Methods for obtaining 32 molecular markers related to kiwifruit traits and their applications

Abstract

The present invention belongs to the field of molecular biology and genetic breeding technology, and more specifically relates to a method for obtaining and applying 32 molecular markers related to kiwifruit traits. The present invention locates 32 SNP sites related to the sugar, acid, chlorophyll and carotenoid content of kiwifruit for the first time. In conventional breeding methods, the identification of the longitudinal diameter phenotype of kiwifruit fruit has to wait until the maturity period, which wastes a lot of time and energy, and the selection efficiency is low. By detecting molecular markers linked to traits, screening can be performed at the seedling stage, which not only saves production costs but also greatly improves the selection efficiency. In the present invention, the position of the kiwifruit trait site is clear, and the detection method of the molecular marker site is convenient and fast, and is not affected by the environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and genetic breeding, and more specifically relates to a method for obtaining and applying 32 kiwifruit trait-related molecular markers. Background Art

[0002] Kiwifruit is rich in vitamin C, dietary fiber and a variety of mineral nutrients. It is a healthy fruit that is deeply favored by consumers. China is the original origin center of the genus Actinidia. Kiwifruit resources are extremely rich, with high morphological and genetic diversity. China has about 96% of the total kiwifruit species resources in the world. With rich natural resources, the selection and breeding of new varieties with excellent traits plays a key role in the healthy and stable development of the kiwifruit industry. Fruit size is one of the important factors affecting fruit quality. The horizontal diameter is an important indicator for measuring fruit size, which directly affects the fruit size, and then affects the unit yield of the orchard and the economic benefits of fruit farmers. Therefore, increasing the horizontal diameter of the fruit and increasing the fruit size are crucial to improving the quality of kiwifruit fruit, and are one of the important goals of kiwifruit breeding.

[0003] In traditional kiwifruit breeding, the long juvenile period and large planting area are the main difficulties affecting breeding efficiency. With the rapid development of molecular biology and sequencing technology, the selection of traits is gradually transitioning from phenotypic selection to genotypic selection. Molecular marker-assisted breeding can use molecular markers that are closely linked or co-segregated with the target trait gene to screen individuals, so as to achieve the purpose of improving the selection efficiency of the target trait and shortening the breeding period. In recent years, molecular markers for important agronomic traits have been successfully developed in fruit trees such as apples, pears, and grapes, and the use of molecular markers for assisted selection has become increasingly mature. However, the positioning of important agronomic traits of kiwifruit and the development of related molecular markers are relatively scarce. In 2013, the publication of the kiwifruit genome draft provided an important foundation for genomic research and kiwifruit molecular-assisted breeding. Subsequently, based on the construction of a high-density genetic map of kiwifruit, three sex identification markers were developed to conduct sex identification in the early stages of breeding, avoiding the waste of manpower and material resources. The use of association analysis has become an effective means to analyze complex quantitative genetics laws, develop associated molecular markers, and conduct early selection of excellent traits. Summary of the invention

[0004] The purpose of the present invention is to provide a method for obtaining 32 kiwifruit trait-related molecular markers.

[0005] Another object of the present invention is to provide the application of the molecular markers obtained by the above method in kiwifruit quality screening and breeding.

[0006] In order to achieve the above object, the present invention adopts the following technical measures: A method for obtaining 32 molecular markers related to kiwifruit traits comprises the following steps:

[0007] ① Collect leaves from individual plants of the natural population of Actinidia chinensis and extract total DNA using the CTAB method;

[0008] ②Use a sonicator to randomly interrupt genomic DNA, perform end repair on DNA fragments, add sequencing adapters and purify, and obtain the final library through PCR amplification; use Qubit 2.0 for preliminary quantification, and use Q-PCR to accurately quantify the effective concentration of the library; use Illumina high-throughput sequencing platform for PE sequencing;

[0009] ③ Filter the raw reads obtained by sequencing, remove low-quality reads and perform comparison analysis. Use GATK to detect mutations in samples by comparing to the reference genome, use the hard-filtering standard to filter the identified SNPs and Indels, and obtain the final mutation file for subsequent association analysis;

[0010] ④Select the ripe fruits of the natural population of Actinidia chinensis in step ①, and determine the sugar and acid components and contents of kiwifruit;

[0011] ⑤Select mature fruits from the natural population of Actinidia chinensis obtained in step ①, and measure the chlorophyll and carotenoid contents.

[0012] ⑥ A mixed linear model was used for trait association analysis, with population genetic structure as a fixed effect and individual kinship as a random effect to correct the effects of population structure and individual kinship; GEMMA was used for GWAS analysis, and the relatedness Matrix between samples was calculated based on genotype data, and linear mixed models were used for association analysis; 32 kiwifruit trait-related SNP molecular markers were finally obtained, and the detection primers designed for the above molecular markers are shown in Table 1;

[0013] The number of portions of the kiwifruit to be tested is ≥258.

[0014] The molecular marker primers obtained by the above method are used to detect the fruit quality of the kiwifruit to be tested, thereby achieving early screening of the kiwifruit fruit quality and assisting kiwifruit breeding.

[0015] The breeding traits include: sucrose, glucose, fructose, total sugar, quinic acid, malic acid, citric acid, total acid, chlorophyll and carotenoid content.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The present invention locates 32 SNP sites related to the sugar, acid, chlorophyll and carotenoid content of kiwifruit for the first time. In conventional breeding methods, the phenotypic identification of kiwifruit fruit quality traits has to wait until the maturity stage, which wastes a lot of time and energy, and the selection efficiency is low. However, by detecting molecular markers linked to the traits, screening can be performed at the seedling stage, which not only saves production costs but also greatly improves the selection efficiency. In the present invention, the location of the kiwifruit trait site is clear, and the detection method of the molecular marker site is convenient and fast, and is not affected by the environment. DETAILED DESCRIPTION

[0018] The technical solutions of the present invention, unless otherwise specified, are conventional technologies; the reagents or materials, unless otherwise specified, are all from commercial channels. In the present invention, the publicly released kiwifruit red5 genome is used as a reference (http: / / kiwifruitgenome.org / organism / 3).

[0019] Embodiment 1:

[0020] Acquisition of 32 molecular markers closely linked to kiwifruit fruit traits:

[0021] ① 258 individual leaves from natural populations of Actinidia chinensis were collected, and total DNA was extracted using the CTAB method. Agarose gel electrophoresis showed that the main band of genomic DNA was intact and clear, without degradation and RNA contamination.

[0022] ②Use a sonicator to randomly interrupt genomic DNA, perform end repair on DNA fragments, add sequencing adapters and purify, and obtain the final library by PCR amplification. Use Qubit 2.0 for preliminary quantification, use Q-PCR to accurately quantify the effective concentration of the library to ensure the quality of the library, and use Illumina high-throughput sequencing platform for PE sequencing.

[0023] ③ Filter the original sequence Raw reads obtained by sequencing, remove the adapter sequence and the polyG and polyX at the end of the sequence, remove low-quality reads, and obtain Clean reads. Perform bwa alignment analysis on each sample, align the filtered Clean reads to the reference genome, use samtools to convert the alignment results from the sam file to the sorted bam file, use python scripts to calculate the alignment rate and coverage, and use GATK to detect variations in the samples based on the alignment result files. Use the hard-filtering standard to filter the identified SNPs and Indels, and then use vcftools to obtain the final variation file for subsequent association analysis.

[0024] ④Select 258 ripe fruits from the natural population of Actinidia chinensis, peel and cut them into pieces, freeze them in liquid nitrogen, and grind them into powder at low temperature. Use an electronic balance to weigh about 1g into a 15ml centrifuge tube, add 5ml ultrapure water, oscillate and ultrasonicate for 30min, centrifuge at 7500g for 10min, filter the supernatant and place it in an injection bottle for high performance liquid chromatography analysis. Use the Agilent 1260HPLC system together with the calcium sugar chromatography column CARBOSep CHO-620Ga, column temperature 90℃, mobile phase ultrapure water, flow rate 0.5ml / min, and automatic injection needle to inject 20ul each time to determine the sugar and acid components and content of kiwifruit.

[0025] ⑤ Select 258 ripe fruits of the natural population of Actinidia chinensis, peel and take about 5g of pulp, add 2ml acetone and 0.3g CaCO3 to grind, then add 5ml 80% acetone to grind into homogenate, filter the homogenate into a 50ml volumetric flask, wash the mortar and pomace with 80% acetone in several times, and finally dilute to 50ml with 80% acetone. Take 80% acetone as the reference solution, measure the absorbance at 440nm, 645nm, and 663nm, and calculate the chlorophyll and carotenoid content.

[0026] ⑥ A mixed linear model was used for trait association analysis, with population genetic structure as a fixed effect and individual kinship as a random effect to correct the effects of population structure and individual kinship. GEMMA was used for GWAS analysis, and the relatedness matrix between samples was calculated based on genotype data. Linear mixed models were used for association analysis, and a number of SNP molecular markers related to kiwifruit traits were finally obtained.

[0027] ⑦ 32 molecular markers with high contribution rates were selected, and 100bp sequences upstream and downstream of the trait-related sites were extracted. According to the primer design principles, SNP marker primers were developed and designed. The primer sequences are shown in Table 1.

[0028] ⑧The amplified fragments were sequenced, the bases at the variant sites were detected, and the traits were screened and classified. The classification results are shown in Table 1.

[0029] In Table 1, in the high-content group, the specific indicators corresponding to each trait are: sucrose content ≥30g / kg, glucose content ≥45g / kg, fructose content ≥45g / kg, total sugar content ≥100g / kg, quinic acid content ≥12g / kg, malic acid content ≥4g / kg, citric acid content ≥12g / kg, total acid content ≥26g / kg, chlorophyll content ≥4μg / g, and carotenoid content ≥4μg / g.

[0030] In the low-content group, the sucrose content is < 30 g / kg, the glucose content is < 45 g / kg, the fructose content is < 45 g / kg, the total sugar content is < 100 g / kg, the quinic acid content is < 12 g / kg, the malic acid content is < 4 g / kg, the citric acid content is < 12 g / kg, the total acid content is < 26 g / kg, the chlorophyll content is < 4 μg / g, and the carotenoid content is < 4 μg / g.

[0031] Example 2:

[0032] Application of 32 molecular markers obtained through Example 1 in kiwifruit trait breeding:

[0033] Randomly select 30 mature fruits from the natural population of Actinidia chinensis, extract DNA, and measure each trait to examine the effective identification rate of the molecular markers screened in the present invention for this trait;

[0034] n: The number of samples with a high-content trait, N: The number of samples with a high-content base type. When n < N, the effective identification rate = n / N; when n > N, the effective identification rate = 1 - (n - N) / N.

[0035] The results are shown in Table 1.

[0036] In Table 1, in the first row of the sucrose trait, by amplifying the sequence fragment to detect the locus at 1297668 on chromosome 25, the base at this locus of the sample with a high sucrose content is A, while the base at this locus of the sample with a low sucrose content is T. Among the 30 sample populations verified, 27 samples have the base A at this locus, and the effective identification rate of this locus is 90.0%.

[0037] The results show that the loci screened in the present invention have a high effective identification rate, achieving the purpose of early screening and breeding for the contents of sugar, acid, chlorophyll, and carotenoid in kiwifruit fruits.

[0038] For the above-mentioned application, the relevant loci of the kiwifruit to be tested can be detected using the existing technology.

[0039] Table 1 32 SNP molecular markers and corresponding phenotypes

[0040]

[0041]

Claims

1. A method for obtaining a detection primer combination for 32 kiwifruit trait-related SNP molecular markers, comprising the following steps: ① Collect leaves from individual plants of the natural population of Actinidia chinensis and extract total DNA using the CTAB method; ②Use a sonicator to randomly interrupt the genomic DNA, perform end repair on the DNA fragments, add sequencing adapters and purify them, and obtain the final library through PCR amplification; Qubit 2.0 was used for preliminary quantification, and Q-PCR was used to accurately quantify the effective concentration of the library; PE sequencing was performed using the Illumina high-throughput sequencing platform; ③ Filter the original sequence Raw reads obtained by sequencing, remove low-quality reads and perform comparison analysis. Use GATK to detect mutations in the samples by comparing them to the reference genome, use the hard-filtering standard to filter the identified SNPs and Indels, and obtain the final mutation file for subsequent association analysis. The reference genome is the kiwifruit red5 genome; ④Select the ripe fruits of the natural population of Actinidia chinensis in step ①, and determine the sugar and acid components and contents of kiwifruit; ⑤Select the ripe fruits of the natural population of Actinidia chinensis in step ① and measure the chlorophyll and carotenoid contents; ⑥ A mixed linear model was used for trait association analysis, with population genetic structure as a fixed effect and individual kinship as a random effect to correct the effects of population structure and individual kinship; GEMMA was used for GWAS analysis, the relatedness Matrix between samples was calculated based on genotype data, and linear mixed models were used for association analysis; 32 kiwifruit trait-related SNP molecular markers were finally obtained, and the detection primer combinations designed for the SNP molecular markers are shown in the following table:

2. Application of the detection primer combination obtained by the method of claim 1 in breeding of Actinidia chinensis, wherein the traits of the breeding of Actinidia chinensis are: sucrose, glucose, fructose, total sugar, quinic acid, malic acid, citric acid, total acid, chlorophyll and carotenoid content; the correspondence between the molecular markers detected in Actinidia chinensis using the detection primer combination and the traits is shown in the following table:

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