WO2019128350A1 - 白来航鸡红羽致因突变基因型鉴定及红羽粉壳蛋鸡配套系培育方法 - Google Patents

白来航鸡红羽致因突变基因型鉴定及红羽粉壳蛋鸡配套系培育方法 Download PDF

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WO2019128350A1
WO2019128350A1 PCT/CN2018/107801 CN2018107801W WO2019128350A1 WO 2019128350 A1 WO2019128350 A1 WO 2019128350A1 CN 2018107801 W CN2018107801 W CN 2018107801W WO 2019128350 A1 WO2019128350 A1 WO 2019128350A1
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red
feather
white
chicken
genotype
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French (fr)
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吴桂琴
杨宁
孙皓
李光奇
孙从佼
李花妮
刘爱巧
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北京市华都峪口禽业有限责任公司
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Priority to US16/958,233 priority Critical patent/US11732309B2/en
Priority to EP18893435.0A priority patent/EP3741873B1/en
Publication of WO2019128350A1 publication Critical patent/WO2019128350A1/zh

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Definitions

  • the invention belongs to molecular biology and poultry breeding technology, and particularly relates to a mutant genotype identification of Bailai Hang chicken red feather cause and a breeding method of the red feather powder shell chicken.
  • Bailaihang chickens Due to China's development history and unique consumption philosophy, the "Red Feather" laying hen is very popular.
  • some of the Bailaihang chickens have a feather color mutation, which can be used as a female parent to cross the red father to produce red feathers.
  • the traditional breeding method adopts the method of cross-testing, and selects the Bailaihang chicken carrying the mutation of the red feather caused by the feather color phenotype of the hybrid offspring. This method is cumbersome and requires a lot of manpower, material and financial resources, and is subject to the expansion of the size. The impact accuracy rate cannot reach 100%.
  • the molecular detection method is used to identify the genotype of the feather color causing mutation site, and then it is applied to the new domestic chicken laying hens for breeding red feather powder shell eggs.
  • the system is necessary.
  • the primer pair provided by the present invention consists of a single-stranded DNA molecule represented by SEQ ID NO: 2 in the Sequence Listing and a single-stranded DNA molecule represented by SEQ ID NO: 3 in the Sequence Listing.
  • Another object of the present invention is to provide a kit for identifying a mutant genotype of the Bailai Hang Chicken Red Feather Cause.
  • the kit provided by the invention comprises the above primer pair and restriction endonuclease
  • the restriction enzyme is NruI.
  • the above primer pair or the above kit is also applicable to the protection of the red-feather-causing mutant homozygous genotype or the red-feather-causing mutant heterozygous genotype of Bailaihang chicken;
  • the above-mentioned primer pair or the above-mentioned kit is also suitable for the protection of the present invention in the selection of the Bailaihang chicken which is crossed with the Luodao red rooster and the hen is red feather;
  • the above primer pair or the above kit is used for identifying the red feather trait of the offspring hens of the white cockatoo and the Luodao red cock to be tested;
  • the red-feather-causing mutant genotype of the white-to-air chicken to be tested includes a homozygous heterozygous mutation of the red feather cause and a heterozygous mutation of the red feather cause.
  • a third object of the present invention is to provide a method for identifying a red feather causing mutant genotype of a white chicken to be tested.
  • the method provided by the invention comprises the following steps:
  • the present invention also provides a method for identifying red feather traits of a hen that is to be tested by a white cock and a Luodao red cock, comprising the following steps:
  • the size of the digested product was identified as the red feather causing mutant genotype of Bailaihang chicken as follows:
  • the white hen is to be tested as a homozygous genotype of the red feather causing mutation
  • the white hen to be tested is a non-red feather causative mutant
  • the hens to be tested are the heterozygous genotypes of the red feather causing mutations.
  • red feather traits of the offspring hens to be tested according to the size of the digested product are as follows:
  • the hens to be tested and the hens of the Luodao red cock are only red feather traits
  • the hen of the white-bred hen and the Luodao red cock are only non-red feather traits
  • the hens of the white hens to be tested and the red cocks of the Luodao red hens are red feather traits and non-red feather traits.
  • the PCR amplified template is the genomic DNA of the chicken to be tested.
  • a fourth object of the present invention is to provide a method for breeding a Baiyin chicken with a homozygous genotype of a red feather causing mutation.
  • the present invention provides a method for breeding a Baiyuhang chicken with a homozygous genotype of a red feather-causing mutation, comprising the steps of: breeding a white-bred chicken of the above-mentioned red-feather-induced mutation homozygous genotype;
  • the present invention provides a method for breeding a white-bred chicken to be tested with a red hen of a hen of a Luohong red rooster, comprising the following steps: breeding the above-mentioned red-feather-causing homozygous genotype of Bailaihang chicken .
  • a fifth object of the present invention is to provide a method for cultivating a red feather powder shell hen.
  • the method provided by the invention comprises the following steps:
  • the white-breasted chicken with the homozygous genotype of the red-yellow mutation is used as the female parent, and hybridized with the red father of Luodao, the hens of the offspring are all red feathers, and the male chicks are all non-red feathers, that is, red Feather shell egg laying system.
  • Bailai Hang Chicken is the Bailai Airlines Department of Beijing Huadu Shoukou Poultry Co., Ltd., including cock and/or hen.
  • the experiments of the present invention prove that primers are designed according to the upstream and downstream nucleotide sequences of the 18th, 288, and 303 deoxynucleotides of the chromosome 18 sense strand of the chicken reference genome Gallus_gallus-4.0 published in NCBI, and the restriction fragment length polymorphism is detected.
  • the locus genotype (SNP) is used to expand the breeding of the Baiyinghang chicken with the homozygous genotype of the red feather causing mutation, and the pure female parent is selected.
  • the female parent and the red father of Luodao are 100% of the offspring.
  • the red feather phenotype can meet the market demand and has broad promotion prospects.
  • the restriction fragment length polymorphism detection method avoids the cumbersome test-crossing selection, shortens the generation interval, accelerates the breeding process, reduces the breeding cost, and overcomes In traditional conventional breeding, the shortcomings of tedious test and long generation interval are long.
  • the method of the invention can directly identify the genotype of the mutation site of the red hen of the male hen, solve the problem that the cockroach red feather genotype cannot be judged by the traditional method of measuring and measuring, increase the accuracy and speed of the cock judging, and improve The use value of the cock.
  • Figure 1 shows the results of three genotype PCR agarose gel electrophoresis.
  • Figure 2 shows the results of three genotype digestions on agarose gel electrophoresis.
  • the chicken vein venous blood collection and phenol imitation methods in the following examples are all routine operations in the art.
  • the red feather is red in plumage.
  • the phenotypes of the hybrid offspring were statistically selected, and the number of hens with more than 5 hens and 100% of red feathers were selected.
  • the red feather group Nine of the hens were used as the red feather group, and 9 of the white hens with more than 5 hens and 100% of the white hens were selected as the white feather group; the two groups were used as the chickens to be tested.
  • the chickens to be tested were subjected to chicken venous blood collection, anticoagulant treatment with anticoagulant, cleavage and protease digestion, and then genomic DNA was extracted by phenol-like method, dissolved in sterile double distilled water, and used to obtain chickens in the red feather group. Genomic DNA and genomic DNA of each chicken in the white feather group.
  • the genomic DNA of each chicken in the red feather group and the genomic DNA of each chicken in the white feather group were re-sequenced using an Illumina HiSeq 4000 sequencer.
  • the SAMtools tool is used to count the alignment results of the two DNA libraries of each individual to form the final alignment result of the individual.
  • the red-spotted SNP locus is the 18th, 288th, and 303th deoxynucleotides of the chicken reference genome 11th chromosome sense strand published in NCBI (Gallus_gallus-4.0 version sequence information), and is also the 253th position of the sequence 1; and the red feather SNP locus
  • the nucleotide is A or G; the genotype of the red-scented SNP site is AA, GG or AG.
  • the red-scented SNP site is on the autosome.
  • Forward primer F 5'-GCCGCCATCCTCAAGAACA-3' (sequence 2)
  • Reverse primer R 5'-AAAAAAAAAAAAAACGCAGCGCATAGAAGATCG-3' (SEQ ID NO: 3).
  • the genomic DNA of the white cockatoo obtained from the above 1 was used as a template, and PCR amplification was carried out by using the forward primer F and the reverse primer R of the above two designs to obtain a PCR amplification product (SEQ ID NO: 1).
  • the above 20 ⁇ L PCR amplification system is as follows:
  • Agarose gel assay 1.5% to 2% agarose gel was used for detection, the voltage was 100V, and the electrophoresis time was 35 minutes.
  • the results are shown in Figure 1.
  • the PCR products are all 288 bp (sequence 1);
  • Marker is DM2000, which is 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 2000 bp from bottom to top.
  • the PCR amplification product described above was digested with NruI endonuclease to obtain a digested product.
  • the enzyme digestion system is as follows:
  • the above-mentioned enzyme-cut products were detected using a 1.5%-2% agarose gel at a voltage of 100 V and an electrophoresis time of 35 minutes.
  • the results are shown in Figure 2.
  • the GG genotypes in lanes 1, 2, and 3 showed a 253 bp band after digestion with NruI endonuclease; the GA genotypes in lanes 4, 5, 6, and 7 were digested with NruI endonuclease.
  • Two bands of 288 bp and 253 bp were shown; the AA genotypes of lanes 8 and 9 showed a 288 bp band after digestion with NruI endonuclease.
  • Marker is DM2000, which is 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp from bottom to top;
  • the GG genotype of the hens was only 253 bp in one band (the lanes of some chickens in this group), and the genotypes of the red-spotted SNPs in the genome of the white hens were all GG (sequenced, And confirmed by hybridization, which is a homozygous gene of the red feather causing mutation, that is, it is 100% red feathered with the hen of the Luodao red rooster; the 253bp digestion product is sequenced as the sequence 1-253bp;
  • the AA genotype has a 288 bp restriction band (the lanes of some chickens in this group are 8-9).
  • the genotypes of the red-suppressed SNPs in the genome of the white hens are AA (sequenced, And it has been verified by hybridization, which is a non-red feather cause mutant individual, that is, the group of white hens and Luodao red cock are 100% non-red feathers.
  • the 288 bp digested product was sequenced to be 1-288 bp of sequence 1;
  • the genotypes of the GA genotypes of the hens were 288 bp and 253 bp in two bands (the lanes of the chickens in this group were 4-7).
  • the genotypes of the red-spotted SNPs in the genome of the white hens were all GA ( Sequencing), which is a heterozygous genotype of the red-feather-causing mutation, that is, the group of the white-breasted hen and the Luodao red rooster hybrid hens have both red feathers and non-red feathers.
  • the 288 bp digested product was sequenced as sequence 1-188 bp; the 253 bp cleavage product was sequenced as sequence 1 1-253 bp.
  • the above method can be used to detect the red feather cause mutation genotype of the white hen to be tested or to detect the red feather trait of the hens of the white hen and the Luodao red cock.
  • the specific method is as follows:
  • NruI digests the PCR amplification product to obtain a digested product.
  • the white hen is to be tested as a homozygous genotype of the red feather causing mutation (the genomic genotype of the red feather SNP in the genome is GG), The red-yellow mutated homozygous genotype of Bailai Hang chicken hen and Luodao red cock hybrid hen is only red feather trait;
  • the white hen that is to be tested is a non-red feather causing mutant genotype (the genotype of the red feather SNP locus in the genome is AA), then The non-red feather cause mutated genotypes of Bailai Hang chicken hen and Luodao red cock are only non-red feather traits;
  • the white hens to be tested are the heterozygous genotypes of the red feather causing mutations (the genotypes of the red feather SNPs in the genome are all AG). Then, the red-yellow mutated heterozygous genotypes of the Bailai Hang chicken hen and the Luodao red cock are offspring hens with red feather traits and non-red feather traits.
  • the red-yellow causative mutant genotype is a homozygous genotype of the red-feather-causing mutation or a heterozygous genotype of the red-feather-causing mutation:
  • red feather causative homozygous genotype is that the genotype of the red-suppressed SNP locus in the genome is GG, and the red-feathering homozygous genotype of the white-breasted hen and the Luodao red rooster are only the offspring of the hen.
  • Red feather trait the genotype of the red-suppressed SNP locus in the genome is GG, and the red-feathering homozygous genotype of the white-breasted hen and the Luodao red rooster are only the offspring of the hen.
  • the above-mentioned red-feather-causing hybrid heterozygous genotype is the genotype of the red-spotted SNP locus in the genome, and the red-feather-causing hybrid heterozygous genotype, the white-carved hen and the Luodao red rooster, is the offspring of the hen. Traits and non-red feather traits.
  • the non-red feather cause mutation genotype is that the red-spotted SNP locus genotype in the genome is AA, and the non-red feather causing mutant genotypes of the white-breasted hen and the Luodao red rooster are only non-red feathers. Traits.
  • Example 2 Breeding homozygous genotype of red-yellow-induced mutants of white-breasted chickens and establishment of matching system of red-feathered eggshells
  • PCR amplification was carried out by using step 1 of the above three methods in Example 1 to obtain a PCR amplification product, using 1573 genomic DNAs of the white hens to be tested as a template.
  • the PCR amplification product of each of the above chickens was digested with NruI to obtain a digested product.
  • the white hen is to be a homozygous genotype of the red feather causing mutation, and the red feather causing the homozygous genotype
  • the hens of chicken hens and Luodao red cocks are only red feather traits
  • the white hen is a heterozygous genotype of the red feather causing mutation, and the red feather causing the heterozygous genotype Chicken hens and Luodao red cocks are crossed and the hens are red feather traits and non-red feather traits.
  • the 962 digested products were only 253 bp in size. It was judged that these hens were homozygous for the red-feather-causing mutation, and the hens in the offspring of the Luodao red rooster were red feathers. ;
  • the 962 red-feased gene mutation homozygous genotypes were identified as the female parent and the Luodao red cock were identified by the above method.
  • the hybrid progeny were detected and 3,620 hens were obtained, of which 3,620 were red feathers.
  • the 592 red-feathering mutant heterozygous genotypes in the above SNP identification were used as the female parent to cross the Luodao red cock, and the hybrid progeny were detected to obtain 2257 hens, of which 1182 were red feathers. 1075 only.
  • the 19 non-red feather cause mutants in the above SNP identification were used as the female parent to cross the Luodao red cock, and the hybrid progeny were detected to obtain 61 hens, of which 0 were red feathers and 61 were non-red feathers. .
  • the primers and the method of the present invention can be used to identify the red feather causative mutant genotype of the white chicken to be tested, and can be used. To judge the feather color traits of the offspring of the white-bred chicken and the Luodao red chicken.
  • the GG genotype Bailai hen chicken hen and Luodao red cock were crossed, the hens of the offspring were all red feathers, and the male chicks were all non-red feathers, which established the matching system of the red feather powder shell.
  • the full-sib cocks of the GG-type and GA-type white-breasted hens can also be judged by the above two methods for the red-feather-causing mutant genotype, and the GG-type white cock is selected.
  • the GG type white cockerel cock and all GG type white hens were expanded and selected. After four generations of breeding, the red feather cause mutation homozygous genotype Bailai hang chicken pure line, red feather SNP position The genotype of the point is GG type.

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Abstract

公开了一种红羽粉壳蛋鸡配套系的培育方法，并且提供了鉴定白来航鸡红羽致因突变基因型的引物对，其由序列表中序列2所示的单链DNA分子和序列表中序列3所示的单链DNA分子组成，该引物对根据NCBI中公布的鸡参考基因组Gallus_gallus-4.0版本序列信息11号染色体正义链第18,288,303位脱氧核苷酸上下游核苷酸序列设计引物，通过限制性片段长度多态性检测该位点基因型，对红羽致因突变基因型纯合的白来航鸡进行扩繁，选育纯系母本，该母本与洛岛红父本杂交后代母鸡100％为红羽表型，能够满足市场需求，具有广阔推广前景。

Description

白来航鸡红羽致因突变基因型鉴定及红羽粉壳蛋鸡配套系培育方法 技术领域
本发明属于分子生物学及家禽育种技术，尤其涉及一种白来航鸡红羽致因突变基因型鉴定及红羽粉壳蛋鸡配套系培育方法。
背景技术
中国是传统美食国家，消费者对鸡蛋的需求具有明显的地域特色，因而对蛋鸡品种的需求也呈现多元化和区域化特点。随着生活水平的提升，消费者对鸡蛋的需求，不仅注重内在的品质，还关注鸡蛋的大小、蛋壳的颜色。鸡蛋需求类型可大致分为奢侈消费需求、高端消费需求、大众消费需求和蛋品加工需求。粉壳蛋鸡品种因所产鸡蛋蛋重偏小、蛋黄比例大，成为居民鸡蛋消费的“新宠”。据统计，近年来市场上粉壳鸡蛋所占比例呈持续上升趋势，达到40％。同时，鸡羽色性状一直都是国内优质鸡育种的一个重要选择指标，也是影响其经济价值的一个重要性状。由于中国的发展历史和独有的消费理念，“红羽”蛋鸡广受欢迎。在生产上发现部分白来航鸡具有羽色基因突变，其作为母本与洛岛红父本杂交可以产生红羽后代母鸡。传统育种方法采用测交的方式，依据杂交后代的羽色表型筛选携带红羽致因突变的白来航鸡，这种方法过程繁琐，需要耗费大量人力、物力和财力，并且受到扩群大小的影响准确率不能达到100％。从供给侧结构性改革，满足国民鸡蛋消费需求新动向的角度而言，利用分子检测方法鉴定羽色致因突变位点基因型，进而应用于培育红羽产粉壳鸡蛋的国产蛋鸡新配套系势十分必要。
发明内容
本发明的一个目的是提供鉴定白来航鸡红羽致因突变基因型的引物对。
本发明提供的引物对，其由序列表中序列2所示的单链DNA分子和序列表中序列3所示的单链DNA分子组成。
本发明的另一个目的是提供鉴定白来航鸡红羽致因突变基因型的试剂盒。
本发明提供的试剂盒，包括上述的引物对和限制性内切酶；
所述限制性内切酶为NruI。
上述的引物对或上述的试剂盒在鉴定待测白来航鸡的红羽致因突变基因型中的应用也是本发明保护的范围。
上述的引物对或上述的试剂盒在选育红羽致因突变纯合基因型或红羽致因突变杂合基因型的白来航鸡中的应用也是本发明保护的范围；
或上述的引物对或上述的试剂盒在选育与洛岛红公鸡杂交后代母鸡为红羽的白来航鸡中的应用也是本发明保护的范围；
或上述的引物对或上述的试剂盒在鉴定待测白来航鸡与洛岛红公鸡杂交后代母鸡的红羽性状中的应用；
或上述的引物对或上述的试剂盒在红羽粉壳蛋鸡配套系的建立中的应用也是本发明保护的范围。
上述中，所述待测白来航鸡的红羽致因突变基因型包括红羽致因突变纯合型和红羽致因突变杂合型。
本发明的第3个目的是提供一种鉴定待测白来航鸡的红羽致因突变基因型的方法。
本发明提供的方法，包括如下步骤：
1)用上述引物对对待测鸡进行PCR扩增，得到PCR扩增产物；
2)将所述PCR扩增产物用上述限制性内切酶酶切，得到酶切产物；
3)检测酶切产物，根据酶切产物大小鉴定待测白来航鸡的红羽致因突变基因型。
或，本发明还提供一种鉴定待测白来航鸡与洛岛红公鸡杂交后代母鸡的红羽性状的方法，包括如下步骤：
1)用上述引物对对待测鸡进行PCR扩增，得到PCR扩增产物；
2)将所述PCR扩增产物用上述限制性内切酶酶切，得到酶切产物；
3)检测酶切产物，根据酶切产物大小鉴定待测白来航鸡与洛岛红公鸡杂交后代母鸡的红羽性状。
上述方法中，
所述酶切产物大小鉴定待测白来航鸡的红羽致因突变基因型为如下：
若待测白来航母鸡的酶切产物大小仅为253bp，则该待测白来航母鸡为红羽致因突变纯合基因型；
若待测白来航母鸡的酶切产物大小仅为288bp，则该待测白来航母鸡为非红羽致因突变型；
若待测白来航母鸡的酶切产物大小为288bp和253bp，则待测白来航母鸡为红羽致因突变杂合基因型。
或所述根据酶切产物大小鉴定待测白来航鸡与洛岛红公鸡杂交后代母鸡的红羽性状为如下：
若待测白来航母鸡的酶切产物大小仅为253bp，则该待测白来航的母鸡与洛岛红公鸡杂交后代母鸡仅为红羽性状；
若待测白来航母鸡的酶切产物大小仅为288bp，则该待测白来航的母鸡与洛岛红公鸡杂交后代母鸡仅为非红羽性状；
若待测白来航母鸡的酶切产物大小为288bp和253bp，则该待测白来航的母鸡与 洛岛红公鸡杂交后代母鸡为红羽性状和非红羽性状。
上述方法中，
所述PCR扩增的模板为待测鸡的基因组DNA。
本发明的第4个目的是提供一种选育红羽致因突变纯合基因型的白来航鸡的方法。
本发明提供选育红羽致因突变纯合基因型的白来航鸡的方法，包括如下步骤：选育上述红羽致因突变纯合基因型的白来航鸡；
或本发明提供一种选育与洛岛红公鸡杂交后代母鸡为红羽的待测白来航鸡的方法，包括如下步骤：选育上述的红羽致因突变纯合基因型的白来航鸡。
本发明的第5个目的是提供一种红羽粉壳蛋鸡配套系的培育方法。
本发明提供的方法，包括如下步骤：
1)选育上述红羽致因突变纯合基因型的白来航鸡；
2)以所述红羽致因突变纯合基因型的白来航鸡为母本，与洛岛红父本进行杂交，后代母鸡均为红羽，公雏均为非红羽，即得到红羽粉壳蛋鸡配套系。
上述待测白来航鸡为北京市华都峪口禽业有限责任公司白来航品系，包括公鸡和/或母鸡。
本发明的实验证明，根据NCBI中公布的鸡参考基因组Gallus_gallus-4.0版本序列信息11号染色体正义链第18,288,303位脱氧核苷酸上下游核苷酸序列设计引物，通过限制性片段长度多态性检测该位点基因型(SNP)，对红羽致因突变纯合基因型的白来航鸡进行扩繁，选育纯系母本，该母本与洛岛红父本杂交后代母鸡100％为红羽表型，能够满足市场需求，具有广阔推广前景；另外，该限制性片段长度多态性检测方法，避免了测交选育的繁琐，缩短世代间隔、加快育种进程、降低育种成本，克服了 传统常规育种中测交繁琐、世代间隔长等缺点。此外，本发明的方法可以直接鉴别公母鸡红羽致因突变位点基因型，解决了无法用传统测交方法判断公鸡红羽基因型的问题，增加公鸡判型的准确性和速度，提高了公鸡使用价值。
附图说明
图1为三种基因型PCR琼脂糖凝胶电泳检测结果。
图2为三种基因型酶切琼脂糖凝胶电泳检测结果。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中鸡翅静脉采血和酚仿法均为本领域的常规操作。
红羽为全身羽毛红色。
实施例1、白来航鸡红羽致因突变基因型鉴定方法所需引物及其方法的建立
一、白来航鸡红羽致因突变位点的发现
1、提取待测鸡的基因组DNA
利用2000只北京市华都峪口禽业有限责任公司白来航品系母鸡与洛岛红公鸡杂交，统计杂交后代羽色表型，选取后代母鸡数大于5只且100％为红羽的白来航母鸡9只作为红羽组，选取后代母鸡数大于5只且100％为白羽的白来航母鸡9只作为白羽组；该2组作为待测鸡。
对待测鸡进行鸡翅静脉采血，用抗凝剂进行抗凝处理后经裂解、蛋白酶消化处理，然后采用酚仿法提取基因组DNA，以灭菌双蒸水溶解后备用，得到红羽组各只鸡基因组DNA和白羽组各只鸡基因组DNA。
2、重测序
对红羽组各只鸡基因组DNA和白羽组各只鸡基因组DNA采用Illumina HiSeq 4000测序仪进行重测序。
对重测序数据进行数据分析，具体分析步骤为：
1)利用NGS QC Toolkit对原始reads进行质量控制，剔除质量较低和含有接头或者引物的reads。
2)利用Burrows Wheeler Aligner(BWA)软件将质控后的reads比对到galGal4参考基因组上。
3)利用Picard软件标记并删除PCR重复。
4)利用SAMtools工具将每个个体的两个DNA文库的比对结果进行统计，组成这个个体的最终比对结果。
5)利用GATK软件从测序数据中进行SNP calling，根据SNP多态性进行固定指数分析(Fst)，确定两组之间的显著差异区间。
6)对显著差异区间内的SNP进行验证，确定该白来航品系红羽致因突变SNP分子标记，命名为红羽SNP。
该红羽SNP位点为NCBI(Gallus_gallus-4.0版本序列信息)中公布的鸡参考基因组11号染色体正义链第18,288,303位脱氧核苷酸，也为序列1第253位；且该红羽SNP位点的核苷酸为A或G；该红羽SNP位点的基因型为AA、GG或AG。
该红羽SNP位点在常染色体。
二、白来航鸡红羽SNP位点的扩增引物的设计合成
根据上述一发现的红羽SNP位置信息设计带有酶切位点的引物，由深圳华大基因科技服务有限公司进行合成，其中：
正向引物F：5'-GCCGCCATCCTCAAGAACA-3'(序列2)
反向引物R：5'-AAAAAAAAAAAAAAAACGCAGCGCATAGAAGATCG-3'(序列3)。
三、限制性片段长度多态性检测待测白来航鸡的红羽致因突变基因型的方法建立
1、提取经测序和杂交试验验证过的具有GG、GA和AA基因型待测白来航母鸡的基因组DNA
2、PCR扩增
以上述1得到的待测白来航鸡的基因组DNA为模板，用上述二设计的正向引物F和反向引物R进行PCR扩增，得到PCR扩增产物(序列1)。
上述20μL PCR扩增体系如下：
2×PCR Mix(北京全式金生物技术有限公司
Figure PCTCN2018107801-appb-000001
上述PCR反应程序如下：
Figure PCTCN2018107801-appb-000002
Figure PCTCN2018107801-appb-000003
琼脂糖凝胶检测：采用1.5％～2％的琼脂糖凝胶进行检测，电压采用100V，电泳时间为35分钟。
结果如图1所示，PCR产物均为288bp(序列1)；Marker为DM2000，从下向上分别为100bp，250bp，500bp，750bp，1000bp，2000bp。
3、酶切
将上述PCR扩增产物用NruI内切酶进行酶切，得到酶切产物。
酶切体系如下：
10μL体系：
Figure PCTCN2018107801-appb-000004
酶切反应程序：
①37℃        1h
②4℃保存
将上述酶切产物采用1.5％-2％的琼脂糖凝胶进行检测，电压采用100V，电泳时间为35分钟。
结果如图2所示，1、2、3泳道GG基因型个体经NruI内切酶酶切后显示253bp一条带；4、5、6、7泳道GA基因型个体经NruI内切酶酶切后显示288bp和253bp两条带；8、9泳道AA基因型个体经NruI内切酶酶切后显示288bp一条带。Marker为DM2000，从下向上分别为100bp，250bp，500bp，750bp，1000bp，2000bp；
可以看出，
GG基因型白来航母鸡的酶切产物仅有253bp一条带(该组部分鸡所在泳道1-3)，该组白来航母鸡基因组中红羽SNP位点基因型均为GG(测序所得，并经杂交验证)，其为红羽致因突变纯合型基因个体，即其与洛岛红公鸡杂交后代母鸡100％为红羽；253bp酶切产物测序为序列1第1-253bp；
AA基因型白来航母鸡的酶切产物仅有288bp一条带(该组部分鸡所在泳道8-9)，该组白来航母鸡基因组中红羽SNP位点基因型均为AA(测序所得，并经过杂交验证)，其为非红羽致因突变型个体，即该组白来航母鸡与洛岛红公鸡杂交后代母鸡100％为非红羽。288bp酶切产物测序为序列1第1-288bp；
GA基因型白来航母鸡的酶切产物有288bp和253bp的2条带(该组部分鸡所在泳道4-7)，该组白来航母鸡基因组中红羽SNP位点基因型均为GA(测序所得)，其为红羽致因突变杂合基因型个体，即该组白来航母鸡与洛岛红公鸡杂交后代母鸡既有红羽又有非红羽。288bp酶切产物测序为序列1第1-288bp；253bp酶切产物测序为序列1第1-253bp。
因此，可以上述方法检测待测白来航母鸡的红羽致因突变基因型或检测待测白来航鸡母鸡与洛岛红公鸡杂交后代母鸡的红羽性状，具体方法如下：
1、提取待测白来航母鸡的基因组DNA：
2、用上述二的引物进行PCR扩增，得到PCR扩增产物；
3、NruI酶切PCR扩增产物，得到酶切产物。
检测酶切产物大小：
若待测白来航母鸡的酶切产物大小仅为253bp，则该待测白来航母鸡为红羽致因突变纯合基因型个体(基因组中红羽SNP位点基因型为GG)，则该红羽致因突变纯合基因型的白来航鸡母鸡与洛岛红公鸡杂交后代母鸡仅为红羽性状；
若待测白来航母鸡的酶切产物大小仅为288bp，则该待测白来航母鸡为非红羽致因突变基因型个体(基因组中红羽SNP位点基因型均为AA)，则该非红羽致因突变基因型白来航鸡母鸡与洛岛红公鸡杂交后代母鸡仅为非红羽性状；
若待测白来航母鸡的酶切产物大小为288bp和253bp，则待测白来航母鸡为红羽致因突变杂合基因型个体(基因组中红羽SNP位点基因型均为AG)，则该红羽致因突变杂合基因型的白来航鸡母鸡与洛岛红公鸡杂交后代母鸡为红羽性状和非红羽性状。
红羽致因突变基因型为红羽致因突变纯合基因型或红羽致因突变杂合基因型：
上述红羽致因突变纯合基因型为基因组中红羽SNP位点基因型为GG，且该红羽致因突变纯合基因型的白来航母鸡与洛岛红公鸡杂交后代母鸡仅为红羽性状；
上述红羽致因突变杂合基因型为基因组中红羽SNP位点基因型为AG，且该红羽致因突变杂合基因型白来航母鸡与洛岛红公鸡杂交后代母鸡为红羽性状和非红羽性状。
上述非红羽致因突变基因型为基因组中红羽SNP位点基因型为AA，且该非红羽致因突变基因型白来航母鸡与洛岛红公鸡杂交后代母鸡仅为非红羽性状。
实施例2、红羽致因突变纯合基因型白来航鸡母本选育及红羽粉壳蛋鸡配套系的建立
一、红羽致因突变纯合基因型白来航鸡母本选育
1、提取基因组DNA
提取1573只待测白来航母鸡的基因组DNA。
2、PCR扩增
分别以1573只待测白来航母鸡的基因组DNA作为模板，用实施例1中上述三的方法中的步骤2进行PCR扩增，得到PCR扩增产物。
3、酶切
NruI酶切上述各只鸡的PCR扩增产物，得到酶切产物。
检测酶切产物大小：
若待测白来航母鸡的酶切产物大小仅为253bp，则该待测白来航母鸡为红羽致因突变纯合基因型个体，则该红羽致因突变纯合基因型的白来航鸡母鸡与洛岛红公鸡杂交后代母鸡仅为红羽性状；
若待测白来航母鸡的酶切产物大小仅为288bp，则该待测白来航母鸡为非红羽致因突变基因型个体，则该非红羽致因突变基因型白来航鸡母鸡与洛岛红公鸡杂交后代母鸡仅为非红羽性状；
若待测白来航母鸡的酶切产物大小为288bp和253bp，则待测白来航母鸡为红羽致因突变杂合基因型个体，则该红羽致因突变杂合基因型的白来航鸡母鸡与洛岛红公鸡杂交后代母鸡为红羽性状和非红羽性状。
结果如下：
1573只待测白来航母鸡中962只酶切产物大小仅为253bp，判断这些母鸡为红羽致因突变纯合基因型个体，其与洛岛红公鸡杂交后代中的母鸡为红羽；
1573只待测白来航母鸡中19只酶切产物大小仅为288bp，判断这些母鸡为非红羽致因突变基因型个体，其与洛岛红公鸡杂交后代中的母鸡为非红羽；
1573只待测白来航母鸡中592只酶切产物大小为288bp和253bp，判断这些母鸡为红羽致因突变杂合基因型个体，其与洛岛红公鸡杂交后代中的母鸡中有红羽和非红羽。
实验验证：
将上述方法鉴定中962只红羽致因突变纯合基因型个体作为母本与洛岛红公鸡杂 交，检测其杂交后代，得到3620只母鸡，其中红羽的为3620只。
将上述SNP鉴定中592只红羽致因突变杂合基因型个体作为母本与洛岛红公鸡杂交，检测其杂交后代，得到2257只母鸡，其中红羽的为1182只，非红羽的1075只。
将上述SNP鉴定中19只非红羽致因突变型个体作为母本与洛岛红公鸡杂交，检测其杂交后代，得到61只母鸡，其中红羽的为0只，非红羽的61只。
从上述杂交结果可以看出，与本发明方法鉴定结果一致，因此，可以看出，本发明的引物及其方法可以用来鉴定待测白来航鸡的红羽致因突变基因型，并且可以用来判断待测白来航鸡与洛岛红鸡杂交后代的羽毛颜色性状。
二、红羽粉壳蛋鸡配套系的建立
将GG基因型白来航鸡母鸡与洛岛红公鸡进行杂交配套，后代母鸡均为红羽，公雏均为非红羽，实现红羽粉壳蛋鸡配套系的建立。
三、红羽致因突变纯合基因型或红羽致因突变杂合基因型白来航公鸡的应用
由于红羽SNP位点位于常染色体，因此，GG型和GA型白来航母鸡的全同胞公鸡也可以用上述二的方法进行红羽致因突变基因型判定，选留GG型白来航公鸡。
将GG型白来航公鸡与所有GG型白来航母鸡个体进行扩繁和选育，经过四个世代的选育后得到红羽致因突变纯合基因型白来航鸡纯系，红羽SNP位点基因型为GG型。

Claims (10)

  1. 鉴定白来航鸡红羽致因突变基因型的引物对，其由序列表中序列2所示的单链DNA分子和序列表中序列3所示的单链DNA分子组成。
  2. 鉴定白来航鸡红羽致因突变基因型的试剂盒，包括权利要求1所述的引物对和限制性内切酶；
    所述限制性内切酶为NruI。
  3. 权利要求1所述的引物对或权利要求2所述的试剂盒在鉴定待测白来航鸡的红羽致因突变基因型中的应用。
  4. 权利要求1所述的引物对或权利要求2所述的试剂盒在选育红羽致因突变纯合基因型或红羽致因突变杂合基因型的白来航鸡中的应用；
    或权利要求1所述的引物对或权利要求2所述的试剂盒在选育与洛岛红公鸡杂交后代母鸡为红羽的白来航鸡中的应用；
    或权利要求1所述的引物对或权利要求2所述的试剂盒在鉴定待测白来航鸡与洛岛红公鸡杂交后代母鸡的红羽性状中的应用；
    或权利要求1所述的引物对或权利要求2所述的试剂盒在红羽粉壳蛋鸡配套系的建立中的应用。
  5. 根据权利要求1所述的引物对或权利要求2所述的试剂盒或权利要求3或4所述的应用，其特征在于：所述待测白来航鸡的红羽致因突变基因型为红羽致因突变纯合基因型或红羽致因突变杂合基因型。
  6. 一种鉴定待测白来航鸡的红羽致因突变基因型的方法，包括如下步骤：
    1)用权利要求1所述引物对对待测鸡进行PCR扩增，得到PCR扩增产物；
    2)将所述PCR扩增产物用权利要求2中的所述限制性内切酶酶切，得到酶切产物；
    3)检测酶切产物，根据酶切产物大小鉴定待测白来航鸡的红羽致因突变基因型；
    或，一种鉴定待测白来航鸡与洛岛红公鸡杂交后代母鸡的红羽性状的方法，包括如下步骤：
    1)用权利要求1所述引物对对待测鸡进行PCR扩增，得到PCR扩增产物；
    2)将所述PCR扩增产物用权利要求2中的所述限制性内切酶酶切，得到酶切产物；
    3)检测酶切产物，根据酶切产物大小鉴定待测白来航鸡与洛岛红公鸡杂交后代母鸡的红羽性状。
  7. 根据权利要求6所述的方法，其特征在于：
    所述酶切产物大小鉴定待测白来航鸡的红羽致因突变基因型为如下：
    若待测白来航母鸡的酶切产物大小仅为253bp，则该待测白来航母鸡为红羽致因突变纯合基 因型；
    若待测白来航母鸡的酶切产物大小仅为288bp，则该待测白来航母鸡为非红羽致因突变基因型；
    若待测白来航母鸡的酶切产物大小为288bp和253bp，则待测白来航母鸡为红羽致因突变杂合基因型；
    或所述根据酶切产物大小鉴定待测白来航鸡与洛岛红公鸡杂交后代母鸡的红羽性状为如下：若待测白来航母鸡的酶切产物大小仅为253bp，则该待测白来航的母鸡与洛岛红公鸡杂交后代母鸡仅为红羽性状；
    若待测白来航母鸡的酶切产物大小仅为288bp，则该待测白来航的母鸡与洛岛红公鸡杂交后代母鸡仅为非红羽性状；
    若待测白来航母鸡的酶切产物大小为288bp和253bp，则该待测白来航的母鸡与洛岛红公鸡杂交后代母鸡为红羽性状和非红羽性状。
  8. 根据权利要求6或7所述的方法，其特征在于：
    所述PCR扩增的模板为待测鸡的基因组DNA。
  9. 一种选育红羽致因突变纯合基因型的白来航鸡的方法，包括如下步骤：选育权利要求6-8任一中的红羽致因突变纯合基因型的白来航鸡；
    或一种选育杂交后代母鸡为红羽的待测白来航鸡的方法，包括如下步骤：选育权利要求6-8任一中的红羽致因突变纯合基因型的白来航鸡。
  10. 一种红羽粉壳蛋鸡配套系的培育方法，包括如下步骤：
    1)选育权利要求6-8任一中的红羽致因突变纯合基因型的白来航鸡；
    2)以所述红羽致因突变纯合基因型的白来航鸡为母本，洛岛红公鸡为父本进行杂交，后代母鸡均为红羽，公雏均为非红羽，即得到红羽粉壳蛋鸡配套系。
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