A molecular marker linked to a QTL for annual branch diameter in kiwifruit and its application

Abstract

The present invention discloses a molecular marker linked to the QTL for the thickness of one-year-old branches of kiwifruit and its application, belonging to the field of molecular biology and genetic breeding technology. The nucleotide sequence of the molecular marker is shown in SEQ ID NO:1, and the molecular marker has one SNP site, which is located at 183bp of the sequence shown in SEQ ID NO:1. The present invention locates the QTL linked to the thickness of one-year-old branches of kiwifruit in kiwifruit, and provides a molecular marker tightly linked to the QTL for the thickness of one-year-old branches of kiwifruit. In conventional breeding methods, the growth traits of kiwifruit trees are difficult to judge at the seedling stage, and the accuracy and breeding efficiency are low. By detecting the molecular marker linked to the QTL for the thickness of one-year-old branches of kiwifruit, the thickness of one-year-old branches of kiwifruit plants can be predicted, and then early screening can be carried out, which not only saves production costs but also greatly improves selection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and genetic breeding, and in particular to a molecular marker linked to a QTL for the thickness of annual branches of kiwifruit and its application. Background Art

[0002] Kiwifruit (Actinidia) is a dioecious perennial vine in the genus Actinidia (Lindl.) of the family Actinidiaceae. It comprises 54 species and 21 varieties and is one of the four most successful wild fruit trees domesticated and cultivated in the 20th century. Varieties are the key to industrial development. The diameter of kiwifruit's annual branches is closely related to the vigor of the tree, forming the foundation for cultivating superior varieties and a crucial factor influencing fruit growth and development. Furthermore, plants with strong vigor have greater overall resistance and are easier to manage later, significantly reducing planting risks and investment costs.

[0003] Kiwifruit breeding methods have gradually shifted from traditional direct selection of wild seeds to hybridization. Selecting superior recombinant genotypes from hybrid offspring is a key step in hybridization. Traditional breeding methods based on phenotypic selection are time-consuming, labor-intensive, and difficult, requiring 10 to 15 years to develop a single new variety. Furthermore, most phenotypic traits are quantitative and susceptible to environmental influences, resulting in unstable performance and reduced selection accuracy. Advances in PCR and modern sequencing technologies have led to the development of marker-assisted selection (MAS). Because this method selects target traits based on genotype rather than phenotype, it has gained widespread favor among breeders and geneticists. Directed hybridization and molecular-assisted selection are combined in breeding efforts to rapidly identify varieties with specific superior traits through association analysis between molecular markers and traits. The key to marker-assisted breeding is to find markers linked to phenotypes, construct genetic linkage maps, and locate quantitative trait loci (QTLs). This can effectively establish the linkage relationship between markers and phenotypes and is an effective way to implement MAS.

[0004] Due to the limitations of traditional breeding methods, it is difficult to carry out kiwifruit breeding at the seedling stage. Combined with QTL positioning interval information, the present invention develops molecular markers linked to the QTL for annual branch thickness. This method can improve breeding efficiency and also provide strong guarantees for cultivation management and comprehensive resistance of kiwifruit after planting. Summary of the Invention

[0005] The purpose of the present invention is to provide a molecular marker linked to the QTL for the thickness of the annual branches of kiwifruit and its application to solve the problems existing in the above-mentioned prior art. The present invention provides a strong guarantee for the early screening of kiwifruit plants by discovering a SNP site on chromosome Group 10 and analyzing its association with the thickness of the annual branches of kiwifruit.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a molecular marker linked to a QTL for annual branch thickness in kiwifruit. The nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1. The molecular marker has a single polymorphism (SNP) site located at 183 bp of the sequence shown in SEQ ID NO: 1, and the SNP site is A or C.

[0008] Furthermore, the genotypes corresponding to the SNP sites include AA and AC.

[0009] Furthermore, when the genotype of the SNP site is AA, the annual branch thickness of the kiwifruit plant is greater than that of the AC genotype plant.

[0010] The present invention also provides a method for detecting the thickness of one-year-old branches of kiwi fruit, comprising the following steps:

[0011] (1) Using the genomic DNA of the kiwifruit to be tested as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO: 2-3 to obtain an amplified product;

[0012] (2) The amplified product was sequenced to detect the genotype of the 183rd bp of the gene sequence of the amplified product. When the genotype was AA, the thickness of the annual branches of the kiwifruit plant was greater than that of the AC genotype plant.

[0013] Furthermore, in step (1), the amplification reaction system is: 2×Hieff Add 25 μL of PCR MasterMix, 2 μL of forward and reverse primers, 3 μL of kiwifruit genomic DNA template, and ddH2O to 50 μL.

[0014] Furthermore, in step (1), the amplification reaction procedure is: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 20 s, extension at 72°C for 30 s, 35 cycles; and extension at 72°C for 5 min.

[0015] The present invention also provides a kit for detecting the thickness of annual branches of kiwifruit, comprising a primer pair as shown in SEQ ID NO: 2-3.

[0016] The invention also provides an application for kiwifruit breeding.

[0017] Furthermore, the application is to predict the annual branch thickness of kiwi plants.

[0018] The present invention discloses the following technical effects:

[0019] The present invention locates a molecular marker in kiwifruit that is linked to the QTL for the annual branch thickness trait of kiwifruit. In conventional breeding methods, kiwifruit cultivation-related traits are difficult to determine at the seedling stage, and the accuracy and breeding efficiency are low, which may result in a large loss of time and cost after planting. By detecting molecular markers linked to cultivation-related traits, not only production costs are saved but also selection efficiency is greatly improved. The QTL site for the annual branch thickness trait of kiwifruit in the present invention is clearly located, and the method for detecting the molecular marker site is convenient and fast, and is not affected by climate, environment, etc. By detecting molecular markers linked to the trait, the annual branch thickness trait of kiwifruit plants can be predicted, and early screening can be carried out. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0021] Figure 1 This is the genetic map of the hybrid population of Actinidia chinensis;

[0022] Figure 2 This is the effect of molecular markers on the thickness of one-year kiwifruit branches. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] 1. Methods

[0030] 1. An F1 hybrid population was constructed using the diploid Chinese kiwifruit 'Hongyang' as the female parent and the matching male plant of 'Boshan Biyu' as the male parent, and 173 individual plants of the F1 population were selected as research objects.

[0031] 2. Take fresh young leaves of 173 progeny plants and hybrid parents from the hybrid population, quickly freeze them in liquid nitrogen, and use the SDS method to extract genomic DNA, and test the DNA quality.

[0032] 3. Qualified genomic DNA was randomly sheared using enzymes, end-repaired, A-tailed, and supplemented with Illumina sequencing adapters. The DNA fragments were amplified and enriched by PCR, and the products were purified. Sequencing libraries were constructed for the male plants of the diploid Chinese kiwifruit varieties 'Hongyang' and 'Boshan Biyu', as well as 173 progeny plants. The sequencing libraries were tested and quantified using real-time PCR. Finally, sequencing was performed on a Novasek 6000 sequencer using the PE 150 sequencing strategy.

[0033] 4. The raw data from the Illumina platform were filtered using FASTP (version 0.18.0) with the following filtering criteria: (1) reads containing unknown nucleotides (N) ≥ 10% were removed; (2) reads containing bases with a phred quality score ≤ 20 ≥ 50% were removed; (3) reads containing sequencing adapters were deleted. The filtered reads were aligned to the reference genome using the alignment software BWA (version 0.7.12) using the mem algorithm with the alignment parameters -k 32-M; (4) After alignment, the results were labeled using the software Picard (version 1.129), and population SNP detection was performed using the variant detection software GATK. The detection results are shown in Table 1

[0034] Table 1 Distribution of SNP detection results on the genome

[0035]

[0036]

[0037] 5. Based on the SNP information obtained from the above analysis, the evolutionary tree was constructed using the treebest software and the neighbor-joining method. Principal component analysis was performed using plink and GCAT64 software. The population structure was analyzed using admixture software to complete the population analysis of the composition population.

[0038] 6. After preliminary quality filtering and variation detection, the filtering results are shown in Table 2, and a VCF file containing variation information was obtained, which was further filtered according to the following criteria: (1) bcftools software was used to filter SNP markers; (2) Plink software was used to remove markers with a genotype missing rate greater than 0.02, samples with a sample missing rate greater than 0.03, and markers with a minor allele frequency less than 0.05; (3) R program was used to retain markers with parental genotypes of "0 / 0×0 / 1", "0 / 1×0 / 0", "0 / 1×0 / 1", "0 / 1×1 / 1", and "1 / 1×0 / 1", and markers that did not separate in the offspring were removed; (4) Chi-square test was performed to remove markers with severe skewed separation.

[0039] Table 2 Summary of SNP filtering results

[0040]

[0041] 7. Use Lep-MAP3 software to construct genetic map, see Figure 1Each linkage group was repeated five times, and the one with the highest likelihood value was selected as the final result. The genetic positions between markers and the LOD value matrix of markers to each genetic position were obtained, and a high-density genetic linkage map of the diploid Chinese kiwifruit hybrid population was constructed.

[0042] 8. The 173 hybrid offspring plants were measured and evaluated. The diameter of the one-year-old branches of each sample at 2 cm from the base was measured using a digital vernier caliper, and the phenotypic traits were statistically analyzed.

[0043] 9. QTL mapping was performed using R / qtl, using the composite interval mapping method cim() function, with a scanning step size of 1 cM and an LOD threshold of 3. Finally, a QTL locus associated with the annual branch thickness trait was detected.

[0044] 10. Molecular marker detection steps

[0045] (1) Fresh young leaves of 173 progeny plants and hybrid parents were taken, quickly frozen in liquid nitrogen, and then genomic DNA was extracted using the SDS method.

[0046] (2) According to 2×Hieff Plus PCR Master Mix (With Dye) kit instructions, design a 50μL reaction system: 2×Hieff Add 25 μL of PCR MasterMix (With Dye), 2 μL of forward and reverse primers, 3 μL of kiwifruit genomic DNA template, and ddH2O to 50 μL.

[0047] (3) PCR amplification program is shown in Table 3:

[0048] Table 3 PCR amplification program

[0049]

[0050] After the PCR amplification process is completed, store it at 4°C.

[0051] (4) The PCR products were subjected to Sanger sequencing to detect the marker sites, and the kiwifruit cultivation-related traits were predicted based on the base types at the molecular markers, thereby achieving the goal of efficient breeding.

[0052] 2. Results

[0053] 1. The molecular marker information related to the annual branch diameter trait of kiwifruit is shown in Table 4.

[0054] Table 4 Molecular marker information related to the annual branch diameter traits of kiwifruit

[0055]

[0056] Amplification product (SEQ ID NO.1):

[0057] The bold underlined portion in SEQ ID NO: 1 is A or C.

[0058] Table 5 Association analysis between molecular markers and annual branch diameter traits of kiwifruit

[0059]

[0060] Table 5 shows the genotyping of kiwifruit hybrid offspring using molecular markers at locus 584605 of Group 10 chromosome. The offspring were divided into two genotypes: 83 offspring had an A / A genotype, with an average annual branch diameter of 6.62 cm; 90 offspring had an A / C genotype, with an average annual branch diameter of 5.60 cm. A significance test was performed on the average annual branch diameter of the 173 hybrid offspring. The results showed that the difference in average annual branch diameter between the two genotypes was 1.02 cm, which was extremely significant (P < 0.005). Figure 2 .

[0061] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for predicting the thickness of annual branches of kiwifruit using molecular markers, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO:

1. The molecular marker has a SNP site located at 183 bp of the sequence shown in SEQ ID NO:

1. The SNP site is A or C. The genotypes corresponding to the SNP site include AA and AC. The method comprises the following steps: (1) Using the genomic DNA of the kiwifruit to be tested as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO: 2-3 to obtain an amplified product; (2) sequencing the amplified product to detect the genotype of the 183rd bp of the gene sequence of the amplified product. When the genotype is AA, the annual branch thickness of the kiwifruit plant is greater than that of the AC genotype plant; The kiwifruits are diploid Chinese kiwifruit 'Hongyang', diploid Chinese kiwifruit 'Boshan Biyu' and an F1 hybrid population constructed with the diploid Chinese kiwifruit 'Hongyang' as the female parent and the matching male plant of 'Boshan Biyu' as the male parent.

2. The method for predicting the thickness of one-year-old branches of kiwi fruit according to claim 1, wherein: In step (1), the amplification reaction system is: 2×Hieff Canace ® Add 25 μL of PCR Master Mix, 2 μL of forward and reverse primers, 3 μL of kiwifruit genomic DNA template, and ddH2O to 50 μL.

3. The method for predicting the thickness of one-year-old branches of kiwi fruit according to claim 1, wherein: In step (1), the amplification reaction procedure is: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 20 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min.

4. Use of a kit for predicting the annual branch diameter of kiwifruit plants, characterized in that: The kit comprises a primer pair as shown in SEQ ID NOs: 2-3; the primer pair is used to detect a molecular marker with a nucleotide sequence as shown in SEQ ID NO: 1; the molecular marker has a single nucleotide polymorphism (SNP) site located at 183 bp of the sequence shown in SEQ ID NO: 1, and the SNP site is A or C; the genotypes corresponding to the SNP site include AA and AC; the application comprises sequencing a PCR amplification product of the primer pair, detecting the genotype of the 183rd bp of the gene sequence of the amplified product; when the genotype is AA, the annual branch thickness of the kiwifruit plant is greater than that of the plant with the AC genotype; The kiwifruits are diploid Chinese kiwifruit 'Hongyang', diploid Chinese kiwifruit 'Boshan Biyu' and an F1 hybrid population constructed with the diploid Chinese kiwifruit 'Hongyang' as the female parent and the matching male plant of 'Boshan Biyu' as the male parent.

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