A SNP marker related to basic density of poplar wood and its application

Abstract

The present invention discloses a SNP molecular marker related to the basic density of poplar wood, and relates to the field of poplar genetic breeding; the marker is located at 1,394,234bp on chromosome 16 of the reference sequence of the Populus trichocarpa genome version 3.0. The present invention determines that it is a key regulatory site affecting the basic density of poplar wood through eQTL analysis, and simultaneously regulates the expression of cellulose synthase and secondary cell wall-related transcription factors. The SNP molecular marker related to the basic density of poplar wood of the present invention provides a new option for the breeding of basic density of poplar wood, and has important application value in accelerating the process of poplar breeding.

Description

Technical Field

[0001] The invention relates to a SNP (single nucleotide polymorphism) marker related to the basic density of poplar wood and application thereof, belonging to the technical field of poplar genetic breeding. Background Art

[0002] As an important biomass energy, wood is increasingly used with the development of social economy. Energy produced from biomass accounts for about 14% of the world's primary energy. Poplar (Populus L.) is a short-rotation tree species and bioenergy raw material due to its fast growth, large biomass and strong adaptability. Its wood can be used for papermaking and bioethanol conversion after lignin degradation. At the same time, because poplar has less fermentation inhibitory extracts, its wood biomass conversion rate is higher than other tree species.

[0003] Wood quality mainly includes physical properties such as wood basic density, microfibril angle, fiber length, fiber width, and chemical properties such as cellulose, holocellulose and lignin content. The basic density of wood affects the physical properties of wood quality. In addition to affecting the transportation of water and nutrients by trees, the basic density of wood also affects the mechanical strength of wood and determines the quality of its derivatives.

[0004] Poplars have a long growth cycle and it is difficult to measure the basic density of wood. It is very important to determine the basic density of wood during the seedling stage, so that excellent poplar varieties can be selected early.

[0005] Therefore, providing a molecular marker related to the basic density of poplar wood and a method for obtaining and applying it to predict the basic density of wood for early selection of poplars, reduce breeding costs, shorten the breeding cycle, and accelerate genetic progress. By directly identifying the marker site, poplar strains with different basic wood density requirements can be screened, and excellent varieties can be accurately and efficiently bred. This has become a technical problem that urgently needs to be solved in this technical field. Summary of the invention

[0006] In order to overcome the above problems, the present invention proposes a molecular marker related to the basic density of poplar wood and a method for obtaining and applying the same, so as to predict the basic density of wood for early selection of poplars, reduce breeding costs, shorten the breeding cycle, and accelerate genetic progress. By directly identifying the marker site, poplar strains with different basic density requirements can be screened, so that excellent varieties can be bred accurately and efficiently.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A SNP molecular marker associated with the basic density of poplar wood is located at 1,394,234bp on chromosome 16 of the reference sequence of the Populus trichocarpa genome version 3.0, and has A / T polymorphism.

[0009] Preferably, the alleles of the SNP molecular marker are A and T.

[0010] Preferably, the genotypes corresponding to the alleles of the SNP molecular marker are AA genotype and AT genotype.

[0011] The present invention also provides a method for obtaining the above-mentioned SNP molecular marker related to the basic density of poplar wood, comprising the following steps:

[0012] Step 1, extracting total RNA from the developmental xylem of poplar hybrid populations and reversely transcribing it into cDNA for constructing a sequencing library for transcriptome sequencing;

[0013] Step 2, determining phenotypic traits;

[0014] Step 3, genotyping;

[0015] Step 4: Perform eQTL (Expression Quantitative Trait Loci) analysis and hotspot analysis to screen key sites.

[0016] Preferably, step 1 is as follows:

[0017] The total RNA from the hair and xylem of the F1 hybrid population of Danhong Poplar × Tongliao No. 1 Poplar was extracted using the RNAprep Pure Plant Plus Kit. 3 μg of RNA from each sample was used to construct sequencing libraries using the NEBNext R UltraTM RNA Library PrepKit. Then, 150 bp paired-end reads were generated on the Illumina Hiseq platform. The raw sequences were filtered and mapped to the reference genome using TopHat v2.0.12.

[0018] Preferably, step 2 is as follows:

[0019] The basic density of wood is determined by the water displacement method.

[0020] Preferably, step 3 is as follows:

[0021] Using GATK4.0 software, the whole genome resequencing data and transcriptome data of the hybrid population were aligned to the reference genome to identify SNPs at unique sites. Subsequently, the two SNP data sets were merged and filtered according to the following criteria: ① the missing rate was less than 0.1; ② the minimum allele frequency was greater than 0.05; ③ the Harwin-Wen equilibrium was greater than 0.001; ④ the individual SNP completeness was greater than 0.95; ⑤ the individual average depth was greater than or equal to 4; finally, 1 504 840 SNP data were obtained for subsequent analysis; ANNOVAR was used to annotate the identified SNPs, namely introns, exons, intergenic regions, and UTR regions; finally, 1 504 840 SNPs were obtained.

[0022] Preferably, step 4 is as follows:

[0023] HTSeq was used to perform quantitative analysis of the gene level for each sample, and the sequencing depth and gene length were corrected. The gene expression value was expressed in FPKM. The linear model in the Matrix eQTL software package was used to perform quantitative trait positioning analysis, and the correlation between the genotype of each sample and the standardized gene expression was calculated. In order to reduce the false positive between SNPs and gene expression, the threshold was determined using Bonferroni correction (P < 1.7*10 -12 ), and the SNP sites significantly associated with gene expression were obtained through P value filtering, i.e., eQTL results, so as to determine the relationship between gene mutation and gene expression. According to the location of the significantly associated SNP and the physical distance of the gene with which it produces an effect on the genome, eQTL was divided into cis-eQTL and trans-eQTL. The gene containing the eQTL site was called eGene. The gene interval distance of adjacent paired genes in the whole genome was calculated, and it was found that 95% of the distances between adjacent genes were ≤25kb. Therefore, if the SNP is located in the corresponding gene or has a transcription start site or the end of the gene is less than 25kb, it is classified as cis-eQTL, otherwise it is trans-eQTL;

[0024] Using hot-scan, the identification window was determined to be 25 kb; the maximum number of regulated genes was 3226, including 131 transcription factors; the hotspots contained 51 SNP sites, 15 of which were located in the intergenic region and the rest were located in the gene; 1 394 on chromosome 16 The 234bp A changed to T, and this site regulated the largest number of genes, regulating 3210 genes in this hotspot; the locus was mainly divided into two genotypes, AA (n=191) and AT (n=43); heatmap cluster analysis was performed on the regulated genes of hotspot12654, and the results showed that 3226 genes were divided into two categories, A and B, containing 2719 and 507 genes respectively, and the clustering between samples was also divided into two categories, which was basically consistent with the genotype division; CesAs genes showed significant differential expression between the AA / AT genotypes, and the AA genotype had a high expression level; laccase (PtrLAC1b) for lignin biosynthesis and PtrMYB3, PtrMYB20, and PtrMYB156 involved in SCW formation were also regulated by hotspot12654, but were all highly expressed in the AT genotype.

[0025] Through the above analysis process, the present invention identified a SNP site that was significantly correlated with the basic density of wood. The alleles of the SNP marker located at 1,394,234 bp on chromosome 16 of the reference sequence of the Populus trichocarpa genome version 3.0 are A and T, and there are two corresponding genotypes, namely, AA genotype and AT genotype.

[0026] The above research results prove that the basic density of wood of the SNP marker located at 1,394,234bp on chromosome 16 of the reference sequence of the Populus trichocarpa genome version 3.0 is significantly lower than that of the poplar with genotype AA.

[0027] Another object of the present invention is to provide the application of the above-mentioned SNP molecular marker related to the basic density of poplar wood.

[0028] The above object of the present invention is achieved through the following technical solutions:

[0029] Application of SNP molecular markers related to basic density of poplar wood in regulating basic density of poplar wood.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention uses a hybrid population of Danhong Poplar × Tongliao No. 1 Poplar as material, determines the SNP marker at 1,394,234 bp on chromosome 16 to affect the basic density of wood through wood property trait determination and eQTL positioning of the hybrid population, shortens the breeding cycle, and accelerates the breeding process; by using the molecular markers of the present invention, a reliable method can be provided for breeding poplars with different basic density of wood.

[0032] The present invention is further described below through specific implementation modes and drawings, but it is not intended to limit the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the whole genome eQTL map of the present invention. The points on the diagonal represent eQTLs located near the regulatory genes, and the other points represent trans-eQTLs. The horizontal axis is the chromosomal position of the eQTL, and the vertical axis is the gene associated with the eQTL.

[0034] Figure 2 This is the distribution diagram of the genes regulated by Hotspot12654 on 19 chromosomes in Example 1 of the present invention. From the outside to the inside, each circle represents: chromosome number and length, trans-eQTLs, cis-eQTL, hotspot12654 and regulated genes.

[0035] Figure 3 This is a clustering heat map of Hotspot12654 regulated genes in Example 1 of the present invention.

[0036] Figure 4 This is a graph showing the expression levels of secondary cell wall-related genes regulated by the A / T marker between the AA / AT genotypes in Example 1 of the present invention.

[0037] Figure 5 This is a comparison diagram of the difference in basic density of two genotypes of wood in Example 1 of the present invention. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with specific embodiments, which are not described in detail in the following embodiments. It should be understood that the following text is only used to describe one or several specific implementations of the present application, and does not strictly limit the scope of protection of the specific requests of the present application. In the absence of conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0039] Unless otherwise specified, the reagents involved in the following examples are all conventional reagents available on the market, and the methods used are all commonly used methods in the art.

[0040] Embodiment 1:

[0041] Example 1 of the present invention uses SNP data from whole genome resequencing and transcriptome sequencing for genotyping, and uses gene expression as a quantitative trait to locate QTL (quantitative trait locus), thereby discovering SNP sites that are significantly associated with target traits and genes; by typing specific sites of the population to be tested, early selection of basic density of poplar wood is achieved, thereby saving breeding costs and time and accelerating genetic progress.

[0042] In a first aspect, Example 1 of the present invention provides a SNP molecular marker related to the basic density of poplar wood, which is located at 1,394,234 bp on chromosome 16 of the reference sequence of the Populus trichocarpa genome version 3.0, and has an A / T polymorphism.

[0043] According to a preferred embodiment of the present invention, the allele sites A and T of the SNP marker located at 1,394,234bp on chromosome 16 of the reference sequence of the Populus trichocarpa genome version 3.0 have two corresponding genotypes, namely AA genotype and AT genotype, and the basic density of wood of poplars with genotype AT at the SNP site is significantly lower than that of poplars with genotype AA.

[0044] The inventors have found that the basic density of wood of individuals with the AT genotype of the SNP marker of poplar is significantly lower than that of individuals with the AA genotype. Therefore, the basic density of wood of poplar can be genetically evaluated according to the genotype of the SNP site. It can also be determined that the SNP marker located at the 1,394,234 bp on chromosome 16 of the reference sequence of the Populus trichocarpa genome version 3.0 is closely related to the basic density of wood trait, and can be effectively used in molecular marker-assisted breeding of poplar, which has the advantages of early screening, time saving, low cost and high accuracy.

[0045] The second aspect of the present invention provides a method for obtaining the above-mentioned SNP molecular marker related to the basic density of poplar wood, comprising the following steps:

[0046] (1) RNA extraction and library construction and sequencing

[0047] Total RNA from the hair and xylem of the hybrid population of Danhong Populus × Tongliao No. 1 Populus was extracted using the RNAprep Pure Plant Plus Kit (Tiangen, Beijing, China). 3 μg of RNA from each sample was used to construct sequencing libraries using the NEBNext RUltraTM RNA Library Prep Kit. Then, 150 bp paired-end reads were generated on the Illumina Hiseq platform. The raw sequences were filtered and mapped to the reference genome using TopHat v2.0.12.

[0048] (2) Genotyping

[0049] Using GATK4.0 software, the whole genome resequencing data and transcriptome data of the hybrid population were aligned to the SNPs of the unique sites identified in the reference genome. Subsequently, the two SNP data sets were merged and filtered according to the following criteria: ① the missing rate was less than 0.1; ② the minimum allele frequency was greater than 0.05; ③ the Harwin-Wen equilibrium was greater than 0.001; ④ the individual SNP completeness was greater than 0.95; ⑤ the individual average depth was greater than or equal to 4; finally, 1504840 SNP data were obtained for subsequent analysis; ANNOVAR was used to annotate the identified SNPs, namely introns, exons, intergenic regions, and UTR regions; finally, 1504840 SNPs were obtained, of which the GG genotype was the most, as shown in Table 1 below:

[0050] Table 1

[0051]

[0052]

[0053] (3) eQTL analysis

[0054] HTSeq was used to perform quantitative analysis of the gene level for each sample, and the sequencing depth and gene length were corrected. FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) was used to represent the gene expression value. The linear model in the MatrixeQTL software package was used to perform quantitative trait positioning analysis, and the correlation between the genotype (SNP) of each sample and the standardized gene expression (FPKM value) was calculated. In order to reduce the false positive between SNPs and gene expression, the threshold was determined using Bonferroni correction (P < 1.7*10 -12 ), and the SNP sites significantly associated with gene expression were obtained through P value filtering, i.e., eQTL results, so as to determine the relationship between gene mutation and gene expression. According to the location of the significantly associated SNP and the physical distance of the gene with which it produces an effect on the genome, eQTL was divided into cis-eQTL and trans-eQTL. The gene containing the eQTL site was called eGene. The gene interval distance of adjacent paired genes in the whole genome was calculated, and it was found that 95% of the distances between adjacent genes were ≤25kb. Therefore, if the SNP is located in the corresponding gene or has a transcription start site or the end of the gene is less than 25kb, it is classified as cis-eQTL, otherwise it is trans-eQTL;

[0055] like Figure 1 As shown in FIG. 1 , it is a genome-wide eQTL map of the present invention. The points on the diagonal represent eQTLs located near regulatory genes, and the other points represent trans-eQTLs. The horizontal axis is the chromosome position of the eQTL, and the vertical axis is the gene associated with the eQTL. Figure 1 Figure 4 shows the eQTL mapping results of the present invention using mixed linear models to analyze the correlation between gene expression and 1 504 840 SNPs distributed on 19 chromosomes. The final eQTL mapping showed that 2 232 908 eQTLs (48 019 cis-eQTLs and 2 184 889 trans-eQTLs) were obtained (P<1.7*10 -12 );

[0056] (4) eQTL hotspot analysis

[0057] eQTL hotspots are helpful for analyzing the complex regulatory relationship between eQTL and eGene. Trans-eQTL is an important eQTL regulatory region (trans-hotspot). Therefore, in order to further explore the regulatory region (trans-hotspot), hot-scan was used for identification, and the identification window was determined to be 25kb;

[0058] like Figure 2 As shown, it is the distribution map of the Hotspot12654 regulated genes on 19 chromosomes in Example 1 of the present invention, and each circle from the outside to the inside represents: chromosome number and length, trans-eQTLs, cis-eQTL, hotspot12654 and regulated genes; Figure 2 The hotspot 12654 regulatory gene network diagram identified in Example 1 of the present invention is located on chromosome 16, and the number of regulated genes is 3226 at most, including 131 transcription factors; the hotspot contains a total of 51 SNP sites, of which 15 are located in the intergenic region and the rest are located in the gene; 1 394 234 bp of A on chromosome 16 changes to T, and the site regulates the largest number of genes, regulating 3 210 genes in the hotspot; the locus is mainly divided into two genotypes, AA (n=191) and AT (n=43); heatmap cluster analysis is performed on the regulatory genes of hotspot 12654, and the results show that: 3226 genes are divided into two categories, A and B, containing 2719 and 507 genes respectively, and the clustering between samples is also divided into two categories, which is basically consistent with the genotype division; among them, the samples of class B high-expression genes (36) are all AT genotypes, and the other group of 199 samples are mainly AA genotypes (191AA+8AT), such as Figure 3As shown, it is a clustering heat map of Hotspot12654 regulated genes in Example 1 of the present invention;

[0059] like Figure 4 As shown, it is an expression diagram of secondary cell wall-related genes regulated by A / T markers between AA / AT two genotypes in Example 1 of the present invention; it is an analysis of the gene expression levels identified according to the A / T genotype, and it is found that the CesAs gene shows significant differential expression between the two AA / AT genotypes, and the expression level of the AA genotype is high; the laccase (PtrLAC1b) of lignin biosynthesis and PtrMYB3, PtrMYB20, and PtrMYB156 involved in SCW formation are also regulated by hotspot12654, but are all highly expressed in the AT genotype;

[0060] In order to reveal whether the marker is related to wood quality, the present invention tested the wood property data of the F1 progeny of the two genotypes and found that the basic density of wood in the hybrid progeny between the two genotypes was significantly different (P=0.028); Figure 5 , which is a comparison diagram of the difference in basic density of two genotypes of wood in Example 1 of the present invention.

[0061] From the above, it can be concluded that the A / T base mutation at 1,394,234bp on chromosome 16 of poplar affects the expression of genes related to secondary wall in poplar, and this mutation affects the phenotypic variation of basic density of poplar wood. This SNP site can be used as a molecular marker for basic density screening of wood and applied in the molecular breeding process of poplar.

[0062] The present invention determines that it is a key regulatory site affecting the basic density of poplar wood through eQTL analysis, and simultaneously regulates the expression of cellulose synthase and secondary cell wall-related transcription factors. The present invention provides a new option for the basic density breeding of poplar wood, and has important application value in accelerating the poplar breeding process.

[0063] The basic density trait of wood is a quantitative trait determined by multiple gene loci. The present invention improves the wood quality of poplar varieties and meets human demand for wood by identifying and screening the loci of the basic density trait of wood.

[0064] Although the purpose, concept and embodiments of the present invention have been described in detail above, a person skilled in the art will recognize that various improvements and modifications may be made to the present invention without departing from the scope of the claims, and such improvements and modifications shall still fall within the scope of protection of the present invention.

Claims

1. A method for obtaining a SNP molecular marker related to the basic density of wood of Danhong Poplar, Tongliao No. 1 Poplar and their hybrid progeny, wherein the SNP molecular marker is located at 1,394,234 bp on chromosome 16 of the reference sequence of the Populus trichocarpa genome version 3.0, and there is an A / T polymorphism. The alleles of the SNP molecular marker are A and T, and the genotypes corresponding to the alleles of the SNP molecular marker are AA genotype and AT genotype; The steps are as follows: Step 1, extracting total RNA from the developmental xylem of poplar hybrid populations and reversely transcribing it into cDNA for constructing a sequencing library for transcriptome sequencing; Step 2, determining phenotypic traits; Step 3, genotyping; Step 4: Use Matrix eQTL to perform eQTL analysis and hotspot analysis to screen key sites; Total RNA from the developing xylem of the hybrid population of Danhong Populus × Tongliao No. 1 Populus was extracted, and gene expression data were obtained by sequencing on the Illumina Hiseq platform.

2. The method for obtaining the SNP molecular markers related to the basic density of Danhong Poplar, Tongliao No. 1 Poplar and their hybrid progeny wood according to claim 1, characterized in that: The basic density of the wood in step 2 is determined by the water displacement method.

3. Application of the method for obtaining SNP molecular markers related to the basic density of Danhong Poplar, Tongliao No. 1 Poplar and their hybrid progeny as described in any one of claims 1-2 in the breeding of basic density of poplar wood, characterized in that: The poplars are Danhong Poplar, Tongliao No. 1 Poplar and their hybrid progeny.

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