SCREENING METHOD FOR HOMOZYGOUS GENOTYPE AGOUTI HAIR COLOR RABBITS

Abstract

The invention provides a selecting method for domestic rabbits with a agouti/Belgian hair color based on homozygous genotype selection. The purification method is applicable to purification of various domestic rabbits with the agouti/Belgian hair color. The method includes the following steps: firstly, selecting the domestic rabbits with a hair color phenotype which is the agouti/Belgian hair color; then detecting an ASIP gene, a MC1R gene and a TYR gene at the same time by utilizing a first primer set, a second primer set and a third primer set; screening the domestic rabbits with genotypes of AA (corresponding to the ASIP gene), EE (corresponding to the MC1R gene) and CC (corresponding to the TYR gene). The domestic rabbits with the genotype of AAEECC, which are obtained by the screening method of the invention, are agouti/Belgian hair color homozygotes and the recessive white gene is eliminated.

Claims

1. A method for selecting a homozygous genotype agouti hair color rabbit, the method comprising the following steps: (1) extracting genomic DNA of a domestic rabbit to be identified, and using a first primer set, a second primer set and a third primer set respectively to amplify to obtain an amplified gene product, the first primer set is used to detect a base sequence of ASIP gene, the second primer set is used to detect a base sequence of MC1R gene, and the third primer set is used to detect a base sequence of TYR gene; and (2) according to the amplified gene product obtained in step (1), identifying a genotype of the domestic rabbit to be identified, and the homozygous phenotype agouti hair color rabbit is obtained by selecting.

2. The method according to claim 1, wherein in step (1), the rabbit to be identified is any one of meat rabbits, rex rabbits, long-hair rabbits, or ornamental rabbits.

3. The method according to claim 1, wherein in step (1), a phenotype of the hair color of the domestic rabbit to be identified is agouti hair color.

4. The method according to claim 1, wherein in step (1), a forward primer of the first primer set is 5′-AAGAAAGCAGGAAGGCACA-3′ as set forth in SEQ ID NO.1, and a reverse primer is 5′-CAAGGCAGGATTGGCTCA-3′ as set forth in SEQ ID NO.2; a forward primer of the second primer set is 5′-ACCTGCTGGTGAGCGTGA-3′ as set forth in SEQ ID NO.3, and a reverse primer is 5′-GTAGCGCAGTGCGTAGAAGA-3′ as set forth in SEQ ID NO.4; and a forward primer of the third primer set is 5′-AAGACAAGGTGAAAGG-3′ as set forth in SEQ ID NO.5, and a reverse primer is 5′-TAAATCCAATAGGCAC-3′ as set forth in SEQ ID NO.6.

5. The method according to claim 1, wherein in step (1), when the first primer set is used for amplification, an amplification system is: a total volume of 20 μL, including Tris-HCL 33.5 mM, MgCl.sub.2 25 mM, dNTPs 0.2 mM, forward primers and reverse primers are 5 ng/μl respectively, Taq DNA polymerase 2.5 U, and template DNA 50 ng; an amplification procedure is: pre-denaturation at 94° C. for 5 minutes; then proceeding 30 cycles of the following steps: denaturation at 94° C. for 45 s, annealing at 52° C. for 45 s, and extending at 72° C. for 30 s; after completing the 30 cycles, extending at 72° C. for 5 min, then a PCR amplification program is completed and storing obtained products at 4° C.

6. The method according to claim 1, wherein in step (1), when an second primer set is used for amplification, an amplification system is: a total volume of 20 μL, including Tris-HCL 33.5 mM, (NH.sub.4).sub.2SO.sub.4 8.0 mM, MgCl.sub.2 1.5 mM, TWEEN-20 0.05%, dNTPs 0.2 mM, forward primers and reverse primers are 3.3 ng/μl respectively, Taq DNA polymerase 2.0 U and template DNA 50 ng; an amplification procedure is: pre-denaturation at 94° C. for 2 min; then proceeding 30 cycles of the following steps: denaturation at 94° C. for 45 s, annealing at 62° C. for 45 s, extending at 72° C. for 60 s; after completing the 30 cycles, extending at 72° C. for 8 min, then a PCR amplification program is completed and storing obtained products at 4° C.

7. The method according to claim 1, wherein in step (1), when the third primer set is used for amplification, an amplification system is: a total volume of 20 μL, including Tris-HCL 33.5 mM, MgCl.sub.2 25 mM, dNTPs 0.2 mM, forward primers and reverse primers are 5 ng/μl respectively, Taq DNA polymerase 2.5 U, and template DNA 50 ng; an amplification procedure is: pre-denaturation at 94° C. for 5 minutes; then proceeding 30 cycles of the following steps: denaturation at 94° C. for 45 s, annealing at 52° C. for 45 s, and extending at 72° C. for 30 s; after completing the 30 cycles, extending at 72° C. for 5 min, then a PCR amplification program is completed and storing obtained products at 4° C.

8. The method according to claim 1, wherein in step (2), a identification method is: sequencing the amplified product obtained by using the first primer set, and if a base “A” is detected as an insertion between a 5th position and a 6th position of a second exon of the ASIP gene, it is recorded as Genotype “a”, and when the base “A” is not detected, it is recorded as Genotype “A”; sequencing the amplified product obtained by using the second primer set, and if there is no deletion of bases at positions 280-285 of the MC1R gene and a 30 bp deletion of bases at positions 304-333 is detected, it is recorded as Genotype “E”; and sequencing the amplified product obtained by using the third primer set, and detecting a base type at position 1119 of a fourth exon of the TYR gene, when the base is “C”, it means that no gene mutation has occurred, thus it is recorded as Genotype “C”; when the base is “A”, it means that a gene mutation has occurred, and it is recorded as Genotype “c”.

9. The method according to claim 1, wherein the genotype of a homozygous agouti hair color rabbit obtained by selecting is AAEECC.

10. A method for breeding agouti hair color rabbits, comprising selecting homozygous genotype agouti hair color rabbits using the method of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to illustrate the examples of the present invention or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings that need to be used in the description of the examples or prior art. Obviously, the drawings in the following description are only some examples of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

[0035] FIG. 1 is a diagram of the amplified fragments of the ASIP gene in Example 1, wherein the sequence (SEQ ID NO.7) in the upper part of FIG. 1 is the corresponding base sequence determined according to the sequencing peak map;

[0036] FIG. 2 is a diagram showing the sequence detection of the MC1R gene in Example 1, wherein 9 base sequences are fragments of the MC1R gene sequence of rabbits with different hair colors, the 9 sequences from top to bottom include SEQ ID NO.9 of white 1, SEQ ID NO.9 of white 2, SEQ ID NO.10 of black 1, SEQ ID NO.12 of black 2, SEQ ID NO.9 of linen 1, SEQ ID NO.9 of linen 2, SEQ ID NO.9 of deep linen 1, SEQ ID NO.9 of deep linen 2, and SEQ ID NO.11 of consensus sequence;

[0037] FIG. 3 is a diagram of amplified fragments of the TYR gene in Example 1, wherein the sequence (SEQ ID NO.8) in the upper part of FIG. 3 is the corresponding base sequence determined according to the sequencing peak map;

[0038] FIG. 4 is a diagram showing that the offspring of the unpurified agouti/Belgian hair color meat rabbits have obvious hair color separation;

[0039] FIG. 5 is a diagram showing that the hair colors of the offspring of the purified agouti/Belgian hair color meat rabbits are purely consistent;

[0040] FIG. 6 is a diagram showing that the hair colors of the offspring of the purified agouti/Belgian hair color meat rabbits and the white meat rabbits are the same.

DETAILED DESCRIPTION OF THE INVENTION

[0041] In order to make the purpose, technical solutions and advantages of the present invention to be clear, the present invention is further described in detail below. Obviously, the described examples are only a part of the examples of the present invention, rather than all the examples. Based on the examples of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

EXAMPLE 1

[0042] The method for selecting homozygous genotype of agouti/Belgian hair color domestic rabbits, comprising the following steps:

[0043] 1. Extracting the genomic DNA of agouti/Belgian hair color meat rabbits, and using the first primer set, the second primer set and the third primer set to amplify to obtain the amplified products;

[0044] The designed primers of this example are shown in Table 1;

TABLE-US-00001 TABLE 1 Primer Set Sequences Length of the Primers Primer sequence product The forward 5′-AAGAAAGCAGGAAGGCACA-3′ 600 bp primer of the 1.sup.st (SEQ ID NO. 1) primer set The reverse 5′-CAAGGCAGGATTGGCTCA-3′ primer of the 1.sup.st (SEQ ID NO. 2) primer set The forward 5′-ACCTGCTGGTGAGCGTGA-3′ 620 bp primer of the 2.sup.nd (SEQ ID NO. 3) primer set The reverse 5′-GTAGCGCAGTGCGTAGAAGA-3′ primer of the 2.sup.nd (SEQ ID NO. 4) primer set The forward 5′-AAGACAAGGTGAAAGG-3′ 488 bp primer of the 3.sup.rd (SEQ ID NO. 5) primer set The reverse 5′-TAAATCCAATAGGCAC-3′ primer of the 3.sup.rd (SEQ ID NO. 6) primer set

[0045] When the first primer set is used for amplification, the amplification system is: a total volume of 20 μL, including Tris-HCL 33.5 mM, MgCl.sub.2 25 mM, dNTPs 0.2 mM, the forward primers and the reverse primers are 5 ng/μl respectively, Taq DNA polymerase 2.5 U, and template DNA 50 ng; the amplification procedure is: pre-denaturation at 94° C. for 5 minutes; then proceeding 30 cycles of the following steps: denaturation at 94° C. for 45 s, annealing at 52° C. for 45 s, and extending at 72° C. for 30 s; after completing the 30 cycles, extending at 72° C. for 5 min, then the PCR amplification program is completed and storing the obtained products at 4° C.

[0046] When the second primer set is used for amplification, the amplification system is: a total volume of 20 μL, including Tris-HCL 33.5 mM, (NH.sub.4).sub.2804 8.0 mM, MgCl.sub.2 1.5 mM, TWEEN-20 0.05%, dNTPs 0.2 mM, the forward primer and the reverse primer are 3.3 ng/μl respectively, Taq DNA polymerase 2.0 U and template DNA 50 ng; the amplification procedure is: pre-denaturation at 94° C. for 2 min; then proceeding 30 cycles of the following steps: denaturation at 94° C. for 45 s, annealing at 62° C. for 45 s, extending at 72° C. for 60 s; after completing the 30 cycles, extending at 72° C. for 8 min, then the PCR amplification program is completed and storing the obtained products at 4° C.

[0047] When the third primer set is used for amplification, the amplification system is: a total volume of 20 μL, including Tris-HCL 33.5 mM, MgCl.sub.2 25 mM, dNTPs 0.2 mM, the forward primer and the reverse primer are 5 ng/μl respectively, Taq DNA polymerase 2.5 U, and template DNA 50 ng; the amplification procedure is: pre-denaturation at 94° C. for 5 minutes; then proceeding 30 cycles of the following steps: denaturation at 94° C. for 45 s, annealing at 52° C. for 45 s, and extending at 72° C. for 30 s; after completing the 30 cycles, extending at 72° C. for 5 min, then the PCR amplification program is completed and storing the obtained products at 4° C.

[0048] 2. According to the amplified product obtained in step (1), identifying the genotype of the agouti/Belgian hair color meat rabbit, and selecting for the homozygous agouti/Belgian hair color meat rabbit. The specific selecting method is as follows:

[0049] sequencing the amplified product obtained by using the first primer set, and if a base “A” is detected as the insertion between the 5th position and the 6th position of the second exon of the ASIP gene, it is recorded as Genotype “a”, and when the base “A” is not detected, it is recorded as Genotype “A”; sequencing the amplified product obtained by using the second primer set, and if there is no deletion of bases at positions 280-285 of the MC1R gene and a 30 bp deletion of bases at positions 304-333 is detected, it is recorded as Genotype “E”; sequencing the amplified product obtained by using the third primer set, and detecting the base type at position 1119 of the fourth exon of the TYR gene, when the base is “C”, it means that no gene mutation has occurred, thus it is recorded as Genotype “C”; when the base is “A”, it means that a gene mutation has occurred, and it is recorded as Genotype “c”.

[0050] 3. After selecting for and obtaining homozygous agouti/Belgian hair color meat rabbits, establishing a basic pool of homozygous agouti/Belgian hair color meat rabbits.

[0051] Using the above primer sets and the identification method, the ASIP gene, the MC1R gene and the TYR gene can be detected at the same time, and the domestic rabbits with the genotypes of “AA” (corresponding to ASIP gene), “EE” (corresponding to MC1R gene) and “CC” (corresponding to TYR gene) are selected. The hybridization of male and female rabbits with genotype “AAEECC” can establish a homozygous agouti/Belgian hair color domestic rabbit population. Correspondingly, the agouti/Belgian hair color heterozygotes with the genotypes “Aa” and “Ee” and the individuals carrying the recessive white gene with the genotypes “Cc” and “cc” can be eliminated to achieve the purification of the agouti/Belgian hair color domestic rabbits. The rabbits obtained by the method of the present invention are homozygous, and the recessive white gene is eliminated. When hybridizing with other breeds, it not only ensures that no white hair color offspring will be born, but also ensures the uniformity of the rabbit phenotype. Under the condition of ensuring the products consistency of the breeding enterprises, the breeding materials of breeding enterprises are well protected.

[0052] The first primer set is used to amplify the ASIP gene fragment, as shown by the position indicated by the arrow in FIG. 1. It can be clearly seen that there are overlapping peaks at the pointing place, indicating that there are multiple bases at this position and there is a genetic mutation with a mutation Genotype “a”.

[0053] The second primer set is used to amplify the MC1R gene fragment. As shown in FIG. 2, the bases at positions 280-285 of the MC1R gene are not deleted, and there are 30 bp bases deletion at positions 304-333, thus it is Genotype “E”. It should be noted that the sequencing is based on the sequence of the MC1R gene, to check whether the bases at positions 280-285 and 304-333 of the MC1R gene are missing, but not to check whether the bases at positions 280-285 and 304-333 of the amplified sequence (the length of the amplified product is 620 bp).

[0054] The third primer set is used to detect the base type at position 1119 of the TYR gene. As shown by the position pointed by the arrow in FIG. 3, it can be clearly seen that there are overlapping peaks at the pointing place, indicating that there are multiple bases at this position and there is a genetic mutation with a mutation Genotype “c”.

EXAMPLE 2

[0055] The difference between Example 2 and Example 1 is only that the selected rabbit breeds are different. In this example, the agouti/Belgian hair color long-hair rabbits are selected as the objects, and other steps are all the same. The method of this example can screen and obtain homozygous genotype Belgian long-haired rabbits to establish a basic group of homozygous genotype Belgian long-haired rabbits.

EXAMPLE 3

[0056] The difference between Example 3 and Example 1 is only that the selected rabbit breeds are different. In this example, the agouti/Belgian hair color rex rabbits are selected as the objects, and other steps are all the same. The method of this example can screen and obtain homozygous genotype agouti/Belgian hair color rex rabbits to establish a basic group of homozygous genotype agouti/Belgian hair color rex rabbits.

EXAMPLE 4

[0057] In this example, a population of homozygous agouti/Belgian hair color meat rabbits is obtained following the same steps as in Example 1, and then using them as the hybridization parents or as the breeding material to cultivate specialized breeds. The offspring of agouti/Belgian hair color meat rabbits with the same hair color can be obtained by economically crossing the parental agouti/Belgian hair color meat rabbits with the white meat rabbit parent or the matching line. The results of this example are shown in Table 2 and FIG. 4-6.

TABLE-US-00002 TABLE 2 Statistics on Hair Color and Genotype of 298 Offspring from unpurified agouti/Belgian hair color meat Rabbit Intercrossing Hair ASIP genotype MC1R genotype TYR genotype color total AA Aa aa EE Ee ee CC Cc cc white  50  12 28 10  29 14  7  0  0 50 black  40  15 19  6  0  0 40 14  26  0 linen 196 133 63  0 140 56  0 76 120  0 yellow  12  0  0 12  8  4  0  3  9  0

[0058] The above examples are only specific examples of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily obtained by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.