in the ethylene release after the fruit matures, it can be divided into ethylene-dependent fruits and ethylene-independent fruits. It is generally believed that sand pear is an ethylene-dependent fruit and is not resistant to storage. However, more and more studies have found that there are extremely significant differences in the ability of different varieties of sand pear to produce ethylene after ripening and harvesting. Most varieties, especially early and mid-ripening varieties, will have obvious ethylene and respiration peaks after ripening and harvesting, with short shelf life and poor storage resistance, showing ethylene-dependent characteristics. However, the ethylene produced by some sand pear varieties during the ripening process has always remained at an extremely low level, showing ethylene-independent characteristics and good storage. The ethylene dependence / independence of different varieties of pear fruits can be clarified by measuring the changes in ethylene release after ripening and harvesting, but the relevant experimental operations are relatively complex and the cycle is long, and the identification results are easily affected by factors such as fruit maturity and storage conditions. The development of molecular markers that can be used to identify the ethylene-dependent / independent types of sand pear fruits at an early stage is of great 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 the ethylene-dependent / independent type of sand pear fruit is located at nucleotide 20023924 of chromosome 15 of the pear genome, and the nucleotide is C or T. When the genotype at this position is T / T or C / T, it is ethylene-dependent, and when the genotype at this position is C / C, it is ethylene-independent. The ethylene-dependent type is dominant over the ethylene-independent type.

[0007] A primer pair for detecting the above 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 the genomic DNA of the pear to be tested;

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

[0012] Step 3: Directly sequence and analyze the PCR amplification product. The ethylene-dependent / independent type of the fruit can be determined based on the SNP site genotype. The SNP site genotype of T / T or C / T indicates ethylene-dependent type, while the genotype of C / C indicates ethylene-independent type.

[0013] Furthermore, 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×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 is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 10 s, annealing temperature 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 type of sand pear fruit, and develops SNP molecular markers that are closely linked to the target trait, which can be effectively used in sand pear molecular marker-assisted selection breeding, improve the accuracy of sand pear fruit ethylene-dependent / independent type selection, and accelerate the molecular breeding process of storability 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 varieties of sand pear. 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 the use of 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, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment 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 fruits of different varieties of sand pear.

[0023] By sampling 100 sand pear varieties at the fruit ripening stage for two consecutive years, the changes in their ethylene release were measured to clarify their ethylene dependence / independence types.

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

[0025] The genome of 450 sand pear varieties was resequenced using the second generation sequencing technology and compared with the '20th Century' sand pear reference genome, identifying 32453152 SNPs and 4079097 InDels. A genome-wide association analysis of the fruit ethylene dependence / independence type was performed using a mixed linear model (MLM), and it was found that the fruit ethylene dependence / independence type was significantly enriched in the gene interval of chromosome 15, 19875007-20069394 (see Figure 1). The association analysis of the SNPs in this interval with different ethylene-dependent / independent types of sand pear varieties found that the SNP at position 20023924 was completely consistent with the ethylene-dependent / independent type phenotype, that is, the sand pear fruit with the genotype of C / C at the SNP site was ethylene-independent, and the sand pear fruit with the genotype of T / T or T / C at the SNP site was ethylene-dependent, and brown was dominant over green. It was determined that the SNP site was closely linked to the ethylene-dependent / independent type 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 conventional CTAB method was used to extract genomic DNA from sand pear leaves. The OD values ​​of the genomic DNA samples at 260nm and 280nm were determined by ultraviolet spectrophotometer. The DNA content and the OD 260 / 280 ratio were calculated. The genomic DNA samples with OD 260 / 280 values ​​between 1.8 and 2.0 were selected and diluted to 100ng / μL for later use.

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

[0030] According to the 150bp sequence upstream and downstream of the SNP site obtained in Example 1, primers were designed using Primer software and developed as SNP markers. Forward primer 5'-ACTGAAACCGCTCTGGAAGA-3' (SEQ ID NO.1) and downstream reverse primer 5'-ATCGCTAATGAGGTGGATGC-3' (SEQ ID NO.2). The amplified product size is 120bp. The total volume of the PCR amplification system is 20μL, including 10ng genomic template DNA 2μL, 2×Es Taq MasterMix 10μL, 10mM forward and reverse primers 2μL each and ddH2O 4μL. The PCR amplification program is 94℃ pre-denaturation for 5min; 94℃ denaturation for 10s, annealing temperature 60℃ for 30s, 72℃ extension for 150s, 35 cycles; finally 72℃ extension for 10min. The PCR product was tested by 1% agarose gel electrophoresis. If there was a bright single electrophoresis band at about 120 bp, it indicated that the amplification was successful. The PCR product was directly sent for sequencing.

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

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

[0033] Example 3: Using SNP molecular markers to identify the ethylene-dependent / independent types of 20 sand pear fruits.

[0034] 20 non-major crop varieties registered in the National Pear Germplasm Resource Garden (Wuhan) were used as materials. The ethylene release amount after fruit maturity was determined to clarify its ethylene dependence / independence type, and then the SNP molecular marker of the present invention was used to identify the ethylene dependence / independence characteristics of its fruit. The specific identification method refers to the method in Example 2 above. The identification results show that the phenotypes of the above 20 Pear varieties are completely consistent with the genotype results, with an accuracy rate of 100%, indicating that the SNP molecular marker of the present invention can accurately identify the ethylene dependence / independence type of Pear fruit. Specific varieties, phenotypes and genotypes are shown in Table 1 below.

[0035] Table 1 Types of fruit ethylene dependence / independence and their genotypes of 20 Pear varieties

[0036] Variety name source Ethylene dependent / independent SNP genotype Fragrance Zhejiang Academy of Agricultural Sciences Ethylene dependent C / T E Pear No.2 Hubei Academy of Agricultural Sciences Ethylene independent C / C Jin Xin Hubei Academy of Agricultural Sciences Ethylene dependent T / T Jin Jing Hubei Academy of Agricultural Sciences Ethylene dependent C / T Jinfengli Hubei Academy of Agricultural Sciences Ethylene independent C / C Jin Xiu Hubei Academy of Agricultural Sciences Ethylene dependent C / T Baojingyang Winter Pear Hunan Academy of Agricultural Sciences Ethylene independent C / C Jade Zhejiang Academy of Agricultural Sciences Ethylene independent C / C Kim Yu-ri Hubei Academy of Agricultural Sciences Ethylene dependent C / T Golden Honey Hubei Academy of Agricultural Sciences Ethylene dependent C / T Dragon Flower Zhijiang City, Hubei Province Ethylene independent C / C Jinbi Hubei Academy of Agricultural Sciences Ethylene dependent C / T Jin Tong Hubei Academy of Agricultural Sciences Ethylene dependent C / T Golden Cui Su Hubei Academy of Agricultural Sciences Ethylene dependent C / T Gold Zaocu Hubei Academy of Agricultural Sciences Ethylene independent C / C Sucui No.1 Jiangsu Academy of Agricultural Sciences Ethylene independent C / C Early Ruby Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences Ethylene dependent C / T Zhongli No.4 Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences Ethylene dependent C / T Sucui No.2 Jiangsu Academy of Agricultural Sciences Ethylene independent C / C New Jade Zhejiang Academy of Agricultural Sciences Ethylene independent C / C

[0037] In summary, the SNP molecular markers provided by the present invention can accurately and quickly identify the ethylene-dependent / independent type of sand pear fruit, improve breeding efficiency, shorten identification time, and are suitable for sand pear molecular-assisted breeding. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A SNP marker for identifying ethylene-dependent / independent types of Pyrus sutchuenensis, characterized in that: The SNP marker is located at nucleotide 20023924 of chromosome 15 of the pear genome, and the nucleotide is C or T.

2. A primer pair for detecting the SNP marker of claim 1, characterized in that: 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'.

3. A method for identifying the ethylene-dependent / independent type of sand pear fruit, characterized in that: The following steps are involved: Step 1, extracting the genomic DNA of the pear to be tested; Step 2: using the pear genomic DNA to be detected as a template, performing PCR amplification using the primer pair described in claim 2 to obtain a PCR amplification product; Step 3: directly sequence and analyze the PCR amplification products; when the genotype of the SNP site described in claim 1 is T / T or C / T, it is ethylene-dependent; when the genotype of the SNP site described in claim 1 is C / C, it is non-ethylene-dependent.

4. The method for identifying the ethylene-dependent / independent type of Pyrus sutchuenensis fruit according to claim 3, characterized in that: 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×Es TaqMasterMix, 2 μl of 10 mM forward and reverse primers, and 4 μl of ddH2O.

5. The method for identifying the ethylene-dependent / independent type of sand pear fruit according to claim 3, characterized in that: The PCR amplification program in step 2 is: 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.

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