Development and application of SNP (Single Nucleotide Polymorphism) marker related to pericarp ground color of Chinese pear

Abstract

The invention relates to the technical field of molecular breeding of Chinese pears, in particular to an SNP (Single Nucleotide Polymorphism) molecular marker related to the peel bottom color of Chinese pears and application of the SNP molecular marker. The SNP site is located at the 4160839th nucleotide of the No.8 chromosome of a Chinese pear genome, and the nucleotide is G or A. The nucleotide polymorphism marker site obtained by the invention can be used for identifying the ground color of the peel of the Chinese pears. The SNP molecular 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 ground color of the pericarp of the Chinese pear. When 74 parts of Chinese pear germplasm resources are subjected to peel ground color identification by using the SNP molecular marker, the result shows that the identification accuracy of the SNP molecular marker on Chinese pears with brown and green peel ground colors is 100%. The method does not need to identify the fruit mature period of the Chinese pears, can accurately and quickly identify the peel bottom color of the Chinese pears in the seedling stage, can improve the breeding efficiency and shorten the identification time, and is suitable for molecular design breeding of the Chinese pears.

Description

Technical Field

[0001] The invention relates to the technical field of pear molecular breeding, and in particular to the development and application of a SNP marker associated with the peel background color of pear. Background Art

[0002] Pear (Pyrus L.) is a plant of the Maleae family, Amygdaloideae subfamily, Rosaceae family. It originated in my country and is cultivated and distributed in all provinces, cities and districts except Hainan Province, Hong Kong, Macao and Taiwan. It is the most widely distributed fruit tree in my country. my country is also the world's largest pear producer, 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.pyrifolia Nakai) 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] Peel color is an important trait that affects the appearance quality of sand pear fruit. Depending on whether the peel is covered with a cork layer, the base color of the sand pear peel can be divided into brown and green. The epidermis of mature fruits of brown-skinned varieties is fully covered with a cork layer, which has important biological significance in protecting the fruit from external stress. Consumers in different countries have different preferences for the base color of sand pear peel. For example, brown-skinned sand pears are favored by Japanese and Korean consumers. Japanese and Korean varieties such as 'Hosui', 'Akizuki', 'Akiro', 'Xin Gao' and 'Yuan Huang' are still the main cultivated varieties of sand pear in East Asia. Chinese consumers mostly prefer green skin, so most of the cultivated sand pear varieties are green-skinned.

[0004] At present, the breeding of sand pear is still mainly based on conventional hybridization. It usually takes more than 15 years to breed a variety, with a long breeding cycle and low breeding efficiency. As an important quality trait of sand pear, there are few relevant markers that can be used for molecular assisted breeding. This method identified a SNP marker related to the fruit peel color through resequencing and genome-wide association analysis (GWAS) of sand pear varieties with different skin colors. It can be effectively used in sand pear molecular marker-assisted selection breeding, improve the accuracy of sand pear fruit peel color selection, and accelerate the breeding process. Summary of the invention

[0005] The present invention provides a development of a SNP marker related to the background color of sand pear peel and its application, so as to solve the problems raised in the above background technology.

[0006] A SNP marker associated with the background color of sand pear peel, wherein the SNP marker is located at position 4160839 of chromosome 8 of the pear genome, and if the base at this position is G, the peel is green, and if the base at this position is A, the peel is brown.

[0007] A method for identifying the base color of fruit peel by SNP markers, comprising the following steps:

[0008] S1, extracting the genomic DNA of the pear to be tested;

[0009] S2, using the pear genomic DNA to be tested as a template, and using primers PPR-F / PPR-R for PCR amplification;

[0010] S3. The PCR amplification products were sequenced and analyzed, and the base color of the sand pear peel could be determined based on the SNP site genotype.

[0011] Furthermore, the sequence of the primer PPR-F in step S2 is

[0012] 5'-ATGGAAATGTTTGTTAGAAG-3' (SEQ ID NO. 1);

[0013] The sequence of primer PPR-R is

[0014] 5'-GGAAAATAATGGACTTGGTG-3' (SEQ ID NO. 1).

[0015] Furthermore, the total volume of the PCR amplification system in step S2 is 20ul, including 2ul of 10ng genomic template DNA, 10μl of 2×Es TaqMasterMix, 2ul each of 10mM primers PPR-F / PPR-R and 4ul of ddH2O.

[0016] Furthermore, the base color of the peel of the sand pear with the SNP site genotype of G / G in step S3 is green, and the base color of the peel of the sand pear with the SNP site base of G / A or A / A is brown.

[0017] Application of a SNP marker in assisted breeding of Pyrus truncatula.

[0018] The advantages and beneficial effects of the present invention are:

[0019] 1. The present invention utilizes genome resequencing and genome-wide association analysis (GWAS) to enrich for gene intervals related to the base color of sand pear peel, and develops SNP molecular markers that are tightly linked to the target trait, which can be effectively used in sand pear molecular marker-assisted selection breeding, improve the accuracy of selection for the base color trait of sand pear peel, and accelerate the breeding process.

[0020] 2. The present invention can identify the base color (green / brown) of the peel of the sand pear based on the single nucleotide polymorphism difference at position 4160839 of chromosome 8 of the sand pear genome, and has high accuracy in identifying the base color of the peel of the sand pear.

[0021] 3. The SNP molecular markers of the present invention were used to identify 74 sand pear germplasm resources with different peel base colors. The results showed that the accuracy of the SNP molecular markers in identifying sand pears with brown and green peels reached 100%. This shows that the identification of sand pear peel base color using the SNP molecular markers of the present invention 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

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 Manhattan plot for GWAS analysis of sand pear fruit peel background color. DETAILED DESCRIPTION

[0024] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0025] like Figure 1 As shown, the present invention provides a development and application of SNP markers related to the background color of sand pear peel, and specifically includes the following embodiments:

[0026] Example 1: Development of SNP markers associated with the base color of sand pear peel

[0027] 1. Identification of the base color of the peel of different varieties of sand pear

[0028] The accurate phenotypes of 450 sand pear varieties were obtained by identifying the base color of the fruit peel at the ripening stage for three consecutive years.

[0029] 2. Genome-wide association analysis to obtain SNP sites

[0030] The genomes of 450 sand pear varieties were resequenced using the second-generation sequencing technology and compared with the "20th Century" sand pear reference genome. 32,453,152 SNPs and 4,079,097 InDels were identified. A genome-wide association analysis of the fruit peel base color was performed using a mixed linear model (MLM). It was found that the fruit peel base color was significantly enriched in the gene interval 3,768,035 to 4,392,187 on the upper part of chromosome 8 (see Figure 1); association analysis between the SNPs in this interval and varieties with different peel base colors revealed that the SNP at position 4160839 was completely consistent with the peel base color phenotype, that is, the peel base color of the sand pear with a base of G at the SNP site was green, and the peel base color of the sand pear with a base of A at the SNP site was brown, and brown was dominant over green; this determined that the SNP site was closely linked to the peel base color trait.

[0031] Example 2: Identification of the base color of sand pear peel using SNP molecular markers

[0032] 1. DNA extraction

[0033] 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.

[0034] 2. SNP marker primer design and PCR amplification

[0035] Based on the 150 bp sequence upstream and downstream of the SNP site obtained in Example 1, primers PPR-F and PPR-R were designed using Primer software to develop SNP markers; the sequence of primer PPR-F is

[0036] 5'-ATGGAAATGTTTGTTAGAAG-3'; the sequence of primer PPR-R is

[0037] 5'-GGAAAATAATGGACTTGGTG-3'; the size of the amplified product was 96 bp; the total volume of the PCR amplification system was 20ul, including 10ng genomic template DNA 2ul, 2×Es TaqMasterMix 10ul, 10mM primers PPR-F / PPR-R 2ul each and ddH2O 4ul; the PCR amplification program was 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 detected by 1% agarose gel electrophoresis. If there was a bright single electrophoresis band at about 100bp, it indicated that the amplification was successful, and the PCR product was directly sent for sequencing.

[0038] 3. SNP site detection and identification of sand pear peel base color

[0039] The sequencing results were compared with the reference genome for sequence analysis, and the base color of the sand pear peel could be determined according to the SNP site bases; the sand pear peel base color with the SNP site genotype of G / G was green, and the sand pear peel base color with the SNP site bases of G / A or A / A was brown.

[0040] Example 3 Identification of the base color of the peel of 74 pears using SNP molecular markers

[0041] 74 sand pear varieties with different peel background colors preserved in the National Sand Pear Germplasm Resource Garden (Wuhan) were used as materials, and the peel background color was identified using the SNP molecular marker of the present invention. The specific identification method was referred to the method in the above Example 2. The identification results showed that the phenotypes and genotypes of the above 74 sand pear germplasms were completely consistent, indicating that the SNP molecular marker of the present invention can accurately identify the peel background color of sand pear with an accuracy rate of 100%. The specific varieties, phenotypes and genotypes are shown in Table 1 below;

[0042] Table 1: Peel base color and genotype of 74 sand pear cultivars

[0043]

[0044]

[0045]

[0046] It can be seen from the above test results that the SNP molecular markers provided by the present invention can accurately and quickly identify the background color of the sand pear peel, can improve breeding efficiency, shorten identification time, and are suitable for sand pear molecular 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.

[0047] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A SNP marker associated with the background color of sand pear peel, characterized in that: The SNP marker is located at position 4160839 of chromosome 8 of the pear genome. If the base at this position is G, the skin will be green, and if the base at this position is A, the skin will be brown.

2. A method for identifying the background color of fruit peel using the SNP marker as claimed in claim 1, characterized in that: Use the following steps: S1, extracting the genomic DNA of the pear to be tested; S2, using the pear genomic DNA to be tested as a template, and using primers PPR-F / PPR-R for PCR amplification; S3. The PCR amplification products were sequenced and analyzed, and the base color of the sand pear peel could be determined based on the SNP site genotype.

3. The method for identifying the background color of fruit peel by SNP markers according to claim 2, characterized in that: The sequence of the primer PPR-F in step S2 is 5'-ATGGAAATGTTTGTTAGAAG-3'; the sequence of the primer PPR-R is 5'-GGAAAATAATGGACTTGGTG-3'.

4. The method for identifying the background color of fruit peel by SNP markers according to claim 2, characterized in that: The total volume of the PCR amplification system in step S2 is 20ul, including 2ul of 10ng genomic template DNA, 10ul of 2×Es Taq MasterMix, 2ul each of 10mM primers PPR-F / PPR-R and 4ul of ddH2O.

5. The method for identifying the background color of fruit peel by SNP markers according to claim 2, characterized in that: The base color of the fruit skin of the sand pear with the SNP site genotype of G / G in step S3 is green, and the base color of the fruit skin of the sand pear with the SNP site base of G / A or A / A is brown.

6. Use of the SNP marker as claimed in claim 1 in assisted breeding of Pyrus communis.

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