AU2020102716A4 - Haplotype markers of KRT14 gene associated with eggshell quality traits and its application - Google Patents

Abstract

OF INSTRUCTION The invention provides the haplotype markers of KRT14 gene associated with eggshell quality traits and its application, the haplotype markers are located in the partial coding sequence of KRT14 gene in laying hens, and the nucleotide sequence of the haplotype markers is shown in SEQ ID NO.1. The haplotype markers comprise the T/C polymorphic site at 184 bp, the T/C polymorphic site at 215 bp, the A/C polymorphic site at 275 bp and the A/G polymorphic site at 277 bp. The invention also provides a detection kit for identifying the SNP, which comprise the pair of primers shown in SEQ ID NO.2 ~ 3. The invention firstly discovered the haplotype markers of KRT14 gene associated with eggshell quality traitsin laying hens, which provide a new molecular breeding marker for marker assisted selection breeding of eggshell quality traits. 1

Description

HAPLOTYPE MARKERS OF KRT14 GENE ASSOCIATED WITH EGGSHELL QUALITY TRAITS AND ITS APPLICATION

FIELD OF THE INVENTION The invention relates to the field of the screening of haplotype markers, and in particular to the haplotype markers of KRT14 gene associated with eggshell quality traits and its application.

BACKGROUND OF THE INVENTION Eggs are rich in protein, fat, lecithin and other nutrients that are necessary for human body. They are important part of the diet structure of Chinese residents. However, there are about 6%-10% of eggs lost due to breakage in the process of purchase, storage, transportation and processing, causing serious economic losses. Previous studies have shown that the egg loss is mainly caused by the eggshell damaged or cracked. Eggshell thickness, eggshell strength, eggshell percentage and yolk ratio are important detection indexes of eggshell quality, and also important factors affecting egg storage and transportation. The correlation coefficients of eggshell thickness, eggshell percentage, yolk ratio with eggshell strength were 0.79, 0.76 and 0.62, respectively. So the egg with greater eggshell strength have the greater the eggshell thickness, eggshell percentage and yolk ratio. With the continuous improvement of the living standards, the demand for eggs is also rising. It is estimated that by 2035, the per capita consumption of eggs will reach 363 per year in our country. Basically, each person has an egg a day. Therefore, how to improve the quality of eggshell, improve the thickness and strength of eggshell, and reduce the loss in the process of storage and transportation is particularly important. Single nucleotide polymorphism (SNP) refers to the polymorphism caused by single nucleotide (A, T, C or G) mutation in genomic DNA sequence, including single base insertion, deletion, transformation and transversion. Genome-Wide Association Study (GWAS) is the application of millions of SNPs in the genome as molecular genetic markers for comparative analysis and correlation analysis at the whole genome level. With the emergence of a large number of SNP markers, the single marker-centered association analysis has gradually changed into the correlation analysis based on haplotype. Haplotype refers to the closely linked SNPs that determine the same trait on the same chromosome or a certain region. SNP markers and their haplotypes play an important role in marker assisted selection (MAS) breeding due to their wide distribution and stability. It can be a feasible technology to accelerate the genetic improvement of eggshell quality traits, especially those sex-limited traits. Based on this, further identification of SNP markers and their haplotype combinations related to economic traits of laying hens, and using them as markers in the assisted selection of laying hens with excellent economic traits will greatly improve the accuracy of breeding selection. This is of great significance for development of laying hens with excellent economic traits. Keratin 14 (KTR]4) is a class I acidic keratin rich in cysteine residues and it forms a keratin pair with keratin 5, which can maintain cell morphology and integrity. It has been shown that KTR14 is closely related to intermediate fibroin, which is an epithelial marker of the occurrence and development of human cancer, and closely related to epidermolysis bullosa simplex and the severity of lung injury of human. However, the study of KRT14 gene in laying hens has not been reported. So far, there is no study on KRT14 gene as a molecular marker of eggshell quality traits. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

SUMMARY OF THE INVENTION The object of the present invention is to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. The invention relates to the haplotype markers of KRT14 gene associated with eggshell quality traits and its application. The invention explored haplotype markers associated with eggshell quality traits in KRT14 gene, and provides a new haplotype marker for marker assisted selection breeding of eggshell quality traits. In one aspect, the present invention provides haplotype markers of KRT14 gene associated with eggshell quality traits, wherein the haplotype markers are located in the partial coding sequence of KRT14 gene in laying hens, wherein the nucleotide sequence of the haplotype marker is shown in SEQ ID NO.1, and the haplotype markers comprise: SNP1 site: a T/C polymorphic site at 184 bp in the sequence shown in SEQ ID NO.1; SNP2 site: a T/C polymorphic site at 215 bp in the sequence shown in SEQ ID NO.1; SNP3 site: a A/C polymorphic site at 275 bp in the sequence shown in SEQ ID NO.1; SNP4 site: a A/G polymorphic site at 277 bp in the sequence shown in SEQ ID NO.1. In another aspect, the present invention provides a pair of primers comprising an upstream primer and a downstream primer for amplifying the haplotype markers of the present invention, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3. In another aspect, the present invention provides a kit for detecting SNPs in KRT14 gene associated with eggshell quality traits, wherein the kit comprises the pair of primers of the present invention. In another aspect, the present invention provides a method for detecting SNPs in KRT14 gene of laying hens associated with eggshell quality traits, wherein the method comprises detecting the SNPs with the pair of primers of the present invention or the kit of the present invention. In another aspect, the present invention provides a construction method of haplotype combination, wherein the method comprises: detecting four SNP sites by the method of the present invention, and then constructing nine haplotypes comprising HI: CCAG, H2: CCCG, H3: CTAA, H4: TCAG, H5: TCAA, H6: TCCG, H7: TTAG, H8: TTAA, H9:TTCG; then analyzing the correlation between haplotype combinations constructed by the nine haplotypes and eggshell quality traits, wherein eggshell strength and eggshell thickness of haplotypes combination H1H9 is highest, and followed by the haplotype combinations H1H6 and H1H8. In another aspect, the present invention provides haplotype combinations comprising the haplotype markers of the present invention, wherein the haplotype combinations comprise the four SNP sites, and the eggshell strength and eggshell thickness of haplotype combination CCAG/TTCG is highest. In another aspect, the present invention provides use of the haplotype markers of the present invention, or the kit of the present invention or the haplotype combinations of the present invention in marker assisted selection of eggshell quality traits. In another aspect, the present invention provides a method for screening high-quality eggshell, wherein the method comprises: detecting four SNP sites of the present invention and predicting the eggshell strength and eggshell thickness according to the haplotype combination, wherein the individual with the haplotype combination of H1H9 whose sequence is CCAG/TTCG has the highest eggshell strength and eggshell thickness. In another aspect, the present invention provides a haplotype marker of KRT14 gene associated with eggshell quality traits, wherein the haplotype markers are located in the partial coding sequence of KRT14 gene, and the nucleotide sequence length of the haplotype marker is 993 bp and it is shown in SEQ ID NO.1, and the haplotype markers comprise: SNP1 site: The T/C polymorphic site at 184 bp in the sequence shown in SEQ ID NO.1; SNP2 site: The T/C polymorphic site at 215 bp in the sequence shown in SEQ ID NO.1; SNP3 site: The A/C polymorphic site at 275 bp in the sequence shown in SEQ ID NO.1; SNP4 site: The A/G polymorphic site at 277 bp in the sequence shown in SEQ ID NO.1. In an embodiment, dominant alleles at 184 bp, 215 bp, 275 bp and 277 bp in the sequence shown in SEQ ID NO.1 are C, C, A and G, and dominant genotypes are CT,

CC, AA and GG, respectively. In a further embodiment, the eggshell quality traits comprises eggshell strength or eggshell thickness or their combination. In another aspect, the present invention provides a pair of primers for amplifying the haplotype marker, wherein the nucleotide sequence of upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of downstream primer is shown in SEQ ID NO.3. In an embodiment, the kit for detecting SNPs in KRT14 gene associated with eggshell quality traits comprises the primers defined above. In an embodiment, the kit also comprises 10xbuffer, dNTPs and Taq DNA polymerase or comprises PCR mix. In some examples, the present invention provides a method for detecting SNPs in KRT14 gene of laying hens associated with eggshell quality traits, wherein the method comprises detecting with the primers or the detection kit defined above. In an embodiment, the method comprises: Step 1: The genomic DNA extracted from the samples is amplified with the primers shown in SEQ ID NO.2-3, and the PCR products are purified; Step 2: The purified PCR products are detected by a genetic analyzer, and the results are analyzed by the gene analysis software. In some examples, the present invention provides a construction method of haplotype combination, and the method comprises: firstly, the four SNP sites are detected by the detection method described above, then, nine haplotypes are constructed comprising HI: CCAG, H2: CCCG, H3: CTAA, H4: TCAG, H5: TCAA, H6: TCCG, H7: TTAG, H8: TTAA, H9:TTCG; then the correlation between the haplotypes combinations constructed by the nine haplotypes and the eggshell quality phenotype are analyzed, wherein the eggshell strength and eggshell thickness of haplotypes combination H1H9 is highest, and followed by the haplotype combinations H1H6 and H1H8. In some examples, the present invention provides a haplotype combination, wherein the haplotype combinations comprises the four SNP sites, and the eggshell strength and eggshell thickness of haplotype combination CCAG/TTCG is highest, while the haplotype combinations CCAG/TCCG and CCAG/TTAA take second place. In some examples, the present invention provides the application of the haplotype marker, or the detection kit or the haplotype combinations in the marker assisted selection of eggshell quality traits. In some examples, the present invention provides a method for screening high-quality eggshell, wherein the method comprises: detecting the four SNP sites defined above and predicting the eggshell strength and eggshell thickness according to the haplotype combination, wherein the individual with the haplotype combination of HlH9 whose sequence is CCAG/TTCG has the highest eggshell strength and eggshell thickness. The invention explored haplotype markers associated with eggshell quality. The haplotype markers are located in the partial coding sequence of KRT14 gene in laying hens (shown in SEQ ID NO.1), and the length of the sequence is 993 bp. There were four allele mutations at 184 bp, 215 bp, 275 bp and 277 bp, which were T/C, T/C, A/C and A/G respectively, and the haplotype combinations composed of them could be the haplotype molecular markers associated with eggshell quality traits, which provides a new molecular breeding marker for marker assisted selection breeding of eggshell quality traits in laying hens.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: The technical route diagram of the invention; Figure 2: The PCR amplification product of KR T]4 gene fragment of laying hens detected by agarose gel, the M is Marker and 1-6 are the amplification result of KRT14 gene fragment; Figure 3: Comparison of sequencing results of four SNPs in KRT14 gene in laying hens with different genotypes, (A) is the T/C mutation site at 184bp; (B) is the T/C mutation site at 215bp; (C) is the A/C mutation site at 275bp; (D) is A/G mutation site at 277bp;

Figure 4: Haplotype block analysis result of KRT14 gene.

DETAILED DESCRIPTION OF THE INVENTION

Embodiment 1: Acquisition of partial coding region of KRT14 gene and establishment of the detection method of SNP 1. Extraction of genomic DNA from laying hens'blood The laying hen variety of the invention was Jianghan chicken, and the sample was collected from the poultry testing farm of institute of animal science and veterinary medicine, Hubei academy of agricultural sciences. The genomic DNA of laying hens was extracted by the Genomic DNA Kit produced by Beijing baiteke Biotechnology Co., Ltd according to the instructions, and the specific steps were as follows: (1) About 1 mL blood was drawn from the inferior vein of hen wing with disposable syringe, and then injected into a 1.5 mL centrifuge tube containing 200 pL sterile ACD anticoagulant which was treated by high pressure steam sterilization, then shaken gently, recorded numbers, and reserved at -20 °C. 10 pL blood with anticoagulant was taken, added 500 pL BB2 and 10 pL protease K with concentration of 20 mg/mL, homogeneously mixed, and incubated at room temperature for 10 min. (2) After a short time of centrifugation, added all the solution into the centrifugal column, centrifuged at 12000 g for 1 min, and discarded the effluent. (3) Added 500 pL CB3 solution, and centrifuged at 12000 g for 30 s, and discarded the effluent. (4) Added 500 pL WB3 solution, and centrifuged at 12000 g for 30 s, and discarded the effluent. (5) Repeated step (4). (6) Centrifuged at 12000 g for 2 min, and removed the residual WB3 completely. (7) Placed the column in a clean centrifuge tube, added 50-200 pL preheated

EB or demonized water in the center of the column, and placed it at room temperature for 1 min, centrifuged at 12000 g for 1 min, and then eluted DNA. The DNA was stored at - 20 °C. 2. Acquisition of partial nucleotide fragment of KRT14 gene from laying hens (1) PCR amplification Designed a pair of primers according to KRT14 gene sequence (Gene ID is NM_001001311.2), and the sequences of the primers are shown as follows (also shown in SEQ ID NO.2 and SEQ ID NO.3): Upstream primer: 5'- GCCGCAACTGTTGAGAAT -3' Downstream primer: 5'- GGGACTGCAGGTCGATCT -3' PCR amplification was carried out by the genomic DNA of Jianghan chicken with the above primers, and the PCR reaction system is shown in Table 1. The PCR reaction condition is shown in Table 2. Table 1 PCR reaction system

Composition Volume PCR mix 10 pL forward primer 0.5 pL reverse primer 0.5 pL genomic DNA 1.0 pL ddH 20 8.0 pL Total volume 20.0 pL

Table 2 PCR reaction condition 94 °C 2 min 1 cycle 94°C 15 s 55 °C 30s 35 cycle 68 °C 90s 69 °C 10 min 1 cycle

The PCR product was detected by agarose gel electrophoresis, and the result is shown in Fig 2. (2) Purification of PCR products The PCR product was purified by Gel Extraction Kit of Sangon Biotech (Shanghai) Co., Ltd, and the specific steps were as follows: Firstly, cut the gel containing target fragments from agarose gel and put it into 1.5 mL centrifuge tube, added 400 pL dissolved solution, heated the gel in a water bath at 50-60 °C until the gel is completely melted, and mix it every 2 minutes while heating, then cooled it to room temperature. Secondly, put a centrifuge column into the collecting tube and transferred the melted gel solution into the centrifuge column, and placed it at room temperature for 2 min, then centrifuged at 12000 r/min for 1 min, and the DNA was adsorbed on the column. Thirdly, discarded the effluent in the centrifuge tube and put the centrifuge column into the same centrifuge tube, added 700 pL eluent into the centrifuge column, and centrifuged at 12000 r/min for 1 min, then discarded the effluent in the centrifuge tube and centrifuged at 12000 r/min for 1 min. Finally, put the centrifuge column into a prepared sterilized 1.5 mL centrifuge tube, added 40 pL eluent or double distilled water (pH>7.0), placed at room temperature or 37 °C for 2-3 min; centrifuged at 12000 r/min for 1 min, and the liquid in the centrifuge tube was the target DNA fragment. 3. Detection of molecular markers by sequencing of PCR products The purified PCR product obtained above was directly sequenced at Beijing AuGCT Biotechnology Co., Ltd. and determined the genotypes of the site in the experimental samples according to the sequencing results. The results of BLAST comparative analysis by DNAStar software are shown in Fig.3. It was found that there were T/C, T/C, A/C and A/G allele mutations at 184 bp, 215 bp, 275 bp and 277 bp of the sequence, respectively. The above allele mutations caused the polymorphism of KRT14 gene, and forming 9 haplotypes, namely CCAG, CCCG, CTAA, TCAG, TCAA, TCCG, TTAG, TTAA and TTCG. The nucleotide sequences of the above four SNP markers are shown in SEQ ID NO.1.

Embodiment 2: Polymorphism detection in Jianghan chicken of the molecular marker of the invention The polymorphism of four sites in the coding region of KRT14 gene in laying hens was detected, and three genotypes were detected at each site. The genotype frequency and allele frequency are shown in Table 3 (the number in parentheses of Genotype column is the number of individuals of that genotype). Table 3 Genotype frequency and allele frequency in KRT14 gene of laying hens SNP sites Genotype Genotype frequency Allele frequency CC (160) 0.39 TT (78) 0.19 C=0.60 g.184bp C>T T=0.40 CT (176) 0.42 CC (256) 0.62 g.215bp C>T TT (15) 0.04 C=0.79 CT (143) 0.34 CC (30) 0.07 g.285bp C>A AA (198) 0.48 A=0.30

CA (186) 0.45 GG (297) 0.72 g.285bp G>A AA (5) 0.01 A=0.86 GA (112) 0.27

As shown in Table 3, three genotypes were detected in all the four mutation sites of the sequence. For the KRT14_SNP1 site to KRT14_SNP4 site, the dominant alleles were C, C, A and G, respectively, and the dominant genotypes were CT, CC, AA and GG respectively.

Embodiment 3: Correlation analysis between molecular markers of the invention and the eggshell quality traits and its application In order to determine the correlation between KRT14_SNP1 to KRT14_SNP4 markers and the difference of egg quality traits, we selected 414 Jianghan chicken, and the blood samples and their egg samples were collected from the poultry testing farm of institute of animal science and veterinary medicine, Hubei academy of agricultural sciences. The egg weight, eggshell strength, eggshell thickness and yolk weight of each egg were detected, and the polymorphism were detected by direct sequencing. The correlation between the different genotypes of partial coding region of KRT14 gene and eggshell quality traits was analyzed. SPSS18.0 software was used to analyze the correlation between genotype and phenotype, and the model used was as follows: Yij=u+Gi+Pj+eij Wherein, Yij was the trait observed value, u was the total average value of traits, Gi was the genotype effect, Pj was the fixed effect, and eij was the Random error. The correlation analysis results between different genotypes of four mutation sites and egg quality traits in Jianghan chicken are summarized in Table 4. Table 4 Correlation analysis results between polymorphism of four mutation sites of KRT14 gene and egg quality traits in laying hens

Egg weigh Eggshell Eggshell Yolk weight SNP sites Genotype (g) strength thickness (g) (kg/cm2 ) (mm) CC(n=160) 3 9 .4 4 ±2.3 1P 16.431.41l 0 .2 4 ±0. 01 b 10 . 5 6 ±0. 8 7b TT(n=78) 4 2 .9 9 2. 17 ab 1 6 .9 6 1. 2 2 ab 0 .2 6 0.0 1 ab 1 1 .9 6 0. 7 8 ab CT(n=176) 49.13±1.82a 21.27+1.17a 0.28±0.02a 14.09±0.67a CC(n=256) 42.68±2.19a 1 8 .2 3 1. 3 4 ab 0.26±0.01a 1 1 .7 3 0. 7 9 ab g.215bp TT(n=15) 34 .7 7 ±2.2 9b 1 6 .0 3 ±0. 7 9b 0.24±0.01l 9.92+0.78b C>T CT(n=143) 49.03+1.84a 20.38+1.13a 0.29+0.02a 14.01+0.66a CC(n=30) 51.43+3.32a 21.40+1.93a 0.30+0.02a 15.46+1.05a g.285bp AA(n=198) 4 1 .6 6 ±2.2 4 b 16.04±1.26b 0.25±0.01l 11.48±0.98b C>A CA(n=186) 4 5 .4 4 2. 13 ab 20.49±1.33a 0 .2 80.0 1 ab 12.61+0.75a GG(n=297) 53.78+3.61 18.84+1.31a 0.27±0.02a 12.10±1.07a g.285bp AA(n=5) 43.19+2.79 16.04±1.26b 0.19±0.01b 8.78±0.75b G>A GA(n=112) 55.29+2.52 17 .8 7 1. 19 ab 0.26±0.02a 12.89±0.75a Notes: the same letters in the above table indicate no significant difference, the letters a, b,c indicate significant difference, and n is the number of individuals of this genotype.

As shown in Table 4, the KRT14_SNP1 to KRT14_SNP4 site polymorphism was significantly correlated with egg weight, eggshell thickness, eggshell strength and yolk weight (P < 0.05), wherein the CT genotype of KRT14_SNP1 site, the CT genotype of KRT14_SNP2 site, the CC genotype of KRT14_SNP3 site, and the GG genotype of KRT14_SNP4 site had higher eggshell strength and eggshell thickness.

Embodiment 4: Identification of the haplotype combinations with excellent eggshell quality traits 1. Construction of haplotype and haplotype combination Haploview software was used for haplotype analysis of KRT14_SNP1 to KRT14_SNP4, and genotype data of KRT14_SNP1 to KRT14_SNP4 sites in all individuals were input into the PHASE program, then calculated the genotype of each individual and the degree of linkage disequilibrium between different sites. The results of haplotype block analysis are shown in Fig. 4. As shown in Fig. 4, according to the linkage disequilibrium analysis of KRT14_SNP1 to KRT14_SNP4 sites, a total of one haplotype block was found. Then the haplotype block was analyzed by haplotype analysis. Nine haplotypes were found in the Jianghan chicken, and are shown in Table 5. Table 5 SNP haplotypes in KRT14 gene Haplotype DNA sequence Quantity HI CCAG 408 H8 TTAA 106 H2 CCCG 92 H6 TCCG 74 H4 TCAG 72 H9 TTCG 62 H5 TCAA 10 H3 CTAA 2 H7 TTAG 2

The haplotype combinations consisting of the above haplotypes were analyzed and a total of 23 haplotype combinations were found (shown in Table 6). Table 6 Haplotype combinations in KRT14 gene Haplotype DNA Quantity Haplotype DNA Quantity combination sequence combination sequence

HIHI CCAG/CCAG 96 H2H9 CCCG/TTCG 8 H1H8 CCAG/TTAA 56 H2H2 CCCG/CCCG 6 H1H2 CCAG/CCCG 52 H2H8 CCCG/TTAA 4 H1H6 CCAG/TCCG 38 H4H4 TCAG/TCAG 4 H1H9 CCAG/TTCG 32 H4H9 TCAG/TTCG 4 H1H4 CCAG/TCAG 30 H1H3 CCAG/CTAA 4 H4H8 TCAG/TTAA 16 H1H7 CCAG/TTAG 2 H4H6 TCAG/TCCG 14 H5H8 TCAA/TTAA 2 H6H8 TCCG/TTAA 12 H6H9 TCCG/TTCG 2 H8H9 TTAA/TTCG 12 H8H8 TTAA/TTAA 2 H2H5 CCCG/TCAA 8 H9H9 TTCG/TTCG 2 H2H6 CCCG/TCCG 8

Some haplotype combinations with small quantity were excluded, and the six haplotypes with larger quantity were selected for correlation analysis. 2. Correlation analysis between haplotype combinations and eggshell quality traits SPSS18.0 software was used to analyze the correlation between haplotype combinations and eggshell quality traits, and the result is shown in Table 7. Table 7 The correlation analysis result between dominant haplotype combinations and eggshell quality traits

Haplotype Egg weight Eggshell Eggshell Yolk weight combination rate (g) strength thickness (g) (kg/cm2 ) (mm) HIHI 0.34 38.09+3.56 15.19±1.40b 0.24±0.0 10.1+0.85 H1H8 0.16 47.28+2.00 17.68 .09b 0. 2 7 ±0. 0 2b 13.29+0.72 H1H2 0.18 39.47+3.81 17 . 16 ±1. 4 4b 0 .2 5 ±0. 01 b 10.51+0.86 H1H6 0.14 48.87+3.72 2 2 .7 5 1. 2 5 ab 0 .3 0 0. 0 2 ab 13.99+0.70 H1H9 0.11 55.44+3.94 26.02±1.09a 0.32±0.2a 15.85+0.51 H1H4 0.07 38.40+3.66 14.641.20b 0 .2 3 ±0. 01 b 10.74+0.91 Notes: the same letters in the above table indicate no significant difference, the letters a, b indicate

significant difference, and n is the number of individuals of this genotype.

As shown in Table 7, the eggshell strength and eggshell thickness of haplotype combination H1H9 whose sequence was CCAG/TTCG were significantly higher than those of other haplotype combinations (P<0.05). In conclusion, the haplotype combination H1H9 whose sequence was CCAG/TTCG has the best eggshell quality traits in the experimental samples of this embodiment. Throughout the description and claims of the specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps. The above embodiments are only preferred embodiments of the invention and do not limit the invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the invention shall be included in the protection scope of the invention.

Claims (10)

1. Haplotype markers of KRT14 gene associated with eggshell

quality traits, wherein the haplotype markers are located in the partial

coding sequence of KRT14 gene in laying hens, wherein the nucleotide

sequence of the haplotype marker is shown in SEQ ID NO.1, and the

haplotype markers comprise:

SNP1 site: a T/C polymorphic site at 184 bp in the sequence shown

in SEQ ID NO.1;

SNP2 site: a T/C polymorphic site at 215 bp in the sequence shown

in SEQ ID NO.1;

SNP3 site: a A/C polymorphic site at 275 bp in the sequence shown

in SEQ ID NO.1;

SNP4 site: a A/G polymorphic site at 277 bp in the sequence shown

in SEQ ID NO.1.

2. The haplotype markers according to claim 1, wherein dominant

alleles at 184 bp, 215 bp, 275 bp and 277 bp in the sequence shown in

SEQ ID NO.1 are C, C, A and G respectively, and dominant genotypes

are CT, CC, AA and GG respectively.

3. A pair of primers comprising an upstream primer and a

downstream primer for amplifying the haplotype markers defined in

claim 1 or claim 2, wherein the nucleotide sequence of the upstream

primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

4. A kit for detecting SNPs in KRT14 gene associated with eggshell

quality traits, wherein the kit comprises the pair of primers defined in

claim 3.

5. The kit according to claim 4, wherein the kit also comprises

xbuffer, dNTPs, and Taq DNA polymerase or the kit also comprises

PCR mix.

6. A method for detecting SNPs in KRT14 gene of laying hens

associated with eggshell quality traits, wherein the method comprises

detecting the SNPs with the pair of primers defined in claim 3 or the kit

defined in claim 4 or claim 5.

7. A construction method of haplotype combination, wherein the

method comprises: detecting four SNP sites by the method defined in

claim 6, and then constructing nine haplotypes comprising HI: CCAG,

H2: CCCG, H3: CTAA, H4: TCAG, H5: TCAA, H6: TCCG, H7: TTAG,

H8: TTAA, H9: TTCG; then analyzing the correlation between haplotype

combinations constructed by the nine haplotypes and an eggshell quality

trait, wherein eggshell strength and eggshell thickness of haplotypes

combination H1H9 is highest, and followed by the haplotype

combinations H1H6 and H1H8.

8. Haplotype combinations comprising the haplotype markers

defined in claim 1, wherein the haplotype combinations comprise the four

SNP sites, and the eggshell strength and eggshell thickness of haplotype

combination CCAG/TTCG is highest.

9. Use of the haplotype markers defined in claim 1 or claim 2, or the

kit defined in claim 4 or the haplotype combinations defined in claim 8 in

marker assisted selection of eggshell quality traits.

10. A method for screening high-quality eggshell, wherein the

method comprises: detecting the four SNP sites defined in claim 1 and

predicting the eggshell strength and eggshell thickness according to the

haplotype combination, wherein the individual with the haplotype

combination of H1H9 whose sequence is CCAG/TTCG has the highest

eggshell strength and eggshell thickness.