A rubber tree genome-wide SNP molecular marker combination and gene chip and its application

Abstract

The invention discloses a rubber tree whole genome SNP molecular marker combination and gene chip and application, belonging to the field of molecular marker development. The rubber tree whole genome SNP molecular marker combination includes 28923 SNP molecular markers, and the 28923 SNP molecular markers include 28923 SNP sites, each SNP site includes two different base variation sites, for detecting the allele change of the site, and the physical location of the 28923 SNP sites is determined based on the genome sequence comparison of the rubber tree wild germplasm MT / VB / 25A 57 / 8; the 28923 SNP sites adopted by the present invention are evenly covered on the rubber tree whole genome, can fully reveal the true level of rubber tree genetic diversity, can be applied to the genetic diversity analysis, population structure analysis, whole gene association analysis, molecular marker assisted breeding and whole genome selection breeding research work of rubber tree. Utilizing the whole genome SNP site combination of the present invention and its gene chip to carry out detection, there is the advantage of being simple, fast and low in cost, and can realize large-scale application in rubber tree genetic breeding research.

Description

Technical Field

[0001] The present invention relates to the field of molecular marker development, and in particular to a rubber tree genome-wide SNP molecular marker combination and a gene chip and applications. Background Art

[0002] Rubber tree is the most important tropical cash crop, and the natural rubber it produces is an important strategic resource for my country. It has an irreplaceable role in the fields of military, aerospace, marine engineering, rail transportation, etc. At present, in the breeding process of rubber tree, what still adopts is the traditional phenotypic character evaluation method, which needs to plant seeds and obtain its character index by observing its bark thickness, cold resistance, stem girth production rate and latex yield. The procedure of traditional rubber tree breeding method is cumbersome, the rubber tree is young and the breeding cycle is as long as 30 years, and the selection method of screening and eliminating according to early phenotype is extremely inaccurate. Along with the continuous improvement of second generation sequencing technology, genome sequencing efficiency is greatly improved, and sequencing cost is also greatly reduced. The rapid development of SNP marker technology, resequencing of different varieties, SNP markers discovered by resequencing, and whole genome association analysis are used to carry out association analysis on important agronomic traits, determine the candidate gene sites related to important agronomic traits, and establish a set of efficient and rapid, mature and stable, low-cost, high-throughput genotyping identification methods, which are the direction that genetic breeding researchers give priority to.

[0003] SNP gene chips have been developed for multiple crop species to provide important high-density molecular markers for applications such as marker-assisted selection of major genes, genetic diversity analysis, backcross breeding and background selection, multi-gene aggregation breeding, seed purity testing, identification of transgenic components, genome-wide association analysis, QTL mapping, and genome-wide selection breeding. However, there are currently no reports of a comprehensive, high-density SNP gene chip covering the entire rubber tree genome. Summary of the Invention

[0004] The purpose of the present invention is to provide a rubber tree whole genome SNP molecular marker combination and gene chip and application to solve the problems existing in the above-mentioned prior art. The present invention establishes a complete set of high-density SNP gene chips covering the whole genome of rubber trees, laying the foundation for later research and genetic breeding of rubber trees.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of a rubber tree genome-wide SNP molecular marker combination in any of the following:

[0007] (1) Application in preparing gene chips for detecting SNP sites in the whole genome of rubber trees;

[0008] (2) Application in identification of genetic relationships of rubber tree germplasm resources;

[0009] (3) Application in identifying the stem girth growth rate of rubber trees;

[0010] (4) Application in whole genome breeding or molecular marker-assisted breeding of rubber trees;

[0011] (5) Application in genetic diversity analysis, QTL mapping, and genome-wide association analysis of rubber trees;

[0012] The rubber tree genome-wide SNP molecular marker combination includes 28,923 SNP molecular markers.

[0013] The present invention also provides an application of a reagent for detecting the whole-genome SNP molecular marker combination of the rubber tree in any of the following:

[0014] (1) Application in preparing gene chips for detecting SNP sites in the whole genome of rubber trees;

[0015] (2) Application in identification of genetic relationships of rubber tree germplasm resources;

[0016] (3) Application in identifying the stem girth growth rate of rubber trees;

[0017] (4) Application in whole genome breeding or molecular marker-assisted breeding of rubber trees;

[0018] (5) Application in genetic diversity analysis, QTL mapping, and genome-wide association analysis of rubber trees.

[0019] The present invention also provides an application of a gene chip for detecting the whole-genome SNP molecular marker combination of the rubber tree in any of the following:

[0020] (1) Application in identification of genetic relationships of rubber tree germplasm resources;

[0021] (2) Application in identifying the stem girth growth rate of rubber trees;

[0022] (3) Application in whole genome breeding or molecular marker-assisted breeding of rubber trees;

[0023] (4) Application in genetic diversity analysis, QTL mapping, and genome-wide association analysis of rubber trees.

[0024] The present invention also provides a reagent comprising the rubber tree whole genome SNP molecular marker combination.

[0025] The present invention also provides a gene chip, comprising the rubber tree whole genome SNP molecular marker combination.

[0026] Preferably, the gene chip comprises a liquid phase gene chip.

[0027] The present invention also provides an application of a whole-genome SNP molecular marker combination of a rubber tree in breeding germplasm resources related to the stem girth growth rate trait of the rubber tree. The whole-genome SNP molecular marker combination of the rubber tree includes 24 SNP molecular markers, and the sites of the 24 SNP molecular markers are represented by a structure of chromosome number: physical position_reference genotype / allele type, and the specific information is as follows: 1:5170612_A / G, 1:5204247_G / T, 1:29940146_C / T, 2:50216040_G / A, 3:112031854_G / A, 4:52518696_C / G, 6:107577233_C / T, 6:10875677 9_C / G, 6:108811432_C / A, 7:53799702_G / A, 7:58543483_A / T, 7:70183332_G / T、8:92459866_C / T、9:2883436_C / T、10:51129608_C / T、10:54540865_T / C、 10:80228946_A / G, 10:80234871_C / T, 11:13685568_C / T, 16:64889608_A / C, 16:64895293_T / C, 16:64902057_T / C, 16:64903791_C / T and 16:64919580_C / T.

[0028] The present invention discloses the following technical effects:

[0029] The 28923 SNP sites adopted by the present invention are mostly gene function association sites, preferably the SNP marker sites on the SNP markers related to the important agronomic traits of rubber trees or functional genes that GWAS is located. More specifically, the present invention locates and filters out 24 SNP sites related to the rubber tree stem girth growth rate, lays the foundation for breeding the variety or strain related to the rubber tree stem girth growth rate trait, is beneficial to improving breeding efficiency and shortening the breeding cycle.

[0030] The 28,923 SNP markers used in the present invention are evenly distributed on the chromosomes of the rubber tree genome, with an average marker spacing of 40 Kb, so that sites will not be lost when the markers are applied to genetic map construction, QTL positioning, GWAS analysis, etc.

[0031] The liquid phase chip used in the present invention adopts the latest targeted sequencing genotyping detection (Genotyping by Target Sequencing, GBTS) technology for marker genotyping detection, which has the advantages of low cost, high accuracy, high detection sensitivity, wide platform adaptability, and high marker flexibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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.

[0033] Figure 1 The figure shows the distribution of 28,923 SNP sites of the whole genome of rubber trees on chromosomes.

[0034] Figure 2 The MAF (A) and PIC (B) distribution diagrams of 28923 rubber tree genome-wide SNP sites of the present invention are shown;

[0035] Figure 3 The figure shows the distribution of heterozygosity (A) and gene diversity (B) of 28923 SNP sites in the whole genome of rubber trees of the present invention;

[0036] Figure 4 It is a phylogenetic tree diagram of the rubber tree germplasm resources in Example 3 of the present invention;

[0037] Figure 5 This is a diagram showing the results of the genome-wide association analysis in Example 4 of the present invention. DETAILED DESCRIPTION

[0038] 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.

[0039] 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. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0040] 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.

[0041] 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.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Example 1 Screening of a Rubber Tree Whole Genome SNP Site Combination

[0044] 1. Experimental Materials

[0045] 142 rubber tree germplasm materials: originated from the National Rubber Tree Germplasm Resource Garden planted in Danzhou City, Hainan Province in 2006-2007.

[0046] 2. Acquisition of SNP marker sites in the whole genome of rubber trees

[0047] High-quality genomic DNA was extracted from young leaves of rubber trees using the CTAB method. This DNA was used for genome resequencing, yielding 4089 Gb of high-quality clean data, with an average of 29 Gb per sample and a sequencing depth of approximately 20×. Sequencing data were aligned to the reference genome of the wild rubber tree accession MT / VB / 25A 57 / 8 (version ASM3005281v1, available at: https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCA / 030 / 052 / 815 / GCA_030052815.1_ASM3005281v1 / ) using BWA software. High-quality single-nucleotide polymorphisms (SNPs) were identified using GATK software. Marker loci with genotype loss less than 10% and a minor allele frequency greater than 5% were filtered out.

[0048] 3. Perform genome-wide association analysis on the obtained rubber tree SNP marker loci and phenotypic traits of rubber trees

[0049] 3.1 Sample collection and phenotype acquisition

[0050] A total of 142 core rubber tree germplasm accessions were planted in the National Rubber Tree Germplasm Nursery in Danzhou City, Hainan Province from 2006 to 2007. Each accession was assigned 5 trees and managed normally in the field. Five agronomic traits, including bark thickness, cold tolerance, stem girth production rate, latex yield, and latex duct number, were obtained for the 142 rubber tree accessions. The average values ​​of the five replicates were used as the phenotypic input files for genome-wide association analysis and construction of genomic selection breeding models.

[0051] Bark thickness: Use a ruler to measure the bark thickness of the rubber tree 1.5m above the ground with an accuracy of 0.1cm.

[0052] Cold resistance: The cold damage level is divided into 5 levels, namely 0, 1, 2, 3, and 4; the average value of 5 repetitions is taken as the phenotypic value of cold resistance.

[0053] Stem girth production rate: For five consecutive years, the stem girth of the rubber tree was measured 1.5 m above the ground with a tape measure, with a measurement accuracy of 0.1 cm. The stem girth growth rate was calculated using linear regression: y = b1xi + b0, where b1 is the slope, xi is the stem girth length in the i-th year, and b0 is the stem girth length at planting. The slope b1 is used to represent the stem girth growth rate.

[0054] Latex production: Latex production was measured for three consecutive years; the annual average latex production per knife was calculated based on the total production as the phenotypic data of latex production.

[0055] The number of laticifers was measured for three consecutive years, and the average value of five replicates was used as the phenotypic value of the number of laticifers.

[0056] 3.2 Genome-wide association analysis

[0057] The obtained SNP marker sites were subjected to genome-wide association analysis with the measured phenotypic information using GAPT analysis software to obtain SNP marker sites that were significantly associated with the agronomic traits of rubber trees.

[0058] 4. Prioritize the marker sites with significant association with GWAS. If there is no such marker within 100Kb, select other markers. Finally, 28,923 SNP marker sites of rubber tree genome were obtained. The SNP molecular markers were evenly distributed with an average spacing of 40Kb (see Figure 1 ).

[0059] 5. Analyze the distribution of 28,923 SNP sites on chromosomes. The results are shown in Figure 2The distribution of the minor allele frequency (MAF) of 28923 SNP sites was statistically analyzed, with a variation range of 0.05 to 0.50 and an average value of 0.24 (see Figure 2 A); The polymorphic information content (PIC) of 28923 SNP sites was statistically analyzed, with a variation range of 0.09 to 0.38 and an average value of 0.27 (see Figure 2 The heterozygosity distribution of 28923 SNP sites was statistically analyzed, with a variation range of 0.00 to 1.00 and an average value of 0.30 (see Figure 3 A); Gene diversity distribution statistics of 28923 SNP sites were performed, with variation ranges of 0.10 to 0.50 and an average value of 0.33 (see Figure 3 Middle B).

[0060] Each of the 28,923 SNPs described above contains two alleles with different bases, which are used to detect allelic variations at that site. The variation information for each SNP is represented in the form of chromosome number: physical location_reference genotype / allele. The variation information for the 28,923 SNPs described above is as follows:

[0061] Figure 4 ). The distance of the relationship between materials can be judged according to the position of the materials in the phylogenetic tree. Example 4 Application of Whole-Genome SNP Marker Sites in Whole-Genome Association Analysis of Rubber Trees This example takes the stem girth growth rate of the agronomic trait of rubber trees as an example for specific explanation. The specific steps are as follows: (1) The liquid phase gene chip of Example 2 is used to perform genotyping detection on 223 germplasm resources and breeding materials to clarify the allele types of the 28,923 whole-genome SNP sites of rubber trees. (2) The 28,923 whole-genome SNP markers of rubber trees obtained are filtered to filter out SNP markers with a missing rate greater than 10% and a minimum allele frequency (MAF) less than 5%. (3) The whole-genome association analysis uses the mixed linear model (MLM) of TASSEL software. In order to avoid the influence of population structure and kinship, the first three principal components (PCs) and kinship are used for covariance. The significance threshold in the association analysis was set at P = 0.0001 to detect SNP markers that were significantly associated with stem girth growth rate. The association analysis results were plotted using the R package CMplot (https: / / github.com / YinLiLin / R-CMplot) to plot the Manhattan plot (see Figure 5 ). (4) The whole genome association analysis detected a total of 24 SNP marker sites associated with the agronomic trait stem growth rate of rubber trees, which are located on chromosomes 1, 2, 3, 4, 6, 7, 8, 9, 10, 11 and 16 respectively (the specific names and chromosome locations of the 24 SNP markers are shown in Table 1). Applying these SNP marker sites associated with the stem growth rate of rubber trees to the molecular marker-assisted breeding of rubber trees can accelerate the breeding process of new rubber tree varieties. Table 1 24 SNP marker sites associated with the agronomic trait stem growth rate of rubber trees

[0062]

[0063] 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. An application of a rubber tree whole genome SNP molecular marker combination in breeding germplasm resources related to the rubber tree stem girth growth rate trait, characterized in that, The rubber tree genome-wide SNP molecular marker combination consists of 24 SNP molecular markers, and the sites of the 24 SNP molecular marker combination are represented by the structure of chromosome number: physical position_reference genotype / allele type, and the specific information is as follows: 1:5170612_A / G, 1:5204247_G / T, 1:29940146_C / T, 2:50216040_G / A, 3:112031854_G / A, 4:52518696_C / G, 6:107577233_C / T, 6:108756779_C / G, 6:108811432_C / A, 7: 53799702_G / A、7:58543483_A / T、7:70183332_G / T、8:92459866_C / T、 9:2883436_C / T、10:51129608_C / T、10:54540865_T / C、10:80228946_ A / G, 10:80234871_C / T, 11:13685568_C / T, 16:64889608_A / C, 16:64895293_T / C, 16:64902057_T / C, 16:64903791_C / T and 16:64919580_C / T; The reference genome of the locus of the 24 SNP molecular marker combination is version numbered ASM3005281V1 on NCBI.

2. an application of a reagent for detecting a combination of SNP molecular markers of the whole genome of rubber trees in breeding germplasm resources related to the rubber tree stem girth growth rate trait, characterized in that, The rubber tree genome-wide SNP molecular marker combination consists of 24 SNP molecular markers, and the sites of the 24 SNP molecular marker combination are represented by the structure of chromosome number: physical position_reference genotype / allele type, and the specific information is as follows: 1:5170612_A / G, 1:5204247_G / T, 1:29940146_C / T, 2:50216040_G / A, 3:112031854_G / A, 4:52518696_C / G, 6:107577233_C / T, 6:108756779_C / G, 6:108811432_C / A, 7: 53799702_G / A、7:58543483_A / T、7:70183332_G / T、8:92459866_C / T、 9:2883436_C / T、10:51129608_C / T、10:54540865_T / C、10:80228946_ A / G, 10:80234871_C / T, 11:13685568_C / T, 16:64889608_A / C, 16:64895293_T / C, 16:64902057_T / C, 16:64903791_C / T and 16:64919580_C / T; The reference genome of the locus of the 24 SNP molecular marker combination is version numbered ASM3005281V1 on NCBI.

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