EGG-TYPE CHICKEN WHOLE-GENOME SNP CHIP AND USE THEREOF

Abstract

Provided are an egg-type chicken whole-genome SNP chip and the use thereof. The SNP loci of the chip are respectively derived from 14,624 SNP loci shared by each line of the major egg-type chickens in China; 3,677 SNP loci associated with disease-resistant traits in egg-type chickens; 16,000 SNP loci associated with economic traits in egg-type chickens; and 9,358 SNP loci making up genomic regions not covered by the foregoing probes. The 43,681 SNPs on the egg-type chicken whole-genome SNP chip have DNA sequences as represented by SEQ ID NO. 1-43,681. The chip can specifically identify the genetic relationship between commercial egg-type chickens and egg-type chickens of local breeds, and can also perform applications such as whole-genome association analysis, genome selective breeding, and QTL location analysis of target traits and population genetic analysis.

Claims

1. (canceled)

2. An egg-type chicken chicken whole-genome SNP chip, wherein, the chip comprises 43,681 SNP molecular markers having the nucleotide sequences represented by SEQ ID NOs. 1 to 43681.

3-10. (canceled)

11. The egg-type chicken whole-genome SNP chip according to claim 2, wherein the SNP loci of the molecular markers is located at the 61.sup.st position of the nucleotide sequences represented by SEQ ID NOs. 1 to 43681.

12. The egg-type chicken whole-genome SNP chip according to claim 2, wherein the molecular markers are associated with disease-resistant traits in egg-type chickens, or associated with economic traits in egg-type chickens; and the economic traits in egg-type chickens include laying performance, egg quality, feed reward, and immunity.

13. A method for genomic selection breeding for egg-type chicken, wherein the method comprises: using the egg-type chicken whole-genome SNP chip according to claim 2.

14. A method of QTL mapping for target traits of egg-type chicken, wherein the method comprises: using the egg-type chicken whole-genome SNP chip according to claim 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows the distribution in the chicken genome of the egg-type chicken whole-genome SNP chip in the present application. The abscissae represent 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 Mb, respectively, and the ordinates represent chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr20, chr21, chr22, chr23, chr24, chr25, chr26, chr27, chr28, chr33, chrLGE64 and chrZ from bottom to top.

[0022] FIG. 2 shows the proportion of all SNP loci on each chromosome of the whole-genome in the present application.

[0023] FIG. 3 shows the principal component analysis diagram of each chicken breed. The numbers in the figure represent 10 breeds, respectively. 1 represents Beijing You chicken, 2 represents White Leghorn, 3 represents Houdan, 4 represents Dongxiang blue-egg shell egg-type chicken, 5 represents Silkie, 6 represents Yellow broiler, 7 represents Rhode Island white, 8 represents Tibetan chicken, 9 represents Dwarf chicken, and 10 represents Rhode Island Red.

[0024] FIG. 4 shows the cluster analysis diagram of each chicken breed. The large branches represent that each breed can be distinguished independently, and the small branches represent each individual.

[0025] FIG. 5 shows the Manhattan graph of the egg weight of 36-week-old Rhode Island Red.

SPECIFIC MODES FOR CARRYING OUT THE EMBODIMENTS

[0026] The following Examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or replacements made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

[0027] Unless otherwise specified, the technical means used in the Examples are conventional means well known to a person skilled in the art.

Example 1: Preparation Method of Egg-Type Chicken Whole-Genome SNP Chip

[0028] The first type of probes of the present invention were obtained as follows: commercial egg-type chicken breeds independently bred in China and Chinese local egg-type chicken breeds were subjected to the whole-genome resequencing, wherein, the egg-type chickens include 7 lines (Line 1 derived from Rhode Island Red, Line 2 and 3 derived from Rhode Island White, Line 4 and Line 5 derived from White Leghorn, Line 6 derived from Houdan, and Line 7 derived from Dongxiang blue-egg shell chicken), totally 479 individuals (Line 1: 92, Line 2: 74, Line 3: 91, Line 4: 60, Line 5: 69, Line 6: 68, and Line 7: 25), the SNP loci of each breed with MAF<0.05 were eliminated, and at the same time the SNPs shared by each line and the SNP loci shared by Line 1 and at least one other lines were screened out, which guarantees the polymorphism and versatility of SNP loci to a large extent. Finally, 64,396 SNPs shared by all lines and 3,782,699 SNP loci shared by Line 1 and any other lines were used as the core candidate SNPs of the chip.

[0029] The second type of probes of the present invention were obtained as follows: based on the applicant's earlier research on disease-resistant traits of chickens, using the Marek's disease-resistant inbred White Leghorn population established by the Poultry Disease and Tumor Laboratory of the United States Department of Agriculture (wherein, there are three individuals each for the disease-resistant line and the susceptible line). Through whole-genome resequencing technology, the genetic variations existing in each line is unearthed, and a total of 1,554,188 specific SNP loci of the lines related to disease-resistant traits were selected, and then subjected to overlapping with the SNPs with good polymorphism obtained by resequencing of 479 individuals in the 7 lines described in the previous step of preparation of the first type of probes, which greatly guarantees the applicability of these SNP loci in other egg-type chicken breeds. Finally, 480,341 disease-resistant specific SNPs were selected.

[0030] The third type of probes of the present invention were obtained as follows: this type of probes were obtained by screening based on the applicant's previous genome-wide association analysis of important economic traits in egg-type chickens. There are two parts: the first part comprises performing association analysis on laying performance (number of eggs laid and egg weight) and egg quality using 385 White Leghorns and 361 Dwarf egg-type chickens from 40 families; and the second part comprises performing whole-genome association analysis on laying performance, feed utilization efficiency, egg quality and reproductive performance of 1,512 hens which are F2 resource population obtained by the reciprocal cross between White Leghorn and Dongxiang blue-egg shell egg-type chicken. The above obtained SNP loci associated with important economic traits in egg-type chickens were analyzed and deduplicated by biostatistics methods, and 2,677 functional loci were obtained and added to the chip.

[0031] The fourth type of probes of the present invention were obtained as follows: QTLs related to egg quality, laying performance, feed efficiency and immunity of the chickens were obtained by searching for related candidate genes in published literature and online QTL database (https://www.animalgenome.org/cgi-bin/QTLdb/GG/index), QTLs with large confidence intervals and insignificant P values (P>0.05) were eliminated, and 1,238 candidate QTL intervals were finally obtained, then SNPs obtained from resequencing data were subjected to overlapping with candidate genes and QTLs, so as to screen out 589,164 SNPs as candidate loci related to traits.

[0032] The fifth type of probes of the present invention were obtained by downloading from the chicken SNP database of NCBI (https://www.ncbi.nlm.nih.gov/snp).

[0033] Identification process of SNP loci: considering that the sizes of different chromosome fragments in chickens are quite different, and the recombination rates of mutation loci on different chromosomes are inconsistent, the level of linkage disequilibrium on each chromosome was firstly detected, so as to set the optimal window/interval size for each chromosome, these windows/intervals were used to make SNPs evenly distributed on each chromosome, and one SNP shared by each line of the first type of probe was preferentially selected for each interval. If there was less than one, the SNP of the second type of probe was selected. The third type of probes were SNPs that were forced to be placed in the chip. If the window cannot be covered after the first type of probes and the second type of probes were selected, the SNP of the fourth type of probes was selected, and the fifth type of probes were used to supplement the interval that cannot be covered by the above-mentioned types of probes.

[0034] The identified SNP loci were submitted to Illumina for scoring with the Infinium XT scoring system (http://www.illumina.com/), and all loci with scores less than 0.8 were removed. For the deleted unqualified SNP loci, the nearest SNP loci were selected for supplementation, and the scoring was performed again. According to the above steps for identification and screening, a total of 50,000 tag-SNP loci were obtained. Since there is a problem of successful conversion rate when making a chip with these 50,000 SNP loci by the Illumina Infinium XT platform, thus, the number of effective SNP loci on the final egg-type chicken whole-genome 50K SNP chip is 43,681 (see FIG. 1 and FIG. 2).

TABLE-US-00001 TABLE 1 The distribution number of loci of the egg-type chicken whole- genome SNP chip of the present invention on each chromosome Chromosome length (bp) number chr1 196,202,544 6,916 chr2 149,560,735 5,111 chr3 111,302,122 3,907 chr4 91,282,656 3,419 chr5 59,825,302 2,287 chr6 35,467,016 1,604 chr7 36,946,936 1,722 chr8 29,963,013 1,248 chr9 24,091,566 1,117 chr10 20,435,342 1,227 chr11 20,218,793 1,148 chr12 19,948,154 1,213 chr13 18,407,460 973 chr14 15,595,052 861 chr15 12,762,846 994 chr16 652,338 47 chr17 10,956,400 718 chr18 11,053,727 799 chr19 9,979,828 735 chr20 14,409,371 915 chr21 6,862,722 513 chr22 4,729,743 300 chr23 5,786,528 313 chr24 6,280,547 439 chr25 2,906,300 131 chr26 5,313,770 654 chr27 5,655,794 441 chr28 4,974,273 524 chr32 78,254 13 chr33 1,648,031 201 chrLGE64 897,576 109 chrZ 82,310,166 3,082 Total 43,681

Example 2: Principal Component and Cluster Analysis Based on the Egg-Type Chicken Whole-Genome SNP Chip of the Present Invention

[0035] DNA genotypes of 20 Rhode Island Red, 20 Rhode Island white, 19 Beijing You chickens, 19 White Leghorn, 19 Houdan, 20 Dongxiang blue-egg shell egg-type chickens, 20 Silkie, 8 Yellow broilers, 20 Tibetan chickens, and 20 Dwarf chickens were detected by using the the whole-genome of egg-type chicken 50K SNP chip provided by the present invention. After the SNP loci with MAF less than 0.01 (641 SNP loci) were firstly eliminated, principal component and cluster analysis were performed with PLINK and SNPhylo software, respectively. The results of principal component analysis and clustering evolutionary tree analysis were shown in FIGS. 3 and 4. All breeds can be distinguished separately, which also illustrates the applicability of the chip of the present invention in different breeds. Among them, the local breeds of Beijing You chickens, Dongxiang and Silkie are clustered into one group, the abroad commercial breeds of Rhode Island White, White Leghorn and Houdan are clustered into one group, and in particular, Rhode Island Red chickens are individually clustered into one group with a genetic distance far from other breeds. This is also because Rhode Island Red has undergone high-intensity artificial selection for multiple generations, and the genetic structure of the genome and nucleic acid polymorphisms have changed. The above-mentioned principal component and cluster analysis results can well prove the applicability of the chip, and the identification results are accurate and reliable.

Example 3: Application of the Whole-Genome SNP Chip of the Present Invention in Genome-Wide Association Analysis

[0036] 680 Rhode Island Red hens provided by Beijing Huaduyukou Poultry Co., Ltd were subjected to genotype detection using the egg-type chicken whole-genome SNP chip provided by the present invention, at the same time, the egg weight phenotype data of these hens at 36 weeks of age (EW36) were collected, and the quality control standards for SNP genotyping were as follows: sample Call rate>95%, MAF>0.01, and Hardy-Weinberg equilibrium test P value is less than 1e-06. After quality control, the remaining 673 individuals and 38,627 SNPs were used for the following genome-wide association (GWAS) analysis. Linear Mixture Model was used as the analyzing model, the P values of 8.1e-06 and 1.62e-04 in number of independent test of SNP were respectively used as the thresholds for genome-wide significance and potential genome-wide significance. The Manhattan graph of the GWAS results was shown in FIG. 5. The results show that there is a significant region associated with egg weight on chromosome 1, which is also consistent with the results reported in previous studies (Liu et al., 2018, Frontiers in Genetics).

Example 4: Use of the Egg-Type Chicken Whole-Genome SNP Chip of the Present Invention in Genomic Selection—Genetic Evaluation

[0037] The egg-type chicken whole-genome SNP chip provided by the present invention was used for genetic evaluation of the genome. The DNA of 2,950 egg-type chickens (285 roosters and 2,665 hens) of three generations of a certain line provided by Beijing Huaduyukou Poultry Industry Co., Ltd were subjected to SNP genotyping. At the same time, genetic evaluation of the body weight at 28 weeks of age (BW28), the egg weight of hens at 28 weeks of age (EW28), and the number of eggs laid at 38 weeks of age (EN38) of these individuals were performed using both pedigree-based selection methods and genome-based selection methods. The specific steps were as follows:

[0038] (1) The typing test results of 2,950 chickens were subjected to quality control, the sample Call rate>95%, MAF>0.01, and Hardy-Weinberg equilibrium test P value is less than 1e-06. After quality control, the remaining 2,950 individuals and 38,413 qualified SNPs were used for genome-wide selection analysis.

[0039] (2) Model: BW28=Date+Hatch+Sex+Animal+Error [0040] EW28/EN38=Date+Hatch+Animal+Error [0041] Date, Hatch, and Sex are the fixed effects of individual birth date, hatching batch, and gender, respectively; [0042] Animal is the random additive genetic effect of an individual animal, that is, individual's breeding value. [0043] Error is random residual.

[0044] (3) Genetic evaluations were performed by pedigree-based selection method (PBLUP model) and genome-based selection method (ssGBLUP: one-step genomic model).

[0045] PBLUP model: the conventional breeding value is estimated by a single-trait animal model:

y=Xb+Zu+e

[0046] wherein y is a vector of phenotypic value, X is a design matrix for fixed-effect, b is a vector of fixed-effect including generation (birth year), hatching batch and gender, Z is a correlation matrix for random effect, u is a vector of breeding value and assumed to follow u˜N(0,Aσ.sub.u.sup.2), wherein A is a matrix for molecular blood relationship, σ.sub.u.sup.2 is an additive genetic variance, e is a vector of residual and assumed to follow e˜N(0,Iσ.sub.e.sup.2), wherein I is a identity matrix, and σ.sub.e.sup.2 is a residual variance.

[0047] ssGBLUP model: the ssGBLUP model is similar in form to the PBLUP model. The difference lies in that: the vector of random effect in the ssGBLUP model follows u˜N(0,Hσ.sub.u.sup.2), wherein H is a matrix that integrates pedigree information and genomic information (Christensen and Lund, 2010; Legarra, et al., 2009). The H matrix is constructed as follows:

[00001]$H=\left(\begin{array}{cc}{H}_{11}& H_{12}\\ H_{21}& H_{22}\end{array}\right)=\left(\begin{array}{cc}{A}_{11}+A_{22}+A_{22}^{-1}\left(G-A_{22}\right)A_{22}^{-1}{A}_{21}& A_{12}{A}_{22}^{-1}G\\ {\mathrm{GA}}_{22}^{-1}{A}_{11}& G\end{array}\right)$

[0048] The subscripts 1 and 2 represent individuals without genotypes and individuals with genotypes, respectively. The inverse matrix of the H matrix is quite simple in form (Aguilar, et al., 2010):

[00002]$H^{-1}=A^{-1}+\left[\begin{array}{cc}0& 0\\ 0& G^{-1}-A_{22}^{-1}\end{array}\right]$

[0049] (4) Results:

TABLE-US-00002 TABLE 2 Comparison of accuracy of different genetic evaluation methods Traits PBLUP SSGBLUP Weight at 28 weeks of age 0.19 0.29 Egg weight at 28 weeks of age 0.17 0.30 Number of eggs laid at 38 weeks of age 0.22 0.38

[0050] (5) It can be seen that, compared with traditional selection methods, the use of the chip of the present invention for genomic selection can effectively improve the accuracy of genetic evaluation.

INDUSTRIAL APPLICABILITY

[0051] The SNP loci on the egg-type chicken whole-genome SNP chip provided by the present invention are respectively derived from 14,624 SNP loci shared by various line of major egg-type chicken breeds in China; 3,677 SNP loci related to disease-resistant traits in egg-type chickens; 16,000 SNP loci associated with economic traits of egg-type chickens; and 9,358 SNP loci for making up the genomic region that are not covered by the aforementioned probes. The 43,681 SNPs on the egg-type chicken whole-genome SNP chip of the present invention have DNA sequences as represented by SEQ ID NOs. 1 to 43681. The chip can specifically identify the genetic relationship between commercial egg-type chickens and local egg-type chicken breeds, and can also be used to perform applications such as genome-wide association analysis, genomic selection, QTL mapping for target traits, and population genetic analysis. The chip has versatility for domestic and abroad chicken breeds, can help accelerate the rapid development of the egg-type chicken industry, and has great economic and practical value and scientific research value.