CN114686598B - Molecular breeding method for improving homogeneity of fine-wool sheep wool - Google Patents

Molecular breeding method for improving homogeneity of fine-wool sheep wool Download PDF

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CN114686598B
CN114686598B CN202011576911.0A CN202011576911A CN114686598B CN 114686598 B CN114686598 B CN 114686598B CN 202011576911 A CN202011576911 A CN 202011576911A CN 114686598 B CN114686598 B CN 114686598B
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邓学梅
王建魁
华国营
陈建飞
马宇浩
张媛媛
杨雪
李锦南
蔡甘先
张冰洁
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Abstract

The invention discloses a molecular breeding method for improving the homogeneity of fine-wool sheep wool. The invention provides a method for identifying the genotype of sheep GTL2-REs, which comprises the following steps: (1) Taking the genome DNA of a test sheep as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F1 shown in a sequence 1 and a primer R1 shown in a sequence 2; (2) And (3) carrying out agarose gel electrophoresis on the product, wherein if a single characteristic band is displayed, the sheep is of a GTL2-REs homozygous genotype, and if two characteristic bands are displayed, the sheep is of a GTL2-REs heterozygous genotype. The invention also provides a method for breeding sheep with the same quality Mao Xingzhuang, which comprises the following steps: taking female sheep of GTL2-REs heterozygous genotype as female parent and male sheep of GTL2-REs homozygous genotype as male parent to obtain offspring. The method can be used in the process of breeding the homogeneous wool of the fine wool sheep and provides a new molecular breeding method for preventing the generation of young heterogeneous wool offspring.

Description

Molecular breeding method for improving homogeneity of fine-wool sheep wool
Technical Field
The invention belongs to the technical field of biology, and relates to a molecular breeding method for improving the homogeneity of wool of a fine-wool sheep, in particular to application of different genotypes of a GTL2-Mirg section of repetitive sequence of a sheep to homogeneity breeding of wool-use fine-wool sheep, which can be used for sheep breeding.
Background
In the breeding of fine wool sheep, the homogeneity of wool fibers is very important. The fine-hair sheep have predominantly secondary follicles and also have a small number of primary follicles, especially in the born lambs, with a significantly larger number of primary follicles present in some lambs. Most of the hair generated from the primary hair follicle is medullary hair, the hair quality becomes hard, mao Zhijing becomes thick, and the quality of the wool is seriously influenced. Homogeneous wool is a prerequisite for wool raw material requirements in the wool spinning industry, and the wool to be shoddy is required to be homogeneous firstly in the breeding of fine wool sheep, and on the basis, the comprehensive quality of wool is further required to be improved. In recent years, due to the change of the specific value of wool and mutton, the selection strength of wool is reduced by improving the meat value, and more seriously, the wool homogeneity of later generations is directly influenced by the gradual 'reverse improvement' trend in the fine wool sheep producing area, namely the hybridization of local ram and fine wool sheep. The 'reverse modification' leads to the increasing difficulty in maintaining excellent properties, so that the yield of high-quality fine wool which cannot meet the requirement of domestic wool finish machining is further reduced, and finally the requirement of domestic wool spinning enterprises on high-quality wool depends on import seriously.
In the actual breeding process of fine-wool sheep, in order to improve the proportion of homogeneous wool sheep, people often adopt a seed selection method for eliminating individual heterogeneous wool sheep, but actually, the elimination method cannot achieve a complete purification effect, and in fact, the heterogeneous wool type phenomenon of the born lambs is not completely eliminated in merino fine-wool sheep varieties all the time. To date, no major genes have been identified that are localized to determine wool homogeneity or heterogeneity. Therefore, a method of utilizing the gene for the directional breeding has not been found.
Disclosure of Invention
The invention aims to provide a molecular breeding method for improving the wool homogeneity of fine-wool sheep, and particularly relates to application of different genotypes of a GTL2-Mirg section of repetitive sequences of sheep to sheep wool homogeneity breeding of fine-wool sheep, which can be used for sheep breeding.
The invention provides a method for identifying the genotype of GTL2-REs of sheep, which comprises the following steps:
(1) Taking the genome DNA of a test sheep as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F1 and a primer R1; the primer F1 is a single-stranded DNA molecule shown in a sequence 1 of a sequence table, and the primer R1 is a single-stranded DNA molecule shown in a sequence 2 of the sequence table;
(2) And (2) carrying out agarose gel electrophoresis on the product obtained in the step (1), wherein if a single characteristic band is displayed, the sheep is of a GTL2-REs homozygous genotype, and if two characteristic bands are displayed, the sheep is of a GTL2-REs heterozygous genotype.
In the reaction system for PCR amplification, the concentration of the primer F1 was 0.25mM, and the concentration of the primer R1 was 0.25mM. The reaction system for PCR amplification may specifically be: primer F1.5. Mu.l, primer R1.5. Mu.l, 2 XTaq PCR Mix 10. Mu.l, genomic DNA 100ng, double distilled water to make up 20. Mu.l.
The reaction procedure of PCR amplification may specifically be: 5 minutes at 95 ℃; 35 cycles of 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, 72 ℃ for 30 seconds; 5min at 72 ℃; storing at 4 ℃.
The agarose gel electrophoresis may be 1.5% agarose gel electrophoresis.
The size of the PCR amplified product is 400-600bp.
The size of the target product amplified by the PCR is 400-600bp.
The size of the specific target product amplified by the PCR is 400-600bp.
Correspondingly, the characteristic band is located in the interval of 400-600bp.
The invention also provides a method for breeding sheep with the same quality Mao Xingzhuang, which comprises the following steps:
identifying the genotype of the sheep GTL2-REs by adopting the method;
and taking a female sheep with the GTL2-REs heterozygous genotype as a female parent to obtain a descendant.
The female parent is a sheep with homoplasmy Mao Xingzhuang.
In the method, a male sheep with a GTL2-REs homozygous genotype is used as a male parent.
The male parent is a sheep with heterogeneity Mao Xingzhuang.
The invention also provides a method for assisting in identifying the sheep wool traits, which comprises the following steps:
identifying the genotype of the sheep GTL2-REs by adopting the method;
if the test sheep is a GTL2-REs homozygous genotype, the test sheep is a candidate sheep with heterogeneity Mao Xingzhuang; if the sheep to be tested is of the GTL2-REs heterozygous genotype, the sheep is selected to have the homogeneous hair character.
The invention also provides a specific primer pair which consists of a primer F1 and a primer R1; the primer F1 is a single-stranded DNA molecule shown in a sequence 1 in a sequence table, and the primer R1 is a single-stranded DNA molecule shown in a sequence 2 in the sequence table.
The invention also protects the application of the specific primer pair in identifying sheep hair traits.
The invention also protects the application of the specific primer pair in breeding sheep with the homogeneous Mao Xingzhuang.
The invention also protects the application of the specific primer pair in breeding sheep with the homogeneous Mao Xingzhuang.
Any of the above sheep is a fine wool sheep.
Any of the sheep described above is a merino fine wool sheep.
Any of the sheep described above is a Australian merino fine wool sheep.
The inventor of the invention finds a sheep GTL2-Mirg gene region with a repeated sequence, the repeated sequence has a plurality of copy forms, and the genotype (homozygous genotype or heterozygous genotype) of the repeated sequence is related to sheep hair traits. Furthermore, the inventor of the present invention found that all offspring of merino fine-hair sheep with heterogeneity Mao Xingzhuang as female parent have heterogeneity Mao Xingzhuang, and all offspring of merino fine-hair sheep with homogeneous hair character as female parent have homogeneous hair character, that is, the offspring is inherited by Mao Xingzhuang (homogeneous hair character or heterogeneity Mao Xingzhuang) as female parent. Based on the method, the hybridization method is developed, the problem of wool heterogeneity caused by 'reverse modification' of the fine-wool sheep is solved, and a new molecular breeding scheme is provided for the homogeneity improvement of the fine-wool sheep. The invention can effectively improve the homogeneity of the fine wool sheep and solve the problem of eliminating heterogeneous wool type individuals in fine wool sheep breeding.
The invention discovers for the first time in the world that a section of repetitive sequence in the GTL2-Mirg gene region of sheep has genotype difference in individuals with different pilus phenotypes. Further discloses the application of the GTL2-REs genotype in breeding of the character for the fine-hair wool for the first time. The method can be used in the process of breeding the homogeneous wool of the fine wool sheep and provides a new molecular breeding method for preventing the generation of young heterogeneous wool offspring.
Drawings
FIG. 1 is an agarose gel electrophoresis image of a portion of the sample.
FIG. 2 is a graph showing the ratio of a GTL2-REs homozygous genotype to a GTL2-REs heterozygous genotype in a homogeneous wool sheep population and a heterogeneous wool sheep population.
FIG. 3 shows the results of the orthogonal test and the inverse orthogonal test.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. All sheep, observed in the lamb stage, were judged to be either homogeneous or heterogeneous by Mao Xingzhuang. The recipient ewes in example 2 were all Wu Zhumu Qin ewes.
Example 1 detection of sheep GTL2-REs genotype
Test sheep: tan sheep (39), small-tailed Han sheep (30), wu Zhumu Qin sheep (37), tan Han miscellaneous sheep (38), heterogeneous wool merino sheep (13), homogeneous wool merino sheep (60), aohan fine wool sheep (50) and Gansu alpine fine wool sheep (88). The Tan Han miscellaneous sheep is the hybrid offspring of Tan sheep and small tailed Han sheep. The offspring of the australian merino fine wool sheep, with some of the coats of the merino fine wool sheep being heterogeneous hairs, are called heterogeneous wool merino sheep, and the remainder of the coats of the merino wool sheep, being homogeneous hairs, are called homogeneous wool merino sheep.
1. The ear tissue of the sheep to be tested was extracted to obtain genomic DNA.
2. And (3) performing PCR amplification by using the genomic DNA obtained in the step (1) as a template and adopting a primer pair consisting of a primer F1 and a primer R1.
Primer F1 (SEQ ID NO: 1 of the sequence table): 5'-GGTGTCTGCTCCTGGATGGT-3';
primer R1 (sequence 2 of the sequence table): 5'-ATCAGCCAACAGTCAGTTCAGC-3'.
The size of the specific amplification product amplified by PCR is 400-600bp.
Reaction system of PCR amplification: primer F1.5. Mu.l, primer R1.5. Mu.l, 2 × taq PCR Mix 10. Mu.l, genomic DNA 100ng, made up to 20. Mu.l in double distilled water. In the reaction system for PCR amplification, the concentration of the primer F1 was 0.25mM, and the concentration of the primer R1 was 0.25mM.
Reaction procedure for PCR amplification: 5 minutes at 95 ℃; 35 cycles of 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, 72 ℃ for 30 seconds; 5min at 72 ℃; storing at 4 ℃.
3. And (3) taking the amplification product obtained in the step (2), carrying out 1.5% agarose gel electrophoresis, and if a single characteristic band is displayed, the sheep is of a GTL2-REs homozygous genotype, and if two characteristic bands are displayed, the sheep is of a GTL2-REs heterozygous genotype. The characteristic band is located in the interval of 400-600bp.
The electrophoretogram of a portion of the test sheep is shown in FIG. 1 (the characteristic bands are marked in the block diagram of the figure). The genotype results for each sheep tested are shown in Table 1 (heterozygous, i.e., individuals heterozygous for GTL2-REs, homozygous for GTL 2-REs). The heterogeneous hair merino sheep are all of GTL2-REs homozygous genotypes. In homogeneous hair merino sheep, 95% are GTL2-REs heterozygous genotypes, and 5% are GTL2-REs homozygous genotypes.
The ratio of the GTL2-REs homozygous genotype to the GTL2-REs heterozygous genotype in the homogeneous wool sheep population (consisting of all homogeneous wool individuals in all sheep tested) and the heterogeneous Mao Mianyang population (consisting of all heterogeneous wool individuals in all sheep tested) is shown in figure 2. For all samples, sheep with heterogeneous Mao Biaoxing were predominantly of the GTL2-REs homozygous genotype, sheep with the homogeneous wool phenotype were predominantly of the GTL2-REs heterozygous genotype.
TABLE 1
Figure BDA0002864508020000041
Example 2 application of different genotypes of GTL2-REs in Mucuna capillaris homogeneity breeding
1. Backcross test
The female parent was the 1 hetero-hair merino ewe (GTL 2-REs homozygous genotype) of example 1, and the male parent was the 1 homo-hair merino ewe (GTL 2-REs heterozygous genotype) of example 1. Carrying out estrus synchronization and superovulation treatment on the female parent, carrying out artificial semen collection on the male parent, then carrying out semen deposition on the female parent, carrying out embryo-flushing treatment on the female parent, and flushing 6 embryos out in total. The punched embryos were transplanted into the uterus of 5 recipient ewes. Pregnancy test was performed 45 days later, 5 recipients were pregnant, and 150 days later after transplantation, 5 lambs were produced, which were heterogenized by Mao Junwei.
2. Quadrature test
1 heterogeneous ram merino ram (GTL 2-REs homozygous genotype) from example 1 was used as male parent, and 7 homogeneous ram merino ram female (GTL 2-REs heterozygous genotype) from example 1 was used as female parent. Carrying out estrus synchronization and superovulation treatment on the female parent, carrying out semen artificial collection on the male parent, then carrying out semen deposition on the female parent, carrying out embryo washing treatment on the female parent, carrying out 71 washed embryos, freezing and storing 18 embryos, and transplanting the rest 53 washed embryos into 27 female ewes. Pregnancy test was carried out 45 days later, 19 subjects were pregnant, and 150 days after transplantation, 26 lambs were produced, and the hair of the quilt was homogeneous.
The orthogonal and inverse orthogonal protocols and results are shown in FIG. 3. The results show that the foreign hair character of the merino fine wool sheep is only inherited to the offspring lambs by the female parent and cannot be inherited to the offspring lambs by the male parent. The sheep with the GTL2-REs heterozygous genotype is used as a female parent, and the sheep with the GTL2-REs homozygous genotype is used as a male parent, so that the generation of young heterogeneous wool offspring can be effectively reduced, and the homogeneity of wool of a colony is improved.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Sequence listing
<110> university of agriculture in China
<120> molecular breeding method for improving homogeneity of fine-wool sheep wool
<130> GNCYX203416
<160> 2
<170> SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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ggtgtctgct cctggatggt 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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atcagccaac agtcagttca gc 22

Claims (2)

1. A method of breeding sheep having homogeneity Mao Xingzhuang comprising the steps of:
(1) Taking the genome DNA of a test sheep as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F1 and a primer R1; the primer F1 is a single-stranded DNA molecule shown in a sequence 1 in a sequence table, and the primer R1 is a single-stranded DNA molecule shown in a sequence 2 in the sequence table;
(2) Carrying out agarose gel electrophoresis on the product obtained in the step (1), if a single characteristic band is displayed, the sheep is a GTL2-REs homozygous genotype, and if two characteristic bands are displayed, the sheep is a GTL2-REs heterozygous genotype; the characteristic band is positioned in a range of 400-600 bp;
(3) Taking a female sheep with GTL2-REs heterozygous genotype and with homogeneous hair character as a female parent, and taking a male sheep with GTL2-REs homozygous genotype and with heterogeneity Mao Xingzhuang as a male parent to obtain offspring;
the sheep are merino fine-wool sheep.
2. A method for assisting in identifying the homogeneous wool character and the heterogeneous wool character of a sheep comprises the following steps:
(1) Taking the genome DNA of a test sheep as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F1 and a primer R1; the primer F1 is a single-stranded DNA molecule shown in a sequence 1 of a sequence table, and the primer R1 is a single-stranded DNA molecule shown in a sequence 2 of the sequence table;
(2) Carrying out agarose gel electrophoresis on the product obtained in the step (1), if a single characteristic band is displayed, the sheep is a GTL2-REs homozygous genotype, and if two characteristic bands are displayed, the sheep is a GTL2-REs heterozygous genotype; the characteristic band is positioned in a range of 400-600 bp;
(3) If the test sheep is a GTL2-REs homozygous genotype, the test sheep is a candidate sheep with heterogeneity Mao Xingzhuang; if the test sheep is a GTL2-REs heterozygous genotype, the test sheep is a candidate sheep with the homogeneous hair character;
the sheep are merino fine-wool sheep.
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刘国庆 ; 代蓉 ; 任航行 ; 王新华 ; 刘守仁 ; 孙业良 ; 杨利国 ; .新疆肉羊18号染色体上与后臀肌发育相关基因的多态性分析.遗传.2006,(07),第815-820页. *
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