MOLECULAR MARKER FOR GENETIC RESISTANCE OF CHICKEN TO INFECTION BY SUBGROUPS A AND K AVIAN LEUKOSIS VIRUS AND USE THEREOF

Abstract

Disclosed is a molecular marker for genetic resistance of chicken to infection by subgroups A and K avian leukosis virus (ALV-A and ALV-K) and use thereof the molecular marker is tva gene with base deletion between 318-323 and/or between 602-607; specifically, bases ACCTCC at positions 318-323 and bases CCGCTG at positions 602-607 are deleted. In the present disclosure, genetic variation of tva receptor gene in Chinese chicken breeds is analyzed, and it is found that the DNA sequence of tva receptor gene in the Chinese chicken breeds has base deletion at positions 318-323 or at positions 602-607. Moreover, a method for breeding of the chicken breeds resistance to ALV-A and ALV-K has been established.

Claims

1. A molecular marker for genetic resistance of chicken to infection by subgroups A and K avian leukosis virus (ALV-A and ALV-K), wherein the molecular marker is tva gene with base deletion between 318-323 and/or between 602-607; wherein, bases ACCTCC at positions 318-323 and bases CCGCTG at positions 602-607 are deleted; and a GenBank accession number of tva gene is AY531262.1.

2. The molecular marker for genetic resistance of chicken to infection by ALV-A and ALV-K according to claim 1, wherein the molecular marker is a DNA sequence of tva gene with base deletion of ACCTCC at positions 318-323.

3. The molecular marker for genetic resistance of chicken to infection by ALV-A and ALV-K according to claim 1, wherein the molecular marker is a DNA sequence of tva gene with base deletion of CCGCTG at positions 602-607.

4-10. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 shows PCR amplification results of three fragments of tva gene; where M indicates DL2000 marker; 1 to 3 indicate PCR amplified products of primers 1, 2, and 3, respectively;

[0032] FIG. 2 is a sequencing map showing different genotype sequences of the tva.sup.318-323delACCTCC site, including the wild type (SEQ ID NO:16), heterozygous mutant (SEQ ID NO:17) and the homozygous mutant (SEQ ID NO:18) sequences;

[0033] FIG. 3 is a sequencing map showing different genotype sequences of the tva.sup.602-607delCCGCTG site, including the wild type (SEQ ID NO:13), heterozygous mutant (SEQ ID NO:14) and the homozygous mutant (SEQ ID NO:15) sequences;

[0034] FIG. 4A-FIG. 4C show a schematic diagram of construction of RCASBP(A)-EGFP and RCASBP(K)-EGFP expression plasmids and their rescue of fluorescent reporter virus; where FIG. 4A indicates a schematic diagram of the construction of RCASBP(A)-EGFP and RCASBP(K)-EGFP expression plasmids; FIG. 4B indicates enzyme digestion identification of the RCASBP(A)-EGFP and RCASBP(K)-EGFP plasmids; and FIG. 4C indicates the rescue of RCASBP(A)-EGFP and RCASBP(K)-EGFP virus;

[0035] FIG. 5 shows a process of RCASBP(A)-EGFP virus infecting CEF cells with different genotypes at the tva.sup.318-323delACCTCC site;

[0036] FIG. 6 shows a process of RCASBP(K)-EGFP virus infecting CEF cells with different genotypes at the tva.sup.318-323delACCTCC site;

[0037] FIG. 7 shows a situation of RCASBP(A)-EGFP virus infecting CEFs with different genotypes at the tva.sup.602-607delCCGCTG mutation site; and

[0038] FIG. 8 shows a situation of RCASBP(K)-GFP virus infecting CEFs with different genotypes at the tva.sup.602-607delCCGCTG mutation site.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] The specific embodiments of the present disclosure are described below to facilitate those skilled in the art to understand the technical scheme of the present disclosure, but it should be clear that the present disclosure is not limited to the scope of the specific embodiments. Various obvious changes made by those of ordinary skill in the art within the spirit and scope of the present disclosure defined by the attached claims should fall within the protection scope of the present disclosure.

Example 1 Screening of Tva.SUP.318-323delACCTCC .Molecular Marker

[0040] 1. Primers Design for PCR Amplification of Tva Receptor Gene

[0041] Referring to the DNA sequence of chicken tva gene in NCBI database (with a GenBank accession number of AY531262.1), 3 pairs of primers were designed to amplify the full-length sequence of tva gene (3607 bp) by PCR in three fragments (fragments 1, 2 and 3), primer sequences, positions, and sizes of PCR amplified fragments were shown in Table 1.

TABLE-US-00001 TABLE 1 PCR amplification information of full-length sequence of tva receptor gene tva gene Target Primer sequence fragment Fragment name Primer sequence (5′-3′) position size (bp) Fragment P1-F GTTCAGCAGATCCTCAT 17-39 1308 SEQ ID NO: 7 1 CTCCCG P1-R GGCCATTGTGCGATCTA 1302-1324 SEQ ID NO: 8 AGAGGG Fragment P2-F AGCCCTCTTAGATCGCA 1300-1319 1253 SEQ ID NO: 9 2 CAA P2-R GTGACACCGAGCACAA 2533-2552 SEQ ID NO: 10 AATG Fragment P3-F GTTGGAGCTGGATGAG 2464-2483 1132 SEQ ID NO: 11 3 CACT P3-R TGAGGGAATTCCTGTCA 3576-3595 SEQ ID NO: 12 CCT

[0042] 2. PCR Amplification of Tva Receptor Gene

[0043] (1) Genomic DNAs of 6570 blood samples were extracted from different Chinese chicken breeds (including 28 local chicken breeds and 57 yellow feathered broiler lines), and the full-length sequence of tva gene was amplified by PCR with the 3 pairs of primers.

[0044] The PCR amplification system included: 1 μL of a DNA template, 2.5 μL of a 10× buffer, 2 μL of dNTPs, 1 μL of each of upstream and downstream primers, 0.5 μL of KOD-FX, and supplementing to 25 μL with ddH.sub.2O.

[0045] The PCR amplification program included: initial denaturation at 94° C. for 3 min; 35 cycles of denaturation at 94° C. for 30 sec, annealing (fragment 1 at 62° C., fragments 2 and 3 at 60° C.) for 30 sec, and extension at 72° C. for 90 sec; then post extension at 72° C. for 10 min, and storage at 4° C.

[0046] (2) PCR products were detected by 2% agarose gel electrophoresis, and the results were shown in FIG. 1; where M: DL2000 marker; 1 to 3: PCR amplified products of primers 1, 2, and 3. As shown in FIG. 1, target bands of the fragments 1, 2 and 3 of tva gene were amplified by PCR, with fragment sizes consistent with expected results.

[0047] (3) The PCR amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for purification and sequencing, sequence comparison was conducted by DNAstar and Mutation Surveyor gene sequence analysis software, genetic variation of tva receptor gene in Chinese chicken breeds was analyzed, and candidate genetic resistance loci were screened for ALV-A and ALV-K.

[0048] By analyzing the genetic variation of tva receptor gene from 28 local chicken breeds and 57 yellow feathered broiler lines (a total of 6570 blood samples), a natural mutation of ACCTCC base sequence deletion (tva.sup.318-323delACCTCC) at positions 318-323 of tva receptor gene sequence of the Chinese chicken breeds were selected and found. The sequence sequencing map was shown in FIG. 2 (in FIG. 2, sequences from top to bottom were reference sequence (wild-type individual), sequence of heterozygous mutant individual, and sequence of homozygous mutant individual successively, in which the box showed the ACCTCC deletion mutation at positions 318 to 323 of tva gene sequence).

Example 2 Screening of Tva.SUP.602-607delCCGCTG .Molecular Marker

[0049] The screening process was the same as that of Example 1. The PCR amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for purification and sequencing, sequence comparison was conducted by DNAstar and Mutation Surveyor gene sequence analysis software, genetic variation of tva receptor gene in Chinese chicken breeds was analyzed, and candidate genetic resistance loci were screened for ALV-A and ALV-K, as shown in FIG. 3. In FIG. 3, sequences from top to bottom were reference sequence (wild-type individual), sequence of heterozygous mutant individuals, and sequence of homozygous mutant individuals successively, in which the box showed the CCGCTG deletion mutation at positions 602 to 607 of tva gene sequence.

[0050] As shown in FIG. 3, by analyzing the genetic variation of tva receptor gene from 28 local chicken breeds and 57 yellow feathered broiler lines (a total of 6570 blood samples), natural mutation of CCGCTG base sequence deletion (tva.sup.602-607delCCGCTG) at positions 602-607 of tva receptor gene sequence of the Chinese chicken breeds were selected and found.

Example 3 Effects of Tva.SUP.318-323delACCTCC .Mutation on Host Resistance

[0051] 1. In Vitro Cell Experiments

[0052] (1) RCASBP(A)-EGFP and RCASBP(K)-EGFP expression plasmids were constructed and transfected into DF-1 cells; 7 days after the transfection, RCASBP(A)-EGFP and RCASBP(K)-EGFP viruses (namely ALV-A and ALV-K reporter viruses carrying EGFPs) were rescued and collected from a supernatant of the DF-1 cells (FIG. 4A-FIG. 4C).

[0053] (2) The fluorescent reporter viruses RCASBP(A)-EGFP and RCASBP(K)-EGFP of ALV-A and ALV-K were used to infect chicken embryo fibroblasts (CEFs) at the tva.sup.318-323delACCTCC mutation sites of the wild-type tva.sup.s/s, the heterozygous mutant tva.sup.s/delACCTCC, and the homozygous mutant tva.sup.delACCTCC/delACCTCC separately (where the CEFs were prepared from 9-day-old chicken embryos hatched from the breeders tested in Example 1); 1 day, 2 days, 4 days, and 7 days after the infection, the CEFs with different genotypes of tva.sup.318-323delACCTCC mutation sites infected by the RCASBP(A)-EGFP and RCASBP(K)-EGFP viruses were detected using flow cytometry; a GPF-positive cell rate (%) represented an infection rate of the virus, and results were shown in FIG. 5 and FIG. 6.

[0054] As shown in FIG. 5 and FIG. 6, the CEFs of wild-type tva.sup.s/s and heterozygous mutant tva.sup.s/delACCTCC were susceptible to the RCASBP(A)-EGFP and RCASBP(K)-EGFP viruses; while the CEFs of homozygous mutant tva.sup.delACCTCC/delACCTCC were resistant to the RCASBP(A)-EGFP and RCASBP(K)-EGFP viruses. This indicated that the natural mutation of tva.sup.318-323delACCTCC led to the host resistance to ALV-A, ALV-K infection.

[0055] 2. In Vivo Experiments

[0056] (1) 1-day-old chicks with tva.sup.318-323delACCTCC of the mutant wild type, heterozygous mutant, and homozygous mutant were randomly divided into groups, reared in isolators, and injected intraperitoneally with equal amounts of ALV-A (GD08 strain) and ALV-K (GDFX0601 strain) separately at 1-day-old and 5-day-old. One month after challenge, blood samples were collected from the chicks and a total RNA was extracted from each blood sample using a TRIZOL kit.

[0057] The upstream and downstream primers were designed for RT-PCR amplification of ALV-A-env:

TABLE-US-00002 env-F: (SEQ ID NO: 3) 5′-GGATGAGGTGACTAAGAAAG-3′; env-R: (SEQ ID NO: 4) 5′-AGAGAAAGAGGGGTGTCTAAGGAGA-3′.

[0058] (2) The encoding sequence of env gene of ALV-A was amplified by RT-PCR, and the amplified fragment by RT-PCR had a length of 692 bp. RT-PCR amplification was conducted by the PrimeScript® One Step RT-PCR Kit Ver. 2, and the PCR reaction program included: reverse transcription at 50° C. for 30 min; at 94° C. for 30 sec, at 56° C. for 30 sec, and at 72° C. for 60 sec, conducting 35 cycles; and post extension at 72° C. for 10 min. PCR products were detected by 2% agarose gel electrophoresis; if a 692 bp target band was observed, the sample was infected with viremia (ALV-A positive); if there was no amplified target band, the sample was not infected with viremia (ALV-A negative), as shown in Table 4.

[0059] (3) The upstream and downstream primers were designed for RT-PCR amplification of ALV-K-env:

TABLE-US-00003 env-F: (SEQ ID NO: 5) 5′- GCACCACCTTGGGAACTGACC-3′; env-R: (SEQ ID NO: 6) 5′-GGCGTGGATCGACAGCACAC-3′.

[0060] The encoding sequence of env gene of ALV-K was amplified by RT-PCR, and the amplified fragment by RT-PCR had a length of 633 bp. RT-PCR amplification was conducted by the PrimeScript® One Step RT-PCR Kit Ver. 2, and the PCR reaction program included: reverse transcription at 50° C. for 30 min; at 94° C. for 30 sec, at 60° C. for 30 sec, and at 72° C. for 60 sec, conducting 35 cycles; and post extension at 72° C. for 10 min. PCR products were detected by 2% agarose gel electrophoresis; if a 633 bp target band was observed, the sample was infected with viremia (ALV-K positive); if there was no amplified target band, the sample was not infected viremia (ALV-K negative), as shown in Table 5.

TABLE-US-00004 TABLE 2 Incidence of ALV-A infection in 1-day-old chicks with different genotypes of tva.sup.318-323delACCTCC mutation sites after being challenged with wild ALV-A virus for 1 month Number of positive Positive tva gene samples/total number infection mutation site Genotype of samples rate (%) tva.sup.318-323delACCTCC Wild-type tva.sup.s/s 28/28 100 tva.sup.s/delACCTCC 25/25 100 tva.sup.delACCTCC/delACCTCC  0/18 0

TABLE-US-00005 TABLE 3 Incidence of ALV-K infection in 1-day-old chicks with different genotypes of tva.sup.318-323delACCTCC mutation sites after being challenged with wild ALV-K virus for 1 month Number of positive Positive tva gene samples/total number infection mutation site Genotype of samples rate (%) tva.sup.318-323delACCTCC Wild-type tva.sup.s/s 28/28 100 tva.sup.s/delACCTCC 25/25 100 tva.sup.delACCTCC/delACCTCC  0/18 0

[0061] As shown in Table 2 and Table 3, the wild-type tva.sup.s/s chicks for the tva.sup.318-323delACCTCC mutation sites (28) each showed ALV-A and ALV-K positive after being challenged with ALV-A and ALV-K wild viruses; the heterozygous mutant tva.sup.s/delACCTCC chicks (25) each showed ALV-A and ALV-K positive after being challenged with ALV-A and ALV-K wild viruses; however, the homozygous mutant tva.sup.delACCTCC/delACCTCC chicks (18) each showed ALV-A and ALV-K negative after being challenged with ALV-A and ALV-K wild viruses. The experimental results showed that the natural mutation of tva.sup.318-323delACCTCC led to the host resistance to ALV-A and ALV-K infection in vivo. The results of the ALV-A and ALV-K challenge tests were consistent with the results of the ALV-A and ALV-K in vitro infection tests. Meanwhile, it was confirmed that the natural mutation of tva.sup.318-323delACCTCC was the molecular marker for genetic resistance to ALV-A and ALV-K in the host chicken.

Example 4 Effects of Tva.SUP.602-607delCCGCTG .Mutation on Host Resistance

[0062] 1. In Vitro Cell Experiments

[0063] (1) RCASBP(A)-EGFP and RCASBP(K)-EGFP expression plasmids were constructed and transfected into DF-1 cells; 7 days after the transfection, RCASBP(A)-EGFP and RCASBP(K)-EGFP viruses (namely ALV-A and ALV-K reporter viruses carrying EGFPs) were rescued and collected from the supernatant of the DF-1 cells (FIG. 4A-FIG. 4C); after measuring the viral infectious unit (IU), the supernatant was aliquoted and stored at −80° C.

[0064] (2) The fluorescent reporter viruses RCASBP(A)-EGFP and RCASBP(K)-EGFP of ALV-A and ALV-K were used to infect CEFs at the tva.sup.602-607delCCGCTG mutation sites of the wild-type tva.sup.s/s, the heterozygous mutant tva.sup.s/delCCGCTG, and the homozygous mutant tva.sup.delCCGCTG/delCCGCTG separately (where the CEFs were prepared from 9-day-old chicken embryos hatched from the breeders tested in Example 1); 1 day, 2 days, 4 days, and 7 days after the infection, the CEFs with different genotypes of tva.sup.602-607delCCGCTG mutation sites infected by the RCASBP(A)-EGFP and RCASBP(K)-EGFP viruses were detected using flow cytometry; the GPF-positive cell rate (%) represented the infection rate of the virus, and results were shown in FIG. 7 and FIG. 8.

[0065] As shown in FIG. 7 and FIG. 8, the CEFs of wild-type tva.sup.s/s and heterozygous mutant tva.sup.s/delCCGCTG were susceptible to the RCASBP(A)-EGFP and RCASBP(K)-EGFP viruses; while the CEFs of homozygous mutant tva.sup.delCCGCTG/delCCGCTG were resistant to the RCASBP(A)-EGFP and RCASBP(K)-EGFP viruses. This indicated that the natural mutation of tva.sup.602-607delCCGCTG led to the host resistance to ALV-A, ALV-K infection.

[0066] 2. In Vivo Experiments

[0067] (1) 1-day-old chicks with tva.sup.602-607delCCGCTG of the mutant wild type, heterozygous mutant, and homozygous mutant were randomly divided into groups, reared in isolators, and injected intraperitoneally with equal amounts of ALV-A (GD08 strain) and ALV-K (GDFX0601 strain) separately at 1-day-old and 5-day-old. One month after challenge, blood samples were collected from the chicks and the total RNA was extracted from each blood sample using the TRIZOL kit.

[0068] The upstream and downstream primers were designed for RT-PCR amplification of ALV-A-env:

TABLE-US-00006 env-F:   (SEQ ID NO: 3) 5′-GGATGAGGTGACTAAGAAAG-3′; env-R: (SEQ ID NO: 4) 5′-AGAGAAAGAGGGGTGTCTAAGGAGA-3′.

[0069] (2) The encoding sequence of env gene of ALV-A was amplified by RT-PCR, and the amplified fragment by RT-PCR had a length of 692 bp. RT-PCR amplification was conducted by the PrimeScript® One Step RT-PCR Kit Ver. 2, and the PCR reaction program included: reverse transcription at 50° C. for 30 min; at 94° C. for 30 sec, at 56° C. for 30 sec, and at 72° C. for 60 sec, conducting 35 cycles; and post extension at 72° C. for 10 min. PCR products were detected by 2% agarose gel electrophoresis; if a 692 bp target band was observed, the sample was infected with viremia (ALV-A positive); if there was no amplified target band, the sample was not infected with viremia (ALV-A negative), as shown in Table 2.

[0070] (3) The upstream and downstream primers were designed for RT-PCR amplification of ALV-K-env:

TABLE-US-00007 env-F: (SEQ ID NO: 5) 5′- GCACCACCTTGGGAACTGACC-3′; env-R: (SEQ ID NO: 6) 5′-GGCGTGGATCGACAGCACAC-3′.

[0071] The encoding sequence of env gene of ALV-K was amplified by RT-PCR, and the amplified fragment by RT-PCR had a length of 633 bp. RT-PCR amplification was conducted by the PrimeScript® One Step RT-PCR Kit Ver. 2, and the PCR reaction program included: reverse transcription at 50° C. for 30 min; at 94° C. for 30 sec, at 60° C. for 30 sec, and at 72° C. for 60 sec, conducting 35 cycles; and post extension at 72° C. for 10 min. PCR products were detected by 2% agarose gel electrophoresis; if a 633 bp target band was observed, the sample was infected with viremia (ALV-K positive); if there was no amplified target band, the sample was not infected with viremia (ALV-K negative), as shown in Table 3.

TABLE-US-00008 TABLE 4 Incidence of ALV-A infection in 1-day-old chicks with different genotypes of tva.sup.602-607delCCGCTG mutation sites after being challenged with wild ALV-A virus for 1 month Number of positive Positive tva gene samples/total number infection mutation site Genotype of samples rate (%) tva.sup.602-607delCCGCTG Wild-type tva.sup.s/s 32/32 100 tva.sup.s/delCCGCTG 23/23 100 tva.sup.delCCGCTG/deICCGCTG  0/27 0

TABLE-US-00009 TABLE 5 Incidence of ALV-K infection in 1-day-old chicks with different genotypes of tva.sup.602-607delCCGCTG mutation sites after being challenged with wild ALV-K virus for 1 month Number of positive Positive tva gene samples/total number infection mutation site Genotype of samples rate (%) tva.sup.602-607delCCGCTG Wild-type tva.sup.s/s 32/32 100 tva.sup.s/delCCGCTG 23/23 100 tva.sup.delCCGCTG/delCCGCTG  0/27 0

[0072] As shown in Table 4 and Table 5, at the tva.sup.602-607delCCGCTG mutation sites, the wild-type tva.sup.s/s chicks (32) each showed ALV-A and ALV-K positive after being challenged with ALV-A and ALV-K wild viruses; the heterozygous mutant tva.sup.s/delCCGCTG chicks (23) each showed ALV-A and ALV-K positive after being challenged with ALV-A and ALV-K wild viruses; however, the homozygous mutant tva.sup.delCCGCTG/delCCGCTG chicks (27) each showed ALV-A and ALV-K negative after being challenged with ALV-A and ALV-K wild viruses. The experimental results showed that the natural mutation of tva.sup.602-607delCCGCTG led to the host resistance to ALV-A and ALV-K infection in vivo. The results of the ALV-A and ALV-K challenge tests were consistent with the results of the ALV-A and ALV-K in vitro infection tests. Meanwhile, it was confirmed that the natural mutation of tva.sup.602-607delCCGCTG was a molecular marker for genetic resistance to ALV-A and ALV-K in the host chicken.

Example 5 Screening of Chickens with Genetic Resistance to ALV-A and ALV-K

[0073] 1. Referring to a DNA sequence of tva gene (with a GenBank accession number of AY531262.1), PCR primers were designed (including a forward primer F: 5′-CGGCCCGCTTTATAGGCGTTG-3′ (SEQ ID NO: 1); a reverse primer R: 5′-CCCACTCGTCCCGTCCATCG-3′ (SEQ ID NO: 2)), and a tva receptor gene region containing tva.sup.602-607delCCGCTG or tva.sup.318-323delACCTCC mutation sites was amplified.

[0074] 2. The genomic DNAs were extracted from 1782 samples to be tested of 15 local chicken breeds and 15 yellow feathered broiler lines.

[0075] 3. PCR detection

[0076] The PCR amplification system included: 1 μL of a DNA template, 2.5 μL of a 10× buffer, 2 μL of dNTPs, 1 μL of each of upstream and downstream detection primers (with a nucleotide sequence set forth in SEQ ID NO: 1), 0.5 μL of KOD-FX, and supplementing to 25 μL with ddH.sub.2O.

[0077] The PCR amplification program included: initial denaturation at 94° C. for 5 min; denaturation at 94° C. for 30 sec, annealing at 58° C. for 30 sec, and extension at 72° C. for 30 sec, conducting 35 cycles; then post extension at 72° C. for 5 min, and storage at 4° C.

[0078] 4. After detection by 2% agarose gel electrophoresis, the PCR amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for purification and sequencing to determine the genotype and whether the sample to be tested was a resistant chicken. The determination criteria were shown in Table 6 and Table 7.

TABLE-US-00010 TABLE 6 Identification criteria for chicken with genetic resistance to ALV-A and ALV-K tva gene Susceptibility to ALV-A mutation site Genotype and ALV-K infection tva.sup.602-607delCCGCTG Wild-type tva.sup.s/s Susceptible tva.sup.s/delCCGCTG Susceptible tva.sup.delCCGCTG/delCCGCTG Resistant

TABLE-US-00011 TABLE 7 Identification criteria for chicken with genetic resistance to ALV-A and ALV-K tva gene Susceptibility to ALV-A mutation site Genotype and ALV-K infection tva.sup.318-323delACCTCC Wild-type tva.sup.s/s Susceptible tva.sup.s/delACCTCC Susceptible tva.sup.delACCTCC/delACCTCC Resistant

[0079] If the genotype of the tva.sup.602-607delCCGCTG or tva.sup.318-323delACCTCC resistance site was wild-type tva.sup.s/s, the sample to be tested was not resistant to ALV-A and ALV-K infection (susceptible), such that the sample to be tested was determined to be the chicken susceptible to ALV-A and ALV-K;

[0080] if the genotype of the tva.sup.602-607delCCGCTG resistance site was tva.sup.s/delCCGCTG or the genotype of the tva.sup.318-323delACCTCC resistance site was tva.sup.s/delACCTCC, the sample to be tested was susceptible to ALV-A and ALV-K infection, but the sample to be tested carried the recessive gene for the genetic resistance of ALV-A and ALV-K; and

[0081] if the genotype of the tva.sup.602-607delCCGCTG resistance site was tva.sup.delCCGCTG/delCCGCTG or the genotype of the tva.sup.318-323delACCTCC resistance site was tva.sup.delACCTCC/delACCTCC, the sample to be tested was resistant to ALV-A and ALV-K infection, such that the sample to be tested was determined to be the chicken resistant to ALV-A and ALV-K.

[0082] 5. Test results

[0083] In Chinese chicken breeds, the genotyping results of tva.sup.318-323delACCTCC resistance site were shown in Table 8, and the genotyping results of tva.sup.602-607delCCGCTG resistance site were shown in Table 9.

[0084] As shown in Table 8, Broiler chicken, Lingshan native chicken, Xuefeng silky chicken, and Xiushui yellow chicken and other local chicken breeds, as well as yellow feathered broiler line 1, yellow feathered broiler line 4, yellow feathered broiler line 10, and yellow feathered broiler line 12 had the resistance genotype tva.sup.delACCTCC/delACCTCC for the tva.sup.318-323delACCTCC resistance site at frequencies of 0.10, 0.33, 0.15, 0.12, 0.20, 0.25, 0.25, and 0.18, respectively. This indicated that these Chinese local chicken breeds and self-bred yellow feathered broiler lines had desirable potential for genetic improvement against ALV-A and ALV-K. Breeding materials for cultivating breeds resistant to ALV-A and ALV-K infection can be screened from these chicken breeds, and used in the breeding of chicken breeds (lines) with genetic resistance to ALV-A and ALV-K, to control the subgroups A and K-caused AL.

[0085] As shown in Table 9, Huaixiang chicken, Hetian chicken, and Chongren ma chicken and other local chicken breeds, as well as yellow feathered broiler line 2, yellow feathered broiler line 5, yellow feathered broiler line 11, and yellow feathered broiler line 14 had the resistance genotype tva.sup.delCCGCTG/delCCGCTG for the tva.sup.602-607delCCGCTG resistance site at frequencies of 0.07, 0.11, 0.17, 0.27, 0.10, 0.20, and 0.13, respectively. This indicated that these Chinese local chicken breeds and self-bred yellow feathered broiler lines had desirable potential for genetic improvement against ALV-A and ALV-K. Breeding materials for cultivating breeds resistant to ALV-A and ALV-K infection can be screened from these chicken breeds, and used in the breeding of chicken breeds (lines) with genetic resistance of ALV-A and ALV-K, to control the ALV subgroups A and K.

TABLE-US-00012 TABLE 8 Genotype frequency distribution of tva.sup.318-323delACCTCC mutation sites in Chinese chicken breeds Sample numbers tva.sup.318-323delACCTCC Breeds (lines) (chicken) tva.sup.s/s tva.sup.s/delACCTCC tva.sup.delACCTCC/delACCTCC Huaixiang chicken 60 0.92 0.08 0 Zhongshan Shalan chicken 60 1 0 0 Beijing Fatty chicken 50 1 0 0 Broiler chicken 60 0.77 0.13 0.10 Longsheng Fengji chicken 58 1 0 0 Lingshan native chicken 36 0.56 0.11 0.33 Taihe silky chicken 50 1 0 0 Hetian chicken 72 1 0 0 Xuefeng silky chicken 60 0.62 0.23 0.15 Wenchang chicken 60 1 0 0 Baier huang chicken 60 1 0 0 Chongren ma chicken 60 1 0 0 Anyiwa gray chicken 60 1 0 0 Xiushui yellow chicken 50 0.72 0.16 0.12 Xiayan chicken 50 1 0 0 Yellow feathered broiler line 1 60 0.55 0.25 0.20 Yellow feathered broiler line 2 72 1 0 0 Yellow feathered broiler line 3 72 1 0 0 Yellow feathered broiler line 4 60 0.45 0.30 0.25 Yellow feathered broiler line 5 60 1 0 0 Yellow feathered broiler line 6 60 0.85 0.15 0 Yellow feathered broiler line 7 60 1 0 0 Yellow feathered broiler line 8 60 1 0 0 Yellow feathered broiler line 9 60 1 0 0 Yellow feathered broiler line 10 60 0.53 0.22 0.25 Yellow feathered broiler line 11 60 1 0 0 Yellow feathered broiler line 12 60 0.54 0.28 0.18 Yellow feathered broiler line 13 60 1 0 0 Yellow feathered broiler line 14 60 1 0 0 Yellow feathered broiler line 15 60 1 0 0

TABLE-US-00013 TABLE 9 Genotype frequency distribution of tva602-607delCCGCTG mutation sites in Chinese chicken breeds Sample numbers tva.sup.602-607delCCGCTG Breeds (lines) (chicken) tva.sup.s/s tva.sup.s/delCCGCTG tva.sup.delCCGCTG/delCCGCTG Huaixiang chicken 60 0.77 0.16 0.07 Zhongshan Shalan chicken 60 1 0 0 Beijing Fatty chicken 50 1 0 0 Broiler chicken 60 1 0 0 Longsheng Fengji chicken 58 1 0 0 Lingshan native chicken 36 1 0 0 Taihe silky chicken 50 1 0 0 Hetian chicken 72 0.56 0.33 0.11 Xuefeng silky chicken 60 1 0 0 Wenchang chicken 60 1 0 0 Baier huang chicken 60 1 0 0 Chongren ma chicken 60 0.60 0.23 0.17 Anyiwa gray chicken 60 1 0 0 Xiushui yellow chicken 50 1 0 0 Xiayan chicken 50 1 0 0 Yellow feathered broiler line 1 60 1 0 0 Yellow feathered broiler line 2 72 0.43 0.30 0.27 Yellow feathered broiler line 3 72 1 0 0 Yellow feathered broiler line 4 60 1 0 0 Yellow feathered broiler line 5 60 0.60 0.30 0.10 Yellow feathered broiler line 6 60 0.83 0.17 0 Yellow feathered broiler line 7 60 1 0 0 Yellow feathered broiler line 8 60 1 0 0 Yellow feathered broiler line 9 60 1 0 0 Yellow feathered broiler line 10 60 1 0 0 Yellow feathered broiler line 11 60 0.63 0.17 0.20 Yellow feathered broiler line 12 60 1 0 0 Yellow feathered broiler line 13 60 1 0 0 Yellow feathered broiler line 14 60 0.4 0.47 0.13 Yellow feathered broiler line 15 60 0.93 0.07 0