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CN-122024828-A - SNP liquid phase chip construction method for forest tree breeding and application

CN122024828ACN 122024828 ACN122024828 ACN 122024828ACN-122024828-A

Abstract

The invention discloses a construction method and application of a SNP liquid phase chip for tree breeding, and belongs to the technical field of tree molecular breeding. The method comprises the steps of obtaining a high-quality candidate background site pool based on whole genome re-sequencing data of a plurality of germplasm resources of a target species through a first set of screening parameters, carrying out probe design feasibility and genome space distribution optimization on the candidate background site pool to form a background site set which is uniformly distributed and feasible to be used for probes, screening out a functional site set directly related to breeding traits based on a whole genome association analysis result, and finally combining the two types of site sets, and carrying out probe design and synthesis to prepare a liquid-phase chip. The invention also provides a chip constructed by the method and application thereof in selective breeding of the tree genome. The invention breaks through the design limitation of the traditional chip, can systematically construct a special chip with high density and high breeding value, and remarkably improves the breeding efficiency.

Inventors

  • CHEN ZHIQIANG
  • FENG QIHANG
  • BIAN LIMING
  • WU XIAMING
  • WU JINZHANG
  • Niu Diewei
  • CHANG WENJING

Assignees

  • 南京林业大学

Dates

Publication Date
20260512
Application Date
20251224

Claims (10)

  1. 1. The construction method of the SNP liquid phase chip for tree breeding is characterized by comprising the following steps: (1) Based on genome-wide resequencing data of a plurality of germplasm resources of a target forest species, to obtain a background site set for population genetics analysis, performing primary screening of SNP sites according to a first set of screening parameters, and obtaining a high-quality candidate background site pool; (2) Based on the whole genome association analysis of the phenotypic character of the target forest species, in order to obtain a functional site set directly associated with the breeding target character, SNP sites obviously associated with the character are screened out from the GWAS result according to a second set of screening parameters, so as to obtain the functional site set; (3) Optimizing the feasibility of probe design and the spatial distribution of the genome of the candidate background site pool obtained in the step (1) to form a background site set which is uniformly distributed on a reference genome and has feasible probe design; (4) Combining the background site set obtained in the step (3) with the functional site set obtained in the step (2), and carrying out probe design and synthesis based on the combined site set to prepare a liquid-phase chip.
  2. 2. The method of claim 1, wherein the first set of screening parameters comprises a sequencing depth having a specified upper and lower limit based on the number of samples, a site deletion rate of no more than 0.5, and a minimum allele frequency of no less than 0.05.
  3. 3. The method of claim 2, wherein the specific upper and lower limits of sequencing depth are determined in such a way that the lower limit is greater than the total number of samples re-sequenced and the upper limit is set based on depth profile statistics of the re-sequenced data.
  4. 4. The method of claim 1, wherein the first set of screening parameters further comprises conforming to the Hardy-Wenberg equilibrium test with a significance p-value of no more than 1e-6 and a distance between the SNP site and the nearest indel marker of at least 5 base pairs.
  5. 5. The method of claim 1, wherein the second set of screening parameters is such that the significance p-value of the association of SNP loci with traits in the whole genome association analysis is no more than 5e-8.
  6. 6. The method of claim 1, wherein the optimizing in step (3) comprises probe design feasibility filtering the pool of candidate background sites, dividing the reference genome into a plurality of windows, and selecting an optimal site within each window based on the probe design score.
  7. 7. The method of claim 1, wherein the plurality of germplasm resources for re-sequencing of step (1) are not less than 100 samples in number and are derived from at least 3 different geographic populations or genetic lineages.
  8. 8. The method of claim 1, wherein the target forest species is fir.
  9. 9. A SNP liquid-phase chip obtained by the method according to any one of claims 1 to 8.
  10. 10. The use of the SNP liquid chip of claim 9 in selective breeding of wood genome.

Description

SNP liquid phase chip construction method for forest tree breeding and application Technical Field The invention relates to the technical field of plant bioinformatics, in particular to a construction method and application of a SNP liquid phase chip for forest breeding. Background Fir improvement starts in the 50 s of the last century, the genetic improvement work of fir is in the first place in the domestic forest genetic improvement, and after decades of genetic improvement, fir has entered into the advanced generation improvement stage. At present, the fourth generation seed garden of the fir in Fujian province is already built, i.e. the new stage of breeding of the fir in the fifth generation is stepped. Traditional tree breeding has long breeding period, low phenotype breeding efficiency, time and labor consuming measurement of filial generation and long breeding time of new varieties, and severely restricts the development of fine variety breeding of fir and artificial forests. The whole genome selective breeding is a novel plant breeding method based on molecular marker and sample phenotype value association analysis, model construction and application, is an early selective breeding technology based on genotype selection, and accelerates the breeding process of fir by constructing a fir GS breeding technology system. Along with the development of a sequencing technology, obtaining high-throughput and high-quality SNP is one of the preconditions for implementing fir GS molecular breeding, however, fir is used as conifer species, the genome is huge, the structure is complex, the application of whole genome sequencing to detect mutation sites faces the dilemma of high cost and difficult implementation, and the SNP chip developed by the targeted sequencing genotype detection (GBTS) technology has higher resolution, lower cost and greater flexibility. Can be widely applied to population genetic diversity analysis, population structure, QTL (quantitative trait loci) positioning and genetic map construction, GWAS and economic trait gene excavation. However, the following problems are common in the design of SNP chips in the forest field: 1) Most chip designs focus only on constructing genetic maps or group structural analysis, and most of the selected sites are evenly distributed in neutral background sites of the genome. Although the sites can provide basic diversity information, the degree of direct association with important economic characters such as growth, materials, adaptability and the like of fir is not high, so that the efficiency and the application value in marker assisted selection and genome prediction are limited; 2) Although some studies may find some sites associated with traits through whole genome association analysis, there is a lack of a systematic approach in the prior art to effectively integrate large-scale, high-quality background sites with strictly validated functional sites to construct an "integrated" solution that both satisfies basic genetic studies and directly serves breeding decisions; 3) The existing chip design flow cannot fully consider the special requirements of breeding application scenes, such as ensuring the accuracy, reliability and cost effectiveness of typing in highly heterogeneous breeding groups. Therefore, an innovative SNP chip construction method is urgently needed in the field, the limitation of traditional neutral site selection is broken, and a special chip with high-density genome coverage and high breeding application value is developed through an optimized breeding-oriented design strategy so as to truly promote the molecular design breeding process of fir and even other forest tree species. Disclosure of Invention Aiming at the problems of single site selection strategy, systematic deletion of functional sites, lack of breeding guidance in design and the like in the existing forest SNP chip design, the invention aims to provide an innovative SNP liquid phase chip construction method. The method constructs the special chip with high-density genome coverage and high breeding application value by systematically integrating genome background information and character function information so as to overcome the defect of low efficiency of the traditional chip in molecular breeding. In order to achieve the above purpose, the technical scheme of the application is as follows: in a first aspect, the invention provides a construction method of an SNP liquid phase chip for tree breeding. The method has the core concept that a 'two-channel screening' strategy is adopted, namely, a background site for basic genetics analysis and a functional site directly serving breeding selection are respectively screened out through two independent and complementary technical paths, and are systematically integrated, so that a special chip with high-density coverage and high breeding value is constructed. The method specifically comprises the following steps: Step (1) acqui