Black brown Chinese merino fine wool sheep breeding method based on editing genotype selection

Abstract

The invention discloses a black brown Chinese merino fine wool sheep breeding method based on editing genotype selection. The method provided by the invention comprises the following steps: 1) mating F0-generation Chinese Merino fine-wool rams with two genotypes and non-white wool color with wild-type Chinese Merino fine-wool ewes to produce F1-generation sheep; (2) selecting ewes of F1-generation ASIP gene edited sheep with any one of the two genotypes and non-white hair color from the F1-generation sheep to mate with F0-generation ASIP gene edited rams to obtain F2-generation target sheep; according to the method, the population with the specific genotype is bred, breeding is guided through artificial insemination or natural mating, gradual propagation of the population is achieved, and after propagation breeding, the black-brown fine-wool sheep population which is neat in wool color and capable of stably inheriting can be bred.

Description

[0001] This application is a divisional application with an application date of April 25, 2022, application number 202210438127.6, and invention name "A breeding method for black-brown Chinese Merino fine-wool sheep based on editing genotype selection". Technical Field

[0002] The invention belongs to the technical field of sheep genetic breeding and molecular biology, and relates to a black-brown Chinese Merino fine-wool sheep breeding method based on editing genotype selection. Background Art

[0003] In the genetic breeding of sheep, coat color, as an important breed characteristic, plays an important role in determining hybrid combinations, identifying breed purity and kinship. At the same time, in sheep breeding, coat color is related to adaptability, disease resistance, and growth and development, and is regarded as one of the important breeding traits. ASIP (agouti signaling protein) is a signal protein encoded by the agouti locus. It is the main candidate gene affecting sheep wool color and plays an important regulatory role in pigment formation. China Merino fine wool sheep (Xinjiang type) (also known as China Merino sheep (Xinjiang type)) is a fine wool sheep used for both wool and meat in Xinjiang. After long-term breeding, the coat color is single, mainly white, and there are very few individuals of other colors. With the development and changes of the market, the improvement of people's quality of life and the awareness of the side effects of chemical printing and dyeing, people's demand for natural colored wool has gradually increased.

[0004] With the rapid development of molecular genetics and new biological technologies in recent years, the emergence of new technologies has not only broken through the genetic problems that cannot be solved in the traditional sheep breeding field, but also achieved the change of specific traits, greatly improving the speed of genetic improvement of sheep breeds. If it is possible to change the animal's coat color by artificially intervening in the expression of the animal's coat color gene, and further establish a stable and uniform coat color group through genotype selection and cross breeding, it will meet this market demand and improve the production value and economic benefits of fur animals. At the same time, it can greatly accelerate the process of coat color trait selection and breeding.

[0005] Because animal coat color is controlled by multiple genes, the coat color separation of hybrid offspring of different breeds of sheep is also different. The ideal coat color separation follows Mendel's law of separation of a pair of traits, that is, all offspring are heterozygous and show dominant characteristics; coat color separation occurs in the second generation, and the separation ratio of dominant and recessive characteristics is close to 3:1. However, due to the complex inheritance of coat color genes and the large number of genes that affect coat color, the types and proportions of coat color separation in individual offspring will also be very different. Moreover, coat color has dominant and recessive characteristics in heredity. Conventional breeding methods often take several or even more than a dozen generations to select and fix a coat color. The breeding cycle is long and the technology is difficult. It is considered to be one of the main technical difficulties in animal genetic breeding. In addition, if sheep are selected and bred only based on the phenotypic traits of wool color, there is also the problem of trait separation in the offspring population.

[0006] In addition, there are multiple copies of the sheep wool color ASIP gene, which may be located at different chromosomal locations or in different regions of the same chromosome. Multiple copies of the ASIP gene may exist in the genome in the form of tandem duplication, scattered distribution, etc., and provide more genetic variation, so that the sheep population can show a rich variety of wool colors, and different copies of the ASIP gene may differ in expression level, spatiotemporal specificity or function, which enables the wool color of sheep to be more finely regulated, thereby producing complex wool color patterns and tones. For example, Norris et al.'s study showed that there are multiple copies of the ASIP gene on the sheep genome (see: Norris Belinda J, Whan Vicki A. A gene duplication affecting expression of the ovine ASIP gene is responsible for white and black sheep. Genome Research, 2008, 18 (8): 1282-1293.); and Chinese patent CN10374082A discloses a kit for detecting the copy variation of the sheep wool color Agouti gene (i.e., ASIP gene). Since there are multiple copies of the ASIP gene in sheep, when detecting the genotype of ASIP gene-edited individuals, it may be that the same individual has multiple different genotypes in the sequence at the same position. However, there is currently little research on how to use different coat color genotypes to breed new sheep breeds. Summary of the invention

[0007] An object of the present invention is to provide a method for multiplying or cultivating non-white Chinese Merino fine-wool sheep.

[0008] The method provided by the present invention comprises the following steps:

[0009] 1) F with the following two genotypes and non-white coat color0 The first generation of Chinese Merino fine wool sheep rams were bred with wild type Chinese Merino fine wool sheep ewes to produce F 1 Sheep;

[0010] 2) From the F 1 Select the F sheep with any of the two genotypes and a non-white coat color. 1 The ewes of the first generation of ASIP gene-edited sheep and F 0 The ASIP gene-edited ram was bred to obtain F 2 The target sheep (black or brown target sheep); 0 The ASIP gene-edited rams are Chinese Merino fine-wool sheep rams with the following two genotypes and non-white coat colors;

[0011] The two genotypes are as follows 1) and 2):

[0012] 1) 27 bp deletion accompanied by 1 bp insertion genotype;

[0013] 2) 4bp base deletion genotype;

[0014] The 4 bp base deletion genotype is that the ASIP gene (nucleotide sequence is sequence 1) in the homologous chromosome of Chinese Merino fine wool sheep is missing 4 bp bases at positions 22338-22341 of sequence 1;

[0015] The 27 bp base deletion accompanied by 1 bp base insertion genotype is that the ASIP gene in the homologous chromosome of Chinese Merino fine wool sheep (nucleotide sequence is sequence 1) has a total deletion of 27 bp bases at positions 22322-22348 of sequence 1, and a 1 bp base T is inserted at positions 22339-22340 of sequence 1;

[0016] The method for selecting Chinese Merino fine wool sheep rams having two genotypes and non-white coat color is as shown in A) below;

[0017] From the F 1 Select F sheep with either genotype from the two generations 1 The method for producing ewes for ASIP gene-edited sheep is as follows A):

[0018] The method shown in A) comprises the following steps: amplifying the sheep genome to be tested using a primer set consisting of the primer shown in Sequence 4 and the primer shown in Sequence 5, and determining the genotype by Sanger sequencing the amplified product.

[0019] In the above method, the non-white Chinese Merino fine wool sheep is black or brown Chinese Merino fine wool sheep; wherein, the black or brown Chinese Merino fine wool sheep has more than 80% of its body hair in black or brown.

[0020] The use of Chinese Merino fine-wool sheep rams having the following two genotypes and non-white wool color in breeding black or brown Chinese Merino fine-wool sheep is also within the scope of protection of the present invention;

[0021] Or the use of Chinese Merino fine wool sheep rams having the following two genotypes and having non-white wool color in the breeding of non-white Chinese Merino fine wool sheep is also within the scope of protection of the present invention;

[0022] The two genotypes are as follows: 1) 27 bp base deletion accompanied by 1 bp insertion genotype; 2) 4 bp base deletion genotype;

[0023] The 4bp base deletion genotype is that the ASIP gene (nucleotide sequence is sequence 1) in the homologous chromosome of Chinese Merino fine wool sheep (Xinjiang type) is missing 4bp bases at positions 22338-22341 of sequence 1;

[0024] The 27 bp base deletion accompanied by 1 bp base insertion genotype is that the ASIP gene (nucleotide sequence is sequence 1) in the homologous chromosome of Chinese Merino fine wool sheep (Xinjiang type) has a 27 bp base deletion at positions 22322-22348 of sequence 1, and a 1 bp base T is inserted at positions 22339-22340 of sequence 1;

[0025] The screening method of Chinese Merino fine wool sheep rams having two genotypes is as shown in A) below;

[0026] The method shown in A) comprises the following steps: amplifying the sheep genome to be tested using a primer set consisting of the primer shown in Sequence 4 and the primer shown in Sequence 5, and determining the genotype by Sanger sequencing the amplified product.

[0027] The application of Chinese Merino fine-wool sheep rams having the following two genotypes and primers used for genotype identification in breeding black-brown Chinese Merino fine-wool sheep also falls within the scope of protection of the present invention;

[0028] The primers are a primer set consisting of the primer shown in Sequence 4 and the primer shown in Sequence 5;

[0029] The two genotypes are as follows: 1) 27 bp base deletion accompanied by 1 bp insertion genotype; 2) 4 bp base deletion genotype;

[0030] The 4bp base deletion genotype is that the ASIP gene (nucleotide sequence is sequence 1) in the homologous chromosome of Chinese Merino fine wool sheep (Xinjiang type) is missing 4bp bases at positions 22338-22341 of sequence 1;

[0031] The 27bp base deletion accompanied by 1bp base insertion genotype is that the ASIP gene (nucleotide sequence is sequence 1) in the homologous chromosome of Chinese Merino fine wool sheep (Xinjiang type) has a 27bp base deletion at positions 22322-22348 of sequence 1, and a 1bp base T is inserted at positions 22339-22340 of sequence 1.

[0032] The Chinese Merino fine wool sheep rams (denoted as F 0 The ASIP gene-edited rams of the next generation can be the ASIP gene of the wild-type Chinese Merino fine-wool sheep (Xinjiang type) that is subjected to site-directed deletion or insertion or gene editing by conventional existing technical means, to obtain rams with the following two genotypes, and the color of the fur on the body is brown and white (brown accounts for more than 80%); or the offspring rams obtained by somatic cell cloning and embryo transplantation using fibroblasts, the offspring rams are still rams with two genotypes, and the color of the fur on the body is brown and white (brown accounts for more than 80%). In the present invention, the Chinese Merino fine-wool sheep are all Xinjiang type, also known as Chinese Merino fine-wool sheep (Xinjiang type).

[0033] At present, CRISPR / Cas9 technology is used to modify and mutate the key gene ASIP that controls the coat color of Chinese Merino fine-wool sheep (Xinjiang type), and gene-edited fine-wool sheep with different coat colors are obtained. Since it is impossible to control the separation and recombination of coat color genes that occur during the breeding process of gene-edited fine-wool sheep, in order to obtain offspring with consistent coat color, the present invention first determines the genotype of breeding coat color, and through the breeding design of the coat color genotype, a population of a specific genotype is selected in advance when formulating a breeding plan, and breeding is guided by artificial insemination or natural mating to achieve gradual expansion of the group. After expansion and selection, a group of black-brown fine-wool sheep with a relatively neat coat color and stable inheritance can be cultivated, which provides a theoretical basis for further determining the direction of coat color breeding, and also provides rare germplasm resources and genetic materials for cultivating new breeds of sheep with different coat colors and studying the genetic mechanism of coat color genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the process of a method for selectively breeding black-brown Chinese Merino fine-wool sheep (Xinjiang type) based on editing genotypes according to an embodiment of the present invention.

[0035] Figure 2 These are the results of Hi-Tom PCR amplification electrophoresis. A is the first round of PCR amplification, and B is the second round of PCR amplification after purification.

[0036] Figure 3 The sequencing peak results of the editing sites of two genotypes of ASIP gene-edited sheep.

[0037] Figure 4Schematic diagram of natural mutations, editing modifications and frameshift mutations in ASIP gene-edited sheep.

[0038] Figure 5 The ASIP gene editing efficiency results were obtained by high-throughput sequencing.

[0039] Figure 6 Sanger sequencing comparison results of two genotypes of ASIP gene-edited sheep. DETAILED DESCRIPTION

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] The nucleotide sequences of the relevant primers in the following examples are shown in Table 1.

[0043] Table 1 shows the nucleotide sequences of primers related to ASIP genes

[0044]

[0045] Note: All primers are single-stranded DNA molecules.

[0046] In the following examples, there are two genotypes of rams (denoted as F 0 The offspring ASIP gene-edited rams can be rams with the following two genotypes obtained by using existing conventional technical means to perform site-specific deletion or insertion or gene editing on the ASIP gene of wild-type Chinese Merino fine-wool sheep (Xinjiang type), and their coat color is brown and white (brown accounts for more than 80%); the offspring rams can also be obtained by somatic cell cloning and embryo transplantation using fibroblasts, and the offspring rams are still rams with two genotypes, and their coat color is brown and white (brown accounts for more than 80%).

[0047] The above two genotypes are a 4bp base deletion genotype and a 27bp base deletion with a 1bp base insertion genotype:

[0048] The 4 bp base deletion genotype is that the ASIP gene (nucleotide sequence is sequence 1) in the homologous chromosome of Chinese Merino fine wool sheep (Xinjiang type) is missing 4 bp bases at positions 22338-22341 of sequence 1;

[0049] The genotype of 27bp base deletion accompanied by 1bp base insertion is that the ASIP gene (nucleotide sequence is sequence 1) in the homologous chromosome of Chinese Merino fine wool sheep (Xinjiang type) has a 27bp base deletion at position 22322-22348 of sequence 1, and a 1bp base T is inserted at position 22339-22340 of sequence 1; the 27bp base deletion spans 3 introns;

[0050] The wild-type Chinese Merino fine-wool sheep (Xinjiang type) is a naturally bred Chinese Merino fine-wool sheep (Xinjiang type) that has not been edited or mutated. The nucleotide sequence of the ASIP gene of the sheep is sequence 1 in the sequence list;

[0051] The above F 0 The preparation method of the first generation ASIP gene-edited ram is obtained by gene editing, which is recorded in the following literature: Xuemei Zhang, Wenrong Li, Chenxi Liu, Xinrong Peng, Jiapeng Lin, Sangang He, Xuejiao Li, Bing Han, Ning Zhang, Yangsheng Wu, Lei Chen, Liqin Wang, Ma Yila, Juncheng Huang, and Mingjun Liu*, Alteration of sheep coat color pattern by disruption of ASIP gene via CRISPR / Cas9, Sci Rep, 2017, 7: 8149. The construction is as follows:

[0052] The CRISPR / Cas9 system includes the following:

[0053] The target sequence is nucleotides 22323 to 22342 from the 5' end of sequence 1 in the sequence list;

[0054] The nucleotide sequence of ASIP-sgRNA is sequence 2;

[0055] Cas9 mRNA is shown as Sequence 3 in the sequence listing;

[0056] After artificial insemination of a Chinese Merino fine-wool sheep (Xinjiang type) donor ewe, a pronuclear embryo is surgically flushed from the oviduct; the ASIP-sgRNA and Cas9 mRNA are mixed and injected into the cytoplasm of the fertilized egg in the single-cell stage, cultured, and the embryo is transplanted into the maternal oviduct. The ewe gives birth to offspring sheep, and the F with the above genotype can be obtained. 0 Generation of ASIP gene-edited rams.

[0057] Example 1: Genotype identification method of gene-edited sheep

[0058] 1. Primer design and synthesis

[0059] According to the sheep ASIP gene sequence published in the NCBI database (accession number: NC_040264), specific primers ASIP-S and ASIP-AS were designed for the sheep ASIP gene exon 2 sequence using Oligo7.0 software to amplify the 426 bp coding region of the ASIP gene. The primer information is detailed in Table 1. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0060] 2. DNA Extraction

[0061] F was extracted using TIANamp Genomic DNA Kit (Beijing Tiangen) 0 Generation or F 1 The ASIP gene was used to edit the genomic DNA of the ram tail tissue. The experimental operation was carried out strictly according to the instructions. The extracted DNA was detected by 0.8% agarose gel electrophoresis, and the DNA concentration was determined by Nanodrop ultra-micro spectrophotometer and stored in a -20°C low-temperature refrigerator. The final absorbance OD value of the DNA extracted by the present invention was between 1.8 and 2.0, and the DNA concentration was diluted to 100 ng / μL for PCR amplification.

[0062] 3. Sequencing

[0063] The following methods can be used for amplification sequencing:

[0064] 1) Sanger sequencing

[0065] The DNA in step 2 was used as a template and PCR amplification was performed using the upstream primer ASIP-S and the downstream primer ASIP-AS in step 1.

[0066] The PCR amplification system was: 2×PCR Master Mix 25 μL, template DNA 100 ng, upstream and downstream primers (concentration 10 μmol / L) 1 μL each, and sterilized ultrapure water was added to 50 μL. The PCR amplification program was: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; and finally 72°C extension for 7 min. The PCR product was detected by 2% agarose gel electrophoresis.

[0067] The PCR products obtained above were subjected to Sanger sequencing and compared with the sequence of the reference genome (NCBI Reference Sequence: NC_040264.1 LOCUS NC_04026427563 bp DNA linear CON 06-FEB-2019), and the information on gene mutation was preliminarily obtained from the sequencing peak graph.

[0068] 2) Hi-Tom high-throughput sequencing

[0069] Hi-Tom high-throughput sequencing was used to perform deep sequencing of the designed ASIP targeted editing region as follows:

[0070] The first round of PCR amplification was performed using the DNA in 2 as a template and ASIP-F (Hi-Tom) (a forward bridging sequence was added before the 5' end of the forward specific primer) and ASIP-R (Hi-Tom) (a reverse bridging sequence was added before the 5' end of the reverse specific primer) as primers.

[0071] The Hi-Tom first-round PCR products were added to the pre-installed Hi-TOM Mix kit (Beijing Novogene Biotechnology Co., Ltd., product number: PT045) for the second-round PCR reaction. The amplified second-round PCR products were mixed, excised, recovered and purified, sealed with dry ice for transportation, and sent to Tianjin Novogene Bioinformatics Technology Co., Ltd. for Illumina sequencing, sequencing 1G data.

[0072] The electrophoresis results of the Hi-Tom first-round PCR products and second-round PCR products after purification are as follows Figure 2 Middle A and Figure 2 As shown in B, it can be seen that the size of the amplified product fragment is consistent with the expectation.

[0073] 4. Comparison and analysis of sequencing results

[0074] After the Hi-Tom sequencing results are returned, the sequencing data is uploaded to the website (http: / / www.hi-tom.net / hi-tom / ), and the types and positions of the samples are edited one by one, including analysis of reads, ratios, mutation types, mutant bases, and DNA sequences.

[0075] The Sanger sequencing results were analyzed by Chromas and Clustalx software for peak plot and sequence alignment.

[0076] F 0 The ASIP genotype peak diagram of the ASIP gene-edited ram is shown in Figure 3 As shown, Figure A is the peak graph of the wild type (wild-type fine-wool sheep as a control), Figure B is the peak graph of the 4bp base deletion genotype, and Figure C is the peak graph of the 27bp base deletion accompanied by 1bp base insertion genotype.

[0077] Natural mutations, editing modifications and frameshift mutations such as Figure 4 and Figure 6 As shown, F 0 、F 1As long as the edited genotypes are consistent, the frameshift mutations are consistent.

[0078] The above Hi-Tom high-throughput sequencing and Sanger sequencing results were combined to accurately determine the genotype, editing efficiency, and natural mutation type. Samples with editing efficiency (target mutation reads / total target site reads) less than 1% were considered negative (no editing of the target site), while samples with mutation rates > 1% were considered mutations.

[0079] Example 2: Breeding method of black-brown Chinese Merino fine-wool sheep (Xinjiang type) based on editing genotype selection

[0080] The schematic diagram of the method for breeding black-brown Chinese Merino fine-wool sheep (Xinjiang type) based on editing genotype selection is as follows Figure 1 The details are as follows:

[0081] 1. Hybridization

[0082] 1. F 0 Selection of ASIP gene-edited rams and wild fine-wool ewes

[0083] According to the genotype determined by Sanger sequencing and Hi-Tom sequencing results, the F-type with brown and white fur (brown accounts for more than 80%) and two genotypes of 4 bp base deletion genotype and 27 bp base deletion with 1 bp base insertion genotype were selected at one year old. 0 The ASIP gene-edited rams were used as breeding semen collection rams. The rams weighed more than 45kg, were more than 60cm tall, had a single ejaculation volume of more than 2.0mL, and had excellent semen test results (sperm motility of more than 0.8). One month before breeding, the rams were fed with carrots and eggs every day, and exercised for 1 hour in the morning and evening.

[0084] Select wild Chinese Merino fine-wool sheep (Xinjiang type) with white body coat, weighing more than 40kg, in good condition and free of reproductive diseases as breeding ewes.

[0085] 2. Synchronized estrus and artificial insemination

[0086] The day when the sponge plug was placed in the ewe's vagina was marked as day 0. On the 12th day, the sponge plug was removed, the ewe's vagina was cleaned, and 330 IU of pregnant mare serum (Ningbo Sansheng Company, China) was injected intramuscularly. Artificial insemination was performed 48 hours after the plug was removed, and 25 μg of ovulation-stimulating agent No. 3 (Ningbo Sansheng Company, China) was injected intramuscularly. Insemination was performed again after an interval of 6-8 hours. The pregnant ewe gave birth and obtained F 1 Generation individual.

[0087] 3. Natural mating

[0088] About 45 days after the artificial insemination, a pregnancy ultrasound examination was performed and the unpregnant ewes were selected and matched with F0 The ASIP gene-edited rams were mixed in the pen for 35 days and allowed to mate freely. The pregnant ewes gave birth to F 1 Generation individual.

[0089] The above steps 2 and 3 yielded a total of 28 F 1 Generation individual.

[0090] 4. Genotype identification and coat color observation

[0091] According to the method of Example 1, F. 1 The genotype of the individuals was tested by Sanger sequencing combined with Hi-Tom sequencing.

[0092] Here are the results:

[0093] Through genotyping testing,

[0094] Among them, 21 F 1 Each generation inherits the genes of its parent F 0 Two edited genotypes of the first generation ASIP gene-edited rams, named F 1 Generation of ASIP gene-edited sheep,

[0095] Counting these 21 F 1 The coat colors and corresponding genotypes of the first generation ASIP gene-edited sheep are as follows:

[0096] 10 F 1 The genotype of the first generation ASIP gene-edited sheep was only a 4bp base deletion genotype, and its coat color was black / / brown and white (black / brown accounted for more than 80%; 2, 1 ewe, 1 ram) and white (8, 4 ewes, 4 rams);

[0097] 11 F 1 The genotype of the first generation ASIP gene-edited sheep is only a 27bp base deletion accompanied by a 1bp base insertion genotype, and its coat color is black and white (black accounts for more than 80%; 1 ewe) and white (10, 5 ewes, 5 rams).

[0098] In addition, 7 individuals did not have parental F 0 The two edited genotypes of the ASIP gene-edited rams of the first generation were not included in further research. Among them, three F 1 The individuals of the first generation did not have the genotype of 4 bp base deletion and 27 bp base deletion with 1 bp base insertion, but their ASIP gene also had deletion and insertion, specifically, the ASIP gene (sequence 1) had a 21 bp deletion at position 22328-22348 and a 2 bp base AA insertion at position 22338-22339; the other 4 F 1The ASIP gene of the first generation individuals was wild type (sequence 1).

[0099] The 21 F 1 The first generation of ASIP gene-edited sheep had the same genotype but different coat colors, which may be due to natural mutations. We continued to analyze 21 F 1 The ASIP gene in sheep was edited and it was found that different coat colors have different natural mutation types:

[0100] 2 F with black / brown and white fur and only 4bp base deletion genotype 1 Among the first generation of ASIP gene-edited sheep, one had a homozygous genotype with neither 5bp nor 9bp deleted (N 5 N 5 , N 9 N 9 ), and one natural mutation occurred to a heterozygous genotype with both 5bp and 9bp deleted (N 5 D 5 , N 9 D 9 );

[0101] 8 white F with only 4 bp base deletion genotype 1 In the first generation of ASIP gene-edited sheep, a heterozygous genotype with a 5bp deletion occurred (N 5 D 5 ) and the homozygous genotype with no 9 bp deletion (N 9 N 9 ).

[0102] 1 F with black and white fur and a genotype of only 27 bp deletion and 1 bp insertion 1 In the first generation of ASIP gene-edited sheep, a natural mutation occurred, resulting in a heterozygous genotype with both 5bp and 9bp missing (N 5 D 5 , N 9 D 9 ).

[0103] 10 white F with genotype of only 27 bp deletion and 1 bp insertion 1 Among the first generation of ASIP gene-edited sheep, 7 had a natural mutation to a heterozygous genotype with a 5bp deletion (N 5 D 5 ) and the homozygous genotype with no 9 bp deletion (N 9 N 9 ), 3 of them had natural mutations and were homozygous for 5bp and 9bp without deletion (N 5 N 5 , N 9 N9 ).

[0104] The homozygous genotype with no deletion of the above 5bp and 9bp (N 5 N 5 , N 9 N 9 ) is that the ASIP gene in the homologous chromosome of fine wool sheep does not have a 5 bp deletion at position 22285-22289 of sequence 1, and does not have a 9 bp deletion at position 22195-22203 of sequence 1;

[0105] The heterozygous genotype with both 5bp and 9bp missing (N 5 D 5 , N 9 D 9 ) is a 5bp deletion in the ASIP gene of one homologous chromosome of fine-wool sheep at position 22285-22289 of sequence 1, and a 9bp deletion in position 22195-22203 of sequence 1, while the ASIP gene in the other homologous chromosome does not have the 5bp and 9bp deletions.

[0106] The heterozygous genotype of the above 5 bp deletion (N 5 D 5 ) and the homozygous genotype with no 9 bp deletion (N 9 N 9 ) is that the ASIP gene in one homologous chromosome of fine-wool sheep has a 5bp deletion at position 22285-22289 of sequence 1, and the ASIP gene in the other homologous chromosome does not have the 5bp deletion, and the ASIP gene in the two homologous chromosomes of fine-wool sheep does not have a 9bp deletion at position 22195-22203 of sequence 1.

[0107] Hi-Tom sequencing results showed that F 1 The mutation degree of the ASIP gene-edited sheep ranged from 1.27% to 65.73%, and the average editing efficiency was 30.34% (the proportion of edited reads to the total number of reads) ( Figure 5 ).

[0108] 2. Backcross

[0109] Section F 1 The first generation of ASIP gene-edited sheep died and could not be further used as F 2 Substitute parent.

[0110] The 4 genotypes above were only 4 bp base deletion genotype F 1 The ewes that survived to 1.5 years of age after ASIP gene editing and 5 ewes that survived to 1.5 years of age with a genotype of only 27 bp base deletion and 1 bp base insertion were in estrus at the same time, and F 0The ASIP gene-edited ram semen was used for artificial insemination to obtain F 2 Generation, a total of 11 F 2 The first generation group, of which 8 are black / brown and white sheep, 3 are white sheep, and the black / brown and white sheep are the target F 2 On behalf of sheep or their group.

[0111] The details are as follows:

[0112] 1 F 0 The ASIP gene-edited rams of the first generation were respectively crossed with an F-type ram with good growth condition, a body weight of more than 40 kg, and a genotype of only 4 bp base deletion, with a coat color of black / brown and white (black / brown accounts for more than 80%). 1 The ASIP gene-edited sheep were bred with ewes, resulting in 2 F 2 The coat color phenotype of the first generation of individuals is black / brown and white all over the body (black / brown accounts for more than 80%), recorded as F 2 Individual of the purpose.

[0113] 1 F 0 The ASIP gene-edited rams of the first generation were respectively crossed with three F rams with white fur color, which were in good growth condition, weighed more than 40 kg and had a genotype of only 4 bp base deletion. 1 The ASIP gene-edited sheep were bred with ewes, resulting in 3 F 2 The first generation of individuals, one of which has black / brown and white fur (black / brown accounts for more than 80%), is recorded as F 2 The other two are white.

[0114] 1 F 0 The ASIP gene-edited rams of the first generation were respectively matched with a ram with good growth condition, a body weight of more than 40 kg, and a genotype with only a 27 bp base deletion and a 1 bp base insertion genotype, with black and white fur (black accounting for more than 80%). 1 The ASIP gene-edited sheep were bred with ewes, and one F sheep was obtained. 2 The coat color phenotype of the first generation individuals is black / brown and white all over the body (black / brown accounts for more than 80%), recorded as F 2 Individual of the purpose.

[0115] 1 F 0 The ASIP gene-edited rams of the first generation were respectively crossed with four F rams with white fur color, which were in good growth condition, weighed more than 40 kg, and had a genotype of only 27 bp base deletion and 1 bp base insertion. 1 The ASIP gene-edited sheep were bred with ewes, resulting in 5 F 2The individuals of the generation group, of which 4 have a coat color phenotype of black / brown and white all over the body (black / brown accounts for more than 80%), are denoted as F 2 This is a substitute individual, the other one is white.

[0116] Control experiment: F 1 The individuals of the first generation did not have the genotype of 4 bp base deletion and 27 bp base deletion with 1 bp base insertion, but had a 21 bp deletion at position 22328-22348 and a 2 bp base AA insertion at position 22338-22339. 0 Breeding of ASIP gene-edited rams to produce 1 F 2 In the first generation, the body is white, indicating that the F 1 The gene deletion and insertion genotypes of the next generation cannot produce the target F 2 A sheep with a non-white coat.

[0117] From the above, it can be seen that F 2 The coat color of the first generation is not affected by F 1 The coat color of the next generation (i.e. natural mutation) is only related to the genotype.

[0118] The above genotype detection is carried out according to the method of Example 1 on the above F 2 The genotype of the target individuals was tested by Sanger sequencing and Hi-Tom high-throughput sequencing.

[0119] Hi-Tom sequencing results showed that F 2 The editing efficiency of different genotypes of the first generation ASIP gene-edited sheep ranged from 1.61% to 98.67%, with an average editing efficiency of up to 62.04% ( Figure 5 ).

[0120] After the determination of coat color genotype and selection of coat color phenotype, body shape and appearance, and production performance, a core breeding group of black-brown Chinese Merino fine-wool sheep (Xinjiang type) was formed to carry out generational breeding. 1 Generation, F 2 The genotype and coat color phenotype of the first generation ASIP gene-edited fine-wool sheep can be stably inherited 0 The editing types of the parents and the diversity of the coat color variations in the offspring also reflect the characteristics of dominant and recessive inheritance of coat color.

[0121] In summary, it can be seen that by selecting genotype F with a 27 bp deletion and a 1 bp insertion 1 The individuals of the first generation may have the 4 bp base deletion genotype F 1 Generation individuals and F 0 After backcrossing, the F1 of non-white coat color was cultivated. 2Generation of individuals or groups, thereby breeding more fine-wool sheep with non-white wool colors.

[0122] The specific method is as follows:

[0123] 1) With F 0 The ASIP gene-edited rams were bred with Chinese Merino fine-wool sheep (Xinjiang type) ewes to produce F 1 Sheep;

[0124] 2) From the F 1 Select F sheep with either of the following 2 genotypes 1 The ewes of the first generation of ASIP gene-edited sheep and F 0 The ASIP gene-edited rams were backcrossed to obtain F 2 The target sheep are those with black / brown and white (black / brown accounts for more than 80%);

[0125] F with the following two genotypes 1 The ewes that reproduce the ASIP gene-edited sheep are any of the following:

[0126] 1) The genotype is a 27 bp deletion accompanied by a 1 bp insertion;

[0127] 2) The genotype is a 4 bp base deletion;

[0128] The 4bp base deletion genotype is that the ASIP gene in the homologous chromosome of Chinese Merino fine wool sheep (Xinjiang type) is missing 4bp bases at positions 22338-22341 of sequence 1;

[0129] The genotype of 27bp base deletion accompanied by 1bp base insertion is that the ASIP gene in the homologous chromosome of Chinese Merino fine-wool sheep (Xinjiang type) has a 27bp base deletion at position 22322-22348 of sequence 1, and a 1bp base T is inserted at position 22339-22340 of sequence 1, wherein the 27bp base deletion spans 3 introns.

[0130] The above selection has the following two genotypes F 1 The genotype identification method for the ewes of the ASIP gene-edited sheep is to extract the genomic DNA from the tail of the sheep to be tested, and use Sanger sequencing primers ASIP-S and ASIP-AS for PCR amplification, and sequence to obtain the genotype; or use Hi-Tom high-throughput sequencing primers ASIP-F (Hi-Tom) and ASIP-R (Hi-Tom) for PCR amplification, and sequence to obtain the genotype.

[0131] Embodiment 3, black brown Chinese Merino fine wool sheep (Xinjiang type) breeding method

[0132] 1. Hybridization

[0133] 1. F 0 Selection of ASIP gene-edited rams and wild fine-wool ewes

[0134] Same as Example 1;

[0135] 2. Synchronized estrus and artificial insemination

[0136] 3. Natural mating

[0137] 4. Genotype identification

[0138] The above steps 2 and 3 yielded a total of 30 F 1 Generation individual.

[0139] According to the method of Example 1, genotype identification,

[0140] 30 F 1 23 F individuals in the generation 1 Each generation inherits the genes of its parent F 0 Any of the two edited genotypes of the ASIP gene-edited ram, named F 1 Generation of ASIP gene-edited sheep,

[0141] Select F with either of the two edited genotypes 1 Generation of ASIP gene-edited sheep ewes as backcross parents:

[0142] 1) The genotype is a 27 bp deletion accompanied by 1 bp;

[0143] 2) The genotype is a 4bp base deletion.

[0144] 2. Backcross

[0145] The 6 genotypes above were only 4 bp base deletion genotype F 1 The ewes that survived to 1.5 years of age and the 6 ewes that survived to 1.5 years of age and had a genotype of only 27 bp base deletion and 1 bp base insertion were in estrus at the same time, and F 0 The ASIP gene-edited ram semen was used for artificial insemination to obtain F 2 A total of 15 F 2 The first generation group, of which there are 12 sheep with black / brown and white body, 3 white sheep, and the sheep with black / brown and white body are the target F 2 On behalf of sheep or their group.

[0146] The details are as follows:

[0147] 1 F 0The ASIP gene-edited rams of the first generation were respectively crossed with two F-type rams with good growth condition, body weight greater than 40 kg and genotypes of only 4 bp base deletion genotypes, with coat colors of black / brown and white (black / brown accounted for more than 80%). 1 The ASIP gene-edited sheep were bred with ewes, resulting in 4 F 2 The coat color phenotype of the first generation of individuals is black / brown and white all over the body (black / brown accounts for more than 80%), recorded as F 2 Individual of the purpose.

[0148] 1 F 0 The ASIP gene-edited rams of the first generation were respectively crossed with four F rams with white coat color, which were in good growth condition, weighed more than 40 kg and had a genotype of only 4 bp base deletion. 1 The ASIP gene-edited sheep were bred with ewes, resulting in 4 F 2 The first generation of individuals, of which two have a coat color phenotype of black / brown and white (black / brown accounts for more than 80%), is recorded as F 2 This is a representative individual, the other two are white.

[0149] 1 F 0 The ASIP gene-edited rams of the first generation were respectively crossed with an F-type ram with good growth condition, body weight greater than 40 kg, and a genotype of only 27 bp base deletion with 1 bp base insertion, with black and white coat color (black accounting for more than 80%). 1 The ASIP gene-edited sheep were bred with ewes, and one F sheep was obtained. 2 The first generation of individuals, whose coat color phenotype is black / brown and white (black / brown accounts for more than 80%), is recorded as F 2 Individual of the purpose.

[0150] 1 F 0 The ASIP gene-edited rams of the first generation were respectively crossed with 5 F rams with white coat color, which were in good growth condition, weighed more than 40 kg, and had a genotype of only 27 bp base deletion and 1 bp base insertion. 1 The ASIP gene-edited sheep were bred with ewes, resulting in 6 F lambs. 2 The five individuals of the first generation with black-brown fur color are denoted as F 2 This is a substitute individual, the other one is white.

[0151] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for propagating or cultivating non-white Chinese Merino fine wool sheep, comprising the following steps: 1) F0 generation Chinese Merino fine wool sheep rams with the following two genotypes and non-white wool color are bred with wild-type Chinese Merino fine wool sheep ewes to produce F1 generation sheep; 2) F1 generation ASIP gene-edited sheep ewes having any one of the two genotypes and a non-white coat color are mated with F0 generation ASIP gene-edited rams to obtain F2 generation target sheep; the F0 generation ASIP gene-edited rams are Chinese Merino fine-wool sheep rams having the following two genotypes and a non-white coat color; The two genotypes are as follows 1) and 2): 1) 27 bp deletion accompanied by 1 bp insertion genotype; 2) 4bp base deletion genotype; The 4bp base deletion genotype is that the ASIP gene in the homologous chromosome of Chinese Merino fine wool sheep is missing 4bp bases at positions 22338-22341 of sequence 1; The 27 bp base deletion accompanied by 1 bp base insertion genotype is that the ASIP gene in the homologous chromosome of Chinese Merino fine wool sheep has a 27 bp base deletion at position 22322-22348 of sequence 1, and a 1 bp base T is inserted at position 22339-22340 of sequence 1; The method for selecting Chinese Merino fine wool sheep rams having two genotypes and non-white coat color is as shown in A) below; The method for selecting F1 generation ASIP gene-edited sheep ewes having any one of the two genotypes from F1 generation sheep is as described in A) below; The method shown in A) comprises the following steps: amplifying the sheep genome to be tested using a primer set consisting of the primer shown in Sequence 4 and the primer shown in Sequence 5, and determining the genotype by Sanger sequencing the amplified product.

2. The method according to claim 1, characterized in that: The non-white Chinese Merino fine-wool sheep is black or brown Chinese Merino fine-wool sheep; Or, the Chinese Merino fine-wool sheep are all of Xinjiang type.

3. Application of Chinese Merino fine wool sheep rams having the following two genotypes in breeding black or brown Chinese Merino fine wool sheep; Or the use of Chinese Merino fine wool sheep rams with the following two genotypes in the multiplication of non-white Chinese Merino fine wool sheep; The two genotypes are as follows: 1) 27 bp base deletion accompanied by 1 bp insertion genotype; 2) 4 bp base deletion genotype; The 4bp base deletion genotype is that the ASIP gene in the homologous chromosome of Chinese Merino fine wool sheep is missing 4bp bases at positions 22338-22341 of sequence 1; The 27 bp base deletion accompanied by 1 bp base insertion genotype is that the ASIP gene in the homologous chromosome of Chinese Merino fine wool sheep has a 27 bp base deletion at position 22322-22348 of sequence 1, and a 1 bp base T is inserted at position 22339-22340 of sequence 1; The screening method of Chinese Merino fine wool sheep rams having two genotypes is as shown in A) below; The method shown in A) comprises the following steps: amplifying the sheep genome to be tested using a primer set consisting of the primer shown in Sequence 4 and the primer shown in Sequence 5, and determining the genotype by Sanger sequencing the amplified product.

4. Application of Chinese Merino fine-wool sheep rams with the following two genotypes and primers used for genotype identification in breeding black or brown Chinese Merino fine-wool sheep; The primers are a primer set consisting of the primer shown in Sequence 4 and the primer shown in Sequence 5; The two genotypes are as follows: 1) 27 bp base deletion accompanied by 1 bp insertion genotype; 2) 4 bp base deletion genotype; The 4bp base deletion genotype is that the ASIP gene in the homologous chromosome of Chinese Merino fine wool sheep is missing 4bp bases at positions 22338-22341 of sequence 1; The 27 bp base deletion accompanied by 1 bp base insertion genotype is that the ASIP gene in the homologous chromosome of Chinese Merino sheep has a 27 bp base deletion at position 22322-22348 of sequence 1, and a 1 bp base T is inserted at position 22339-22340 of sequence 1.

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