WO2022151020A1 - 与山羊角型相关的核酸分子及其应用 - Google Patents

与山羊角型相关的核酸分子及其应用 Download PDF

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WO2022151020A1
WO2022151020A1 PCT/CN2021/071422 CN2021071422W WO2022151020A1 WO 2022151020 A1 WO2022151020 A1 WO 2022151020A1 CN 2021071422 W CN2021071422 W CN 2021071422W WO 2022151020 A1 WO2022151020 A1 WO 2022151020A1
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goat
seq
nucleic acid
primers
junction
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PCT/CN2021/071422
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English (en)
French (fr)
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杨漫漫
李勇
魏强
苗泽圃
王然
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深圳华大生命科学研究院
深圳市华大农业应用研究院
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Priority to CN202180075073.5A priority Critical patent/CN116601306A/zh
Priority to PCT/CN2021/071422 priority patent/WO2022151020A1/zh
Publication of WO2022151020A1 publication Critical patent/WO2022151020A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to the field of biotechnology and agriculture, in particular, to nucleic acid molecules related to goat horn type and applications thereof, more particularly, to a method, primer set, kit, method for determining goat horn type, and determination of nucleic acid typing Goat horn type device, goat breeding system.
  • Hornless sheep have many advantages: it is easy to manage, reduces the loss caused by the ram's horny fight, and the hornless sheep is not easy to damage the fence; when feeding, the number of hornless sheep in the same sheep house is more than the number of horned sheep; From the perspective of feed conversion efficiency, sheep horns also need to consume a lot of nutrients. For horned sheep, searing and caustic soda are generally used for dehorning. These two methods are easy to cause infection and cause various infectious diseases of goats, which are not in line with the principle of animal welfare, and will cause certain economic losses at the same time.
  • the present invention provides a nucleic acid molecule.
  • the nucleic acid molecule has an insertion mutation at the g.129424780bp site compared with the chromosome 1 of the goat genome, and the nucleic acid fragment of the insertion mutation is at least the region g.150334286-150818095bp of the chromosome 1 of the goat genome. Part of the reverse fragment.
  • the inventor found through research that there is a copy number variation in the hornless goat at the position of g.129424780bp of chromosome 1 of the goat genome, and the above copy number variation is the reverse insertion of the sequence of the region of g.150334286-150818095bp of the chromosome 1 of the goat genome. At the above g.129424780bp position.
  • the present invention proposes the use of the reagent for detecting the above-mentioned nucleic acid molecule in the preparation of a kit.
  • the kit is used to determine goat horn type.
  • the reagent according to the embodiment of the present invention can effectively detect the presence or absence of the above-mentioned nucleic acid molecule, or detect its copy number, so as to quickly and accurately determine the horn type of the detected goat.
  • the present invention provides a primer set.
  • the primer set is used to determine whether there is an insertion mutation at the g.129424780bp site of chromosome 1 of the genome of the sample to be tested, and the inserted nucleic acid fragment is at least the region of chromosome 1 of the goat genome g.150334286-150818095bp Part of the reverse fragment.
  • simple biotechnological means such as PCR can be used to accurately and effectively detect whether the genome of the goat to be tested contains the above-mentioned variation.
  • the results of PCR and gel electrophoresis based on the band information, the variation can be accurately determined The presence or absence of , makes the detection more convenient and direct.
  • the present invention provides a probe set.
  • the probe set is used to determine whether there is an insertion mutation at the chromosome 1 g.129424780bp site of the sample genome to be tested, and the inserted nucleic acid fragment is the goat genome chromosome 1 region g.150334286-150818095bp region at least a portion of the reverse fragment.
  • the breakpoints formed in the genome of the goat with the above-mentioned insertion mutation and the upstream and downstream sequences of the junction point can be specifically identified, so as to accurately and effectively detect and screen the goat with the above-mentioned mutation.
  • the present invention provides a kit.
  • the kit is used to determine whether there is an insertion mutation at the g.129424780bp site of chromosome 1 of the genome of the sample to be tested, and the inserted nucleic acid fragment is at least the region of chromosome 1 of the goat genome g.150334286-150818095bp Part of the reverse fragment.
  • the kit according to the embodiment of the present invention contains the reagents and instructions for detecting the above-mentioned variation, which can quickly and efficiently detect whether the genome of the goat to be detected contains the above-mentioned variation, and then determine the horn type of the goat to be detected.
  • the present invention provides a method for determining the goat horn type.
  • the method includes: obtaining nucleic acid of a goat sample to be tested; determining whether there is an insertion mutation at the chromosome 1 g.129424780bp site of the nucleic acid, and the inserted nucleic acid fragment is the goat genome chromosome 1 g .150334286-150818095bp region at least a part of the reverse fragment; when there is an insertion mutation, it is an indication that the goat horn type to be tested is hornless; when there is no insertion mutation, it is an indication that the goat horn type to be tested is horned .
  • the method according to the embodiment of the present invention is based on the nucleic acid of the goat to be detected, uses biotechnology to obtain the information of the goat to be detected from the gene level, and determines whether the gene copy number variation occurs at a specific location on chromosome 1 of the goat to be detected. Determine the horn type of the goat.
  • the method can realize the non-invasive detection of goat horn type. It only needs to collect a very small amount of tissue, peripheral blood, excrement, etc. of the goat to be detected, and obtain the nucleic acid of the goat to be detected, so that the horn type of the goat can be accurately judged when the goat is not horned. And then realize the distinction and screening of horned goats and hornless goats.
  • the present invention proposes the application of the aforementioned nucleic acid molecules, primer sets, probe sets and kits in breeding goat hornless breeds.
  • Fig. 1 is a CNV insertion pattern diagram at 129.4Mb of goat chromosome 1 (chr1) according to an embodiment of the present invention
  • Fig. 2 is the electrophoresis result of the left and right junctions of the horned/hornless individuals of the green goat in the embodiment of the present invention, wherein in Fig. 2A, lanes 1-3 and 7-9 are the amplification of the left and right junctions of the horned individuals, and no amplified strips can be obtained.
  • swimming lanes 4-6 and 10-12 are the amplification of the left and right junctions of hornless individuals, and the target bands are 284bp and 492bp respectively;
  • Figure 2B is the electrophoresis result of the internal reference gene, all individuals in lanes 1-12 can amplify the target Band 197bp;
  • FIG. 3 is a flowchart of a method for determining a goat horn type according to an embodiment of the present invention
  • L represents the electrophoresis results of the left junction point detection, wherein a 284bp band is amplified from a polled individual, and a horn ( horned) individuals have no bands
  • R represents the electrophoresis results of the right junction detection, wherein the polled individuals have a 492 bp band, and the horned individuals have no bands
  • HSP90 is an internal reference, and the amplification length is 197 bp.
  • CDG Cashmere Goat
  • MSG Horse Head Goat
  • JGG Jintang Black Goat
  • WSG Wushan White Goat
  • Ujimqin Goat UMG
  • GCG Guizhou Black Goat
  • Figure 5 shows the results of sanger sequencing of an hornless individual according to an embodiment of the present invention.
  • the sequences of the left and right junctions are consistent with expectations: the non-shaded part is the 129M region, the shaded part represents the g.150334286-150818095bp region, and the middle sequence is omitted and not listed out;
  • FIG. 6 shows the results of PCR electrophoresis of the left and right junction points of the three breeds of goats according to an embodiment of the present invention.
  • first”, “second”, “third” and other similar terms used in this document are for the purpose of distinguishing between description and convenience, and do not imply or expressly differ from each other for any purpose. There are differences in order or importance between them, and it does not mean that the content defined by “first”, “second”, “third” and other similar terms is composed of only one component.
  • the copy number variation (Copy number variation, CNV) used in this paper refers to the increase or decrease in the copy number of large segments of the genome caused by the rearrangement of the genome, mainly manifested as deletions at the submicroscopic level. and repeat.
  • the "copy number variation" of the present invention includes the reverse insertion of the sequence of the region g.150334286-150818095 bp of chromosome 1 of the goat genome at the position of g.129424780 bp of the chromosome 1 of the goat genome.
  • goats without horns from birth to adulthood are defined as hornless, and goats from birth to adulthood are defined as having horns regardless of sickle horns, plate horns, and right angles.
  • nucleic acid when referring to nucleic acid, those skilled in the art should understand that it actually includes any one, or two, of complementary double strands. For convenience, in this specification and claims, although only one chain is given in most cases, another chain complementary to it is actually disclosed. For example, reference to SEQ ID NO: 1 actually includes its complementary sequence, and those skilled in the art will also understand that using one strand can detect the other strand, and vice versa.
  • the present invention provides a nucleic acid molecule.
  • the nucleic acid molecule has an insertion mutation at the g.129424780bp site compared with the goat genome chromosome 1, and the inserted nucleic acid fragment is at least a part of the goat genome chromosome 1 region g.150334286-150818095bp region reverse fragment.
  • the inventors performed simplified genome sequencing of a population of 477 goats (398 with horns, 79 without horns), while performing deep whole-genome resequencing of another 10 goats (6 without horns, 4 with horns), including third- and second-generation And first-generation sequencing, through comparative analysis, it was found that there is a CNV (copy number variation, copy number variation) in the 129.4Mb region on chromosome 1 (chr1) in hornless green goat. The chromosome 1 of the goat genome was assembled locally, and then the 129.4Mb region was fine-mapped to determine the exact position of the breakpoint.
  • CNV copy number variation, copy number variation
  • the goat is selected from at least one of the following: Guizhou black goat, Ujimqin goat, Jintang black goat, horse head goat or Boer goat.
  • the nucleic acid molecule according to the embodiment of the present invention is universal in goat population. The inventors determined the correlation between the nucleic acid molecule and the goat horn type based on the results of simplified genome sequencing of 477 goats and deep whole genome resequencing of 10 goats, The above-mentioned nucleic acid molecule specifically exists in the genome of the hornless goat, while the above-mentioned nucleic acid molecule does not exist in the horned goat. There is no particular limitation on the breed of the goat. The inventors have confirmed that the CNV marker of the present invention has are conservative.
  • the phenotypic characteristics of goat horns can be identified, thereby guiding livestock production and landrace protection.
  • the CNV marker site also has great scientific research value, and some genetic evolution laws can be studied through this obvious angular model.
  • the present invention proposes the use of the reagent for detecting the nucleic acid molecule proposed in the first aspect of the present invention in the preparation of a kit.
  • the kit is used to determine goat horn type.
  • the reagent for detecting nucleic acid molecules can specifically detect the above-mentioned nucleic acid molecule or its copy number, or the mutation existing in the above-mentioned nucleic acid molecule, and then judge the horn type of the detected goat according to the detection result.
  • a breakpoint is formed at the position of g.129424780bp on the chromosome 2 of the goat genome, and the sequence of the g.150334286-150818095bp region on the chromosome 1 of the goat genome is reversely inserted, thereby forming two junctions on the left and right point, the upstream sequence immediately adjacent to the left junction is the normal sequence of chromosome 1 of the goat genome (the upstream sequence at the breakpoint when the reverse sequence is not inserted), and the downstream sequence immediately adjacent to the left junction is the reversely inserted goat genome
  • the sequence of the g.150334286-150818095bp region on chromosome 1, the upstream sequence immediately adjacent to the right junction is the reversely inserted sequence of the g.150334286-150818095bp region on chromosome 1 of the goat genome, and the downstream sequence immediately adjacent to the right junction is Goat genome chromosome 1 normal sequence (sequence downstream of the breakpoint when the reverse sequence is not
  • the positions of g150334286bp, g150818095bp and g129424780bp of chromosome 1 of the goat genome referred to in the present invention all refer to physical coordinates (Accession number: NC_030808.1) on the chromosome.
  • the reverse insertion of the sequence of the g150334286-150818095bp region of chromosome 1 will form two junctions at the position of g129424780bp, namely L (left junction) and R (right junction), as long as any junction is
  • the primer pairs are designed upstream and downstream (ie, both sides), and the junction can be amplified, and the goat horn type can be judged accordingly.
  • primers are designed on both sides of the two junctions to amplify the two junctions respectively, so that the goat horn type can be more accurately determined.
  • the nucleic acid sequence and the detection primer of the present invention provide a basis for early and accurate identification for the cultivation of hornless goats.
  • the identification of goat horn type can be realized as long as appropriate primers are designed on both sides of the breakpoint-junction. There are many primers that can achieve this purpose.
  • the primers can be designed near or far from the junction point.
  • the forward primer and the reverse primer are respectively 20bp, 50bp, 80bp, 100bp, 120bp, 150bp, 160bp, Distances of 180bp, 200bp, 250bp, 300bp, 450bp, 500bp, 600bp, 800bp, 1000bp, 1200bp, 1500bp, 1800bp, 2000bp, 3000bp, 5000bp, or 50-500bp.
  • the reagents include primers suitable for amplifying the breakpoint formed at the g.129424780bp position by the insertion mutation and at least 100bp upstream and downstream of its junction.
  • the primers are suitable for amplifying the breakpoint formed at the position of g.129424780bp by the insertion mutation and at least 200bp upstream and downstream of its junction.
  • the reagent includes a probe suitable for recognizing the breakpoint formed at the g.129424780bp position by the insertion mutation and at least 10bp upstream and downstream of its junction.
  • the probe is suitable for identifying the breakpoint formed at the g.129424780bp position by the insertion mutation and at least 20bp upstream and downstream of its junction.
  • the probe may include a label such as a fluorescent group, a biotin group, etc., for tracing or characterizing the detected nucleic acid molecule, wherein the fluorescent group may also include a luminescent group and a quenching group clumps used to characterize whether the nucleic acid molecule was detected.
  • the probe can be one of DNA, RNA, or a combination thereof, and the probe can be designed at a position near or far from the junction on both sides. For example, the probe can be designed at a specific recognition distance of 20bp, 50bp, or 80bp from the breakpoint.
  • the inventors designed primers according to the sequences on both sides of the breakpoint after inserting the reverse sequence, as shown in Table 1, the primer sequences SEQ ID NO: 5-10, wherein, such as SEQ ID NO: 5 and SEQ ID NO: 6
  • the primers shown are used to amplify the second breakpoint (namely the right breakpoint) formed at the position of g.129424780bp by the insertion mutation and the sequences upstream and downstream of the junction, and the target band of 492bp is obtained by amplification ;
  • Primers as shown in SEQ ID NO: 7 and SEQ ID NO: 8 are used to amplify the first breakpoint (ie the left breakpoint) formed at the g.129424780bp position by the insertion mutation and its junction
  • the upstream and downstream sequences were amplified to obtain a target band of 284bp.
  • SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 were used simultaneously to detect the left breakpoint and its junction respectively. point and the right breakpoint and its junction site.
  • SEQ ID NO: 7 and SEQ ID NO: 8 can also be used with SEQ ID NO: 9 and SEQ ID NO: 10, wherein the primers shown in SEQ ID NO: 9 and SEQ ID NO: 10 are used for amplification
  • the second breakpoint ie, the right breakpoint
  • formed at the position of g.129424780bp by the insertion mutation and the sequences upstream and downstream of the junction point were added to amplify to obtain a target band of 285bp.
  • the primer sequences SEQ ID NOs: 11-12 are the amplification sequences of the internal reference gene, and the prediction of the CNV is verified by PCR amplification and electrophoresis detection.
  • the target fragments of L (left junction) and R (right junction) can be detected by the above primers, it indicates that there is CNV in the 129.4Mb region, and the goat phenotype is hornless; when L and R electrophoresis have no bands , indicating that there is no CNV in the 129.4Mb region, and the goat phenotype is horned, specifically, the fragment lengths are 284bp and 492bp, respectively.
  • lanes 1-3, 7-9 are the amplification of the left and right junctions of horned individuals, and no amplified band can be obtained; lanes 4-6, 10-12 are the amplification of the left and right junctions of hornless individuals, and the target band
  • the bands are 284bp and 492bp respectively;
  • Figure 2B shows the electrophoresis results of the internal reference gene. All individuals in lanes 1-12 can amplify the target band of 197bp.
  • the first breakpoint formed at the g.129424780bp position by the insertion mutation and the sequence upstream and downstream of the junction point are shown in SEQ ID NO: 1 or SEQ ID NO: 3.
  • underlines indicate breakpoints and junctions.
  • the second breakpoint formed at the g.129424780bp position by the insertion mutation and the upstream and downstream sequences of its junction are shown in SEQ ID NO:2 or SEQ ID NO:4.
  • underlines indicate breakpoints and junctions.
  • SEQ ID NOs: 1 to 4 are the breakpoints formed at the g.129424780bp position by the insertion mutation and the sequences upstream and downstream of their junctions, wherein SEQ ID NO: 1 and SEQ ID NO: 1 and SEQ ID NO: 1 and SEQ ID NO: 1 and SEQ ID NO: 1 ID NO:3 is the left breakpoint-junction sequence, SEQ ID NO:3 sequence includes all SEQ ID NO:1 sequence, SEQ ID NO:2 and SEQ ID NO:4 are the right breakpoint-junction sequence, SEQ ID NO:2 and SEQ ID NO:4 are the right breakpoint-junction sequence, SEQ ID NO:3 The entire sequence of SEQ ID NO:2 is included in the sequence of ID NO:4.
  • the primer has at least one of the sequences shown in SEQ ID NO: 5SEQ ID NO: 6SEQ ID NO: 7SEQ ID NO: 8SEQ ID NO: 9SEQ ID NO: 10.
  • SEQ ID NO: 5 and SEQ ID NO: 6 are used to detect the right junction and upstream and downstream sequences of the breakpoint
  • SEQ ID NO: 9 and SEQ ID NO: 10 can also be used to detect the breakpoint
  • SEQ ID NO: 7 and SEQ ID NO: 8 are used to detect the left junction and upstream and downstream sequences of the breakpoint.
  • SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 to detect the right junction and upstream and downstream of the breakpoint, the left junction and upstream and downstream, or Use SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 7 and SEQ ID NO: 8 to detect the right junction and upstream and downstream of the breakpoint, the left junction and upstream and downstream, SEQ ID NO: 11 and SEQ ID NO: 12 was used to detect gDNA (genomic DNA) quality. If the target fragments on both sides of the junction point are positive, it is determined that the nucleic acid molecule is detected, and the horn type of the detected goat can be determined to be hornless.
  • gDNA genomic DNA
  • the present invention provides a primer set.
  • the primer set is used to determine whether there is an insertion mutation at the g.129424780bp site of chromosome 1 of the genome of the sample to be tested, and the inserted nucleic acid fragment is at least the region of chromosome 1 of the goat genome g.150334286-150818095bp Part of the reverse fragment.
  • simple biotechnological means such as PCR can be used to accurately and effectively detect whether the genome of the goat to be tested contains the above-mentioned variation.
  • the results of PCR and gel electrophoresis based on the band information, the variation can be accurately determined The presence or absence of , makes the detection more convenient and direct.
  • the primer set includes a first pair of primers and a second pair of primers, and the nucleic acid sequences thereof are as follows:
  • the upstream sequence of the first pair of primers 5'-CTACAGCATAAGATTCCGCATA-3' (SEQ ID NO: 5);
  • the downstream sequence of the first pair of primers 5'-CATCAGGGAGTTCACAGTTCAT-3' (SEQ ID NO: 6);
  • the upstream sequence of the second pair of primers 5'-GGCCATAGCTAAATGGTACTTAG-3' (SEQ ID NO: 7);
  • the downstream sequence of the second pair of primers 5'-CCTGGACTTGCTGATTCTGAG-3' (SEQ ID NO: 8).
  • nucleic acid sequences of the first pair of primers are as follows:
  • the upstream sequence of the first pair of primers 5'-CCATTTCTTCTACTAACCCATC-3' (SEQ ID NO: 9);
  • the downstream sequence of the first pair of primers 5'-TTGAGCACTACTTTACTACCGT-3' (SEQ ID NO: 10).
  • SEQ ID NO: 5 and SEQ ID NO: 6 are used to detect the right junction and upstream and downstream sequences of the breakpoint
  • SEQ ID NO: 9 and SEQ ID NO: 10 can also be used to detect the breakpoint
  • SEQ ID NO: 7 and SEQ ID NO: 8 are used to detect the left junction and upstream and downstream sequences of the breakpoint.
  • SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 to detect the right junction and upstream and downstream of the breakpoint, the left junction and upstream and downstream, or Use SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 7 and SEQ ID NO: 8 to detect the right junction and upstream and downstream of the breakpoint, the left junction and upstream and downstream, both sides of the junction target fragment If both are positive, it is determined that the above nucleic acid mutation is detected, and the horn type of the detected goat can be determined to be hornless.
  • the present invention provides a probe set.
  • the probe set is used to determine whether there is an insertion mutation at the chromosome 1 g.129424780bp site of the sample genome to be tested, and the inserted nucleic acid fragment is the goat genome chromosome 1 region g.150334286-150818095bp region at least a portion of the reverse fragment.
  • the probe set includes a first probe and/or a second probe, the nucleotide sequences of which are as follows: the first probe: Second probe: Wherein, the first probe is used to identify the second breakpoint and its junction, the second probe is used to identify the first breakpoint and its junction, and the junction of the breakpoint is represented by bold fonts, so The first probe and the second probe are used to identify hornless goats.
  • the probe set includes a third probe: Breakpoint junctions are indicated in bold, and the third probe was used to identify horned goats.
  • the probe is 10 bp, 15 bp or 20 bp around the junction of the breakpoint, and the probe can be used in conjunction with the above-mentioned primer set, or can be used alone to identify the breakpoint and its junction.
  • the probe set according to the embodiment of the present invention can carry a fluorescent group, a biotin group, etc., which is convenient for indication.
  • the present invention provides a kit.
  • the kit is used to determine whether there is an insertion mutation at the g.129424780bp site of chromosome 1 of the genome of the sample to be tested, and the inserted nucleic acid fragment is at least the region of chromosome 1 of the goat genome g.150334286-150818095bp Part of the reverse fragment.
  • the reagents for detecting the above-mentioned variation and the instructions for use can be quickly and efficiently detected whether the genome of the goat to be detected contains the above-mentioned variation, and then the horn type of the goat can be determined.
  • the kit includes the primer set proposed in the third aspect of the present invention and/or the probe set proposed in the fourth aspect of the present invention.
  • the kit further comprises at least one of PCR amplification enzyme, PCR amplification buffer and dNTPs.
  • the present invention provides a method for determining the goat horn type.
  • the method includes: referring to FIG. 3 , S100 , obtaining the nucleic acid of the goat sample to be tested; S200 , determining whether there is an insertion mutation in the nucleic acid chromosome 1 g.129424780bp site, the inserted nucleic acid
  • the fragment is a reverse fragment of at least a part of the goat genome chromosome 1 g.150334286-150818095bp region; when there is an insertion mutation, it is an indication that the goat horn type to be tested is hornless; when there is no insertion mutation, it is the The goat horn type is a horned indication.
  • the method according to the embodiment of the present invention can non-invasively detect the horn type of the goat at an early stage, avoid diseases such as infection caused by the surgical removal of the goat horn, thereby avoiding losses to the goat breeding industry, and avoiding the breeding of goats due to horns. The cost increases and the loss of goat breeding is avoided.
  • the method includes the following steps: using the genomic DNA of the goat to be tested as a template, performing PCR amplification with the primer pair of the present invention to obtain a PCR product; performing electrophoresis detection on the PCR product, and according to the electrophoresis result Determine the goat horn type. If an amplified band is obtained, the goat horn type to be tested is hornless, and if no amplified band is obtained, the goat horn type to be tested is horned.
  • a nucleic acid sample is obtained from a biological sample of a goat to be detected, and the type of the biological sample is not particularly limited, as long as a nucleic acid molecule reflecting whether the biological sample has a mutation can be extracted from the biological sample.
  • the biological sample may be at least one selected from the blood, skin, and subcutaneous tissue of the goat to be tested, preferably peripheral blood. Thereby, sampling and detection can be conveniently performed.
  • the nucleic acid sample can be analyzed and amplified, and the fragment size of the amplified product can be detected, so as to determine the goat horn type.
  • the amplification products obtained by using SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 are 492bp and 284bp, indicating that the horn type of the goat to be tested is hornless;
  • the obtained amplification product is an indication that the horn type of the goat to be tested is horny;
  • the amplification products obtained by amplification using SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 7 and SEQ ID NO: 8 are 285bp and 284bp is an indication that the horn type of the goat to be tested is without horns; if no amplification product is obtained, it is an indication that the horn type of the goat to be tested is horned.
  • the goat is selected from at least one of the following: Guizhou black goat, Ujimqin goat, Jintang black goat, horse head goat or Boer goat.
  • the method according to the embodiment of the present invention can identify the horn type of all kinds of goats, and the inventor found that the above-mentioned species of goat can better distinguish the horn type.
  • Figure 4 shows the results of breakpoint-junction PCR electrophoresis of 6 breeds of goats, among which the electrophoresis results of the junction point detection on the left side show that the hornless individual is a 284bp band, and the horned individual has no band; the junction point detection electrophoresis results on the right side, The hornless individuals have a 492bp band, and the horned individuals have no band.
  • Figure 6 shows the PCR electrophoresis results of the left and right junction points of the three breeds of goats, wherein the electrophoresis results of the right junction point detection show that the hornless individuals have a 285bp band, and the horned individuals have no band; the left junction point detection electrophoresis results, no Horn individuals have a 284bp band, and horn individuals have no band.
  • the inserted nucleic acid fragment is at least a part of the reverse fragment of the goat genome chromosome 1 g.150334286-150818095bp region, the horn type of the goat can be judged;
  • the primers provided by the present invention it is possible to simply and accurately determine whether the chromosome 1 of the goat genome has the above mutation, and then determine the horn type of the goat.

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Abstract

提供了一种山羊角型相关的核酸分子，与山羊基因组1号染色体相比，所述核酸分子在g.129424780bp位点存在插入突变，插入突变的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。还提供了所述核酸分子，及其引物组、探针组和试剂盒在选育山羊无角品种中的应用。

Description

与山羊角型相关的核酸分子及其应用
优先权信息
无
技术领域
本发明涉及生物技术领域和农业领域，具体地，涉及山羊角型相关的核酸分子及其应用，更具体地，涉及核酸分型的方法、引物组、试剂盒、确定山羊角型的方法、确定山羊角型的装置、山羊选育系统。
背景技术
我国是世界上山羊品种最多的国家，在山羊饲养过程中，羊角易对羊造成频繁、严重的撞伤，以致影响羊的体质量和皮张质量。无角羊有诸多的优点：便于管理，减少公羊角斗伤残造成的损失，无角羊不易破坏围栏；在舍饲时，相同羊舍内容纳无角羊数要多于有角羊数；从饲料的转化效率来讲，羊角也需要消耗很多的营养。对于有角羊，一般会采用烧烙法和苛性碱法进行去角。这两种方法均易引起感染，造成山羊各种感染性疾病，不符合动物福利原则，同时会造成一定的经济损失。
因此开发一种能够有效并准确判断山羊角型的无创检测方法对指导无角山羊品种选育和畜牧业生产显得十分必要。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
目前关于羊角型的研究主要集中在绵羊上，目前已有研究发现松弛素/胰岛素样肽受体(Relaxin/insulin-like familypeptidereceptor2，RXFP2)基因对绵羊角的大小具有调控作用。Montgomery等首次发现了微卫星标记OarVH58与羊的角型连锁，并且重新将羊的无角基因锁定在10号染色体上；Xiaolong Wang等研究发现RXFP2基因第14外显子的一个同义突变c.1125A/G与绵羊的角型显著相关。但目前还未见关于能够准确预测、确定山羊角型相关的报道。
在本发明的第一方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子与山羊基因组1号染色体相比，在g.129424780bp位点存在插入突变，所述插入突变的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。发明人经过研究发现，无角山羊在山羊基因组1号染色体的g.129424780bp位置处存在一个拷贝数变异，上述拷贝数变异是山羊基因组1号染色体的g.150334286-150818095bp区域的序列反向插入在上述g.129424780bp位置处。
在本发明的第二方面，本发明提出了检测上述核酸分子的试剂在制备试剂盒中的用 途。根据本发明的实施例，所述试剂盒用于确定山羊角型。根据本发明实施例的试剂可以有效检测上述核酸分子是否存在，或检测其拷贝数，从而快速准确判断被检测山羊的角型。
在本发明的第三方面，本发明提出了一种引物组。根据本发明的实施例，所述引物组用于确定待测样品基因组1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。根据本发明实施例的引物组，可以利用PCR等简单的生物技术手段准确有效地检测待检测山羊的基因组中是否含有上述变异，根据PCR及凝胶电泳结果，基于条带信息，可以准确判断变异的存在与否，使检测更加方便直接。
在本发明的第四方面，本发明提出了一种探针组。根据本发明的实施例，所述探针组用于确定待测样品基因组1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。根据本发明实施例的探针组，可以特异性识别具有上述插入突变的山羊基因组中所形成的断点及其接合点上下游序列，进而准确有效地检测并筛选出具有上述变异的山羊。
在本发明的第五方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒用于确定待测样品基因组1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。根据本发明实施例的试剂盒中包含与检测上述变异的试剂及使用说明，可以快速高效地检测待检测山羊基因组中是否含有上述变异，进而判断待检测山羊的角型。
在本发明的第六方面，本发明提出了一种确定山羊角型的方法。根据本发明的实施例，所述方法包括：获取待测山羊样品的核酸；确定所述核酸1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段；当存在插入突变，是所述待测山羊角型为无角的指示；当不存在插入突变，是所述待测山羊角型为有角的指示。根据本发明实施例的方法基于待检测山羊的核酸，利用生物技术手段，从基因层面上获取待检测山羊的信息，通过判断待检测山羊1号染色体上特定位点是否出现基因拷贝数变异，来判断山羊的角型。该方法可以实现对山羊角型的无创检测，只需要采集待检测山羊极少量组织、外周血、排泄物等，获取待检测山羊的核酸即可在山羊未出角时准确判断山羊的角型，进而实现对有角山羊和无角山羊的区分与筛选。
在本发明第七方面，本发明提出了前述核酸分子、引物组、探针组、试剂盒在选育山羊无角品种中的应用。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1为根据本发明实施例的山羊1号染色体(chr1)129.4Mb处CNV插入模式图；
图2为本发明实施例中青山羊有角/无角个体左右侧接合点电泳结果，其中图2A泳道1～3、7～9为有角个体左右侧接合点扩增，不能得到扩增条带；泳道4～6、10～12为无角个体左右侧接合点扩增，目的条带分别为284bp，492bp；图2B为内参基因电泳结果，泳道1-12所有个体均能扩增出目的条带197bp；
图3为根据本发明实施例的确定山羊角型的方法的流程图；
图4为根据本发明实施例的6个品种山羊左右侧接合点PCR电泳结果，其中L表示左侧接合点检测电泳结果，其中，无角(polled)个体扩增出284bp条带，有角(horned)个体无条带；R表示右侧接合点检测电泳结果，其中，无角(polled)个体为492bp条带，有角(horned)个体无条带；HSP90为内参，扩增长度为197bp。从上到下依次为柴达木绒山羊(CDG)、马头山羊(MTG)、金堂黑山羊(JGG)、巫山白山羊(WSG)、乌珠穆沁山羊(UMG)、贵州黑山羊(GBG)；
图5为根据本发明实施例的无角个体sanger测序结果，左、右侧接合点的序列与预期一致：非阴影部分为129M区域，阴影部分表示g.150334286-150818095bp区域，中间序列省略未列出；
图6为根据本发明实施例的3个品种山羊左右侧接合点PCR电泳结果。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
术语解释
如无特别说明，在本文中所使用的“第一”、“第二”、“第三”等类似术语均为用于描述方便而进行区分的目的，并不以任何目的暗示或者明示互相之间存在顺序或者重要性等差异，同时并不意味着由“第一”、“第二”、“第三”等类似术语所限定的内容仅有一种成分构成。
需要说明的是在本文中所使用的拷贝数变异((Copy number variation,CNV))指由基因组发生重排而导致的基因组大片段的拷贝数增加或者减少，主要表现为亚显微水平的缺失和重复。具体地，本发明的“拷贝数变异”包括山羊基因组1号染色体的g.150334286-150818095bp区域的序列反向插入山羊基因组1号染色体的g.129424780bp位置处。
需要说明的是，在本文中，山羊从出生到成年均无角定义为无角，山羊从出生到成年，无论镰刀角、板角、直角等均定义为有角。
对于本发明说明书和权利要求书中，提及核酸，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。例如，提及SEQ ID NO:1，实际包括其互补序列，本领域技术人员还可以理解，利用一条链可以检测另一条链，反之亦然。
需要说明的是，在本文中所提到的山羊基因组为Capra hircus ARS1(NCBI基因组序列编号:GCA_001704415.1，https://www.ncbi.nlm.nih.gov/genome/？term＝goat)。
需要说明的是，文中“第一断点及其接合点”与“左侧断点及其接合点”具有相同含义；“第二断点及其接合点”与“右侧断点及其接合点”具有相同含义。
核酸分子
在本发明的第一方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子与山羊基因组1号染色体相比，在g.129424780bp位点存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。
发明人通过对477只山羊(398有角，79无角)群体进行简化基因组测序，同时对另外10只山羊(6只无角，4只有角)进行深度全基因组重测序，包括三代、二代和一代测序，通过对比分析发现1号染色体(chr1)上129.4Mb区域在无角青山羊中存在一个CNV(copy number variation，拷贝数变异)，进而通过对1只无角山羊进行三代测序，对山羊基因组的1号染色体进行局部组装，再对129.4Mb区域进行精细定位，确定断点的确切位置，如图1所示，推断无角山羊基因组1号染色体上g.150334286-150818095bp区域的序列反向插入到基因组1号染色体的g.129424780bp位置处，进而破坏了山羊角的形成。
根据本发明的实施例，所述山羊选自下列至少之一：贵州黑山羊、乌珠穆沁山羊、金堂黑山羊、马头山羊或波尔山羊。根据本发明实施例的核酸分子在山羊群体中具有普适性，发明人根据477只山羊简化基因组测序及10只山羊深度全基因组重测序的结果，确定该核酸分子与山羊角型的相关性，在无角山羊的基因组中特异性存在上述核酸分子，而有角山羊并不存在上述核酸分子，对山羊的品种并无特别限制，发明人已经证实在多种山羊品种中，本发明的CNV标记均具有保守性。
利用本发明所提供的CNV标记，可以鉴定山羊角的表型特征，从而指导畜牧生产和地方品种保护。同时，该CNV标记位点还具有比较大的科研价值，通过这种明显的角型模型，来研究一些遗传进化规律。
试剂在制备试剂盒中的用途
在本发明的第二方面，本发明提出了检测本发明第一方面所提出的核酸分子的试剂在制备试剂盒中的用途。根据本发明的实施例，所述试剂盒用于确定山羊角型。根据本发明的 实施例，检测核酸分子的试剂可以特异性检测上述核酸分子或其拷贝数，或上述核酸分子中所存在的突变，进而根据检测结果判断被检测山羊的角型。
根据本发明的实施例，在山羊基因组2号染色体的g.129424780bp位置处形成断点，并反向插入山羊基因组1号染色体上g.150334286-150818095bp区域的序列，进而形成左、右两个接合点，左侧接合点紧邻的上游序列为山羊基因组1号染色体正常序列(未插入所述反向序列时断点处的上游序列)，左侧接合点紧邻的下游序列为反向插入的山羊基因组1号染色体上g.150334286-150818095bp区域的序列，右侧接合点紧邻的上游序列为反向插入的山羊基因组1号染色体上g.150334286-150818095bp区域的序列，右侧接合点紧邻的下游序列为山羊基因组1号染色体正常序列(未插入所述反向序列时断点处的下游序列)。
需要说明的是，本发明所称的山羊基因组1号染色体的g150334286bp、g150818095bp、g129424780bp位置均指的是染色体上的物理坐标(Accession number:NC_030808.1)。另外，1号染色体的g150334286-150818095bp区域的序列反向插入在g129424780bp位置处会形成两个接合点，即L(左侧接合点)和R(右侧接合点)，只要在任何一个接合点的上下游(即两侧)设计引物对，即可扩增到接合点，并据此判断山羊角型。在优选实施例中，在两个接合点的两侧均设计引物，分别扩增两个接合点，能够更精确地判断山羊角型。本发明的核酸序列及其检测引物，为无角山羊的培育提供了一种早期、准确鉴定的依据。
本发明中，由于1号染色体g150334286-150818095bp区域的序列反向插入在g129424780bp位置处具有一定的保守性，因此只要在断点-接合点的两侧设计合适的引物即可实现山羊角型鉴定。有很多引物均可实现这样的目的，引物可以设计在接合点或近或远的位置，例如，正向引物和反向引物分别距离断点20bp、50bp、80bp、100bp、120bp、150bp、160bp、180bp、200bp、250bp、300bp、450bp、500bp、600bp、800bp、1000bp、1200bp、1500bp、1800bp、2000bp、3000bp、5000bp、或50-500bp的距离。
根据本发明的实施例，所述试剂包括引物，所述引物适用于扩增所述插入突变在所述g.129424780bp位置处形成的断点及其接合点上下游至少100bp。
根据本发明的实施例，所述引物适用于扩增所述插入突变在所述g.129424780bp位置处形成的断点及其接合点上下游至少200bp。
根据本发明的实施例，所述试剂包括探针，所述探针适用于识别所述插入突变在所述g.129424780bp位置处形成的断点及其接合点上下游至少10bp。
根据本发明的实施例，所述探针适用于识别所述插入突变在所述g.129424780bp位置处形成的断点及其接合点上下游至少20bp。
根据本发明的实施例，所述探针可以包含荧光基团、生物素基团等标记，用于示踪或表征所检测的核酸分子，其中荧光基团还可以包括发光基团和淬灭基团，用于表征是否检测到所述核酸分子。探针可以为DNA、RNA的一种或其组合，探针可以设计在两侧接合点 两侧或近或远的位置，例如，探针可以设计在特异性识别距离断点20bp、50bp、80bp、100bp、120bp、150bp、160bp、180bp、200bp、250bp、300bp、450bp、500bp、600bp、800bp、1000bp、1200bp、1500bp、1800bp、2000bp、3000bp、5000bp、或50-500bp的位置。
发明人根据插入所述反向序列之后的断点两侧序列设计引物，如表1中所示的引物序列SEQ ID NO：5-10，其中，如SEQ ID NO：5和SEQ ID NO：6所示的引物用于扩增所述插入突变在所述g.129424780bp位置处形成的第二断点(即右侧断点)及其接合点上下游的序列，扩增得到492bp的目的条带；如SEQ ID NO：7和SEQ ID NO：8所示的引物用于扩增所述插入突变在所述g.129424780bp位置处形成的第一断点(即左侧断点)及其接合点上下游的序列，扩增得到284bp的目的条带，SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8同时使用，分别检测左侧断点及其接合位点及右侧断点及其接合位点。此外，SEQ ID NO：7和SEQ ID NO：8也可以与SEQ ID NO:9和SEQ ID NO:10一起使用，其中，SEQ ID NO:9和SEQ ID NO:10所示的引物用于扩增所述插入突变在所述g.129424780bp位置处形成的第二断点(即右侧断点)及其接合点上下游的序列，扩增得到285bp的目的条带。引物序列SEQ ID NO：11-12为内参基因扩增序列，通过PCR扩增和电泳检测，验证了该CNV的推测。当可以利用上述引物检测到L(左侧接合点)和R(右侧接合点)的目的片段时，表明129.4Mb区域存在CNV，山羊表型为无角；当L和R电泳无条带时，表明129.4Mb区域不存在CNV，山羊表型为有角，具体地，片段长度分别为284bp和492bp。如图2A泳道1～3、7～9为有角个体左右侧接合点扩增，不能得到扩增条带；泳道4～6、10～12为无角个体左右侧接合点扩增，目的条带分别为284bp，492bp；图2B为内参基因电泳结果，泳道1-12所有个体均能扩增出目的条带197bp。
表1：角型鉴定序列
Figure PCTCN2021071422-appb-000001
Figure PCTCN2021071422-appb-000002
根据本发明的实施例，所述插入突变在所述g.129424780bp位置处形成的第一断点及其接合点上下游的序列如SEQ ID NO:1或SEQ ID NO:3所示。
Figure PCTCN2021071422-appb-000003
其中，下划线表示断点与接合点。
根据本发明的实施例，所述插入突变在所述g.129424780bp位置处形成的第二断点及其接合点上下游序列如SEQ ID NO:2或SEQ ID NO:4所示。
Figure PCTCN2021071422-appb-000004
Figure PCTCN2021071422-appb-000005
其中，下划线表示断点与接合点。
根据本发明的实施例，SEQ ID NO:1～4均为所述插入突变在所述g.129424780bp位置处形成的断点及其接合点上下游的序列，其中，SEQ ID NO:1和SEQ ID NO:3是左侧断点-接合序列，SEQ ID NO:3序列中包含全部SEQ ID NO:1序列，SEQ ID NO:2和SEQ ID NO:4是右侧断点-接合序列，SEQ ID NO:4序列中包含全部SEQ ID NO:2序列。
根据本发明的实施例，所述引物具有SEQ ID NO：5SEQ ID NO：6SEQ ID NO：7SEQ ID NO：8SEQ ID NO：9SEQ ID NO：10所示的序列至少之一。
5’-CTACAGCATAAGATTCCGCATA-3’(SEQ ID NO：5)；
5’-CATCAGGGAGTTCACAGTTCAT-3’(SEQ ID NO：6)；
5’-GGCCATAGCTAAATGGTACTTAG-3’(SEQ ID NO：7)；
5’-CCTGGACTTGCTGATTCTGAG-3’(SEQ ID NO：8)；
5’-CCATTTCTTCTACTAACCCATC-3’(SEQ ID NO：9)；
5’-TTGAGCACTACTTTACTACCGT-3’(SEQ ID NO：10)。
根据本发明的实施例，SEQ ID NO：5和SEQ ID NO：6用于检测断点的右侧接合点及上下游序列，SEQ ID NO：9和SEQ ID NO：10也可以用于检测断点的右侧接合点及上下游序列，SEQ ID NO：7和SEQ ID NO：8用于检测断点的左侧接合点及上下游序列。检测时，需要同时使用SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8检测断点的右侧接合点及上下游、左侧接合点及上下游，或者使用SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：7和SEQ ID NO：8检测断点的右侧接合点及上下游、左侧接合点及上下游，SEQ ID NO：11和SEQ ID NO：12用于检测gDNA(基因组DNA)质量。两侧接合点目标片段均为阳性，则判定为检测到上述核酸分子，并可判定被检测山羊的角型为无角。
引物组、探针组、试剂盒
在本发明的第三方面，本发明提出了一种引物组。根据本发明的实施例，所述引物组用 于确定待测样品基因组1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。根据本发明实施例的引物组，可以利用PCR等简单的生物技术手段准确有效地检测待检测山羊的基因组中是否含有上述变异，根据PCR及凝胶电泳结果，基于条带信息，可以准确判断变异的存在与否，使检测更加方便直接。
根据本发明的实施例，所述引物组包括第一对引物和第二对引物，其核酸序列如下所示：
第一对引物上游序列：5’-CTACAGCATAAGATTCCGCATA-3’(SEQ ID NO：5)；
第一对引物下游序列：5’-CATCAGGGAGTTCACAGTTCAT-3’(SEQ ID NO：6)；
第二对引物上游序列：5’-GGCCATAGCTAAATGGTACTTAG-3’(SEQ ID NO：7)；
第二对引物下游序列：5’-CCTGGACTTGCTGATTCTGAG-3’(SEQ ID NO：8)。
根据本发明的实施例，所述第一对引物的核酸序列如下所示：
第一对引物上游序列：5’-CCATTTCTTCTACTAACCCATC-3’(SEQ ID NO：9)；
第一对引物下游序列：5’-TTGAGCACTACTTTACTACCGT-3’(SEQ ID NO：10)。
根据本发明的实施例，SEQ ID NO：5和SEQ ID NO：6用于检测断点的右侧接合点及上下游序列，SEQ ID NO：9和SEQ ID NO：10也可以用于检测断点的右侧接合点及上下游序列，SEQ ID NO：7和SEQ ID NO：8用于检测断点的左侧接合点及上下游序列。检测时，需要同时使用SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8检测断点的右侧接合点及上下游、左侧接合点及上下游，或者使用SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：7和SEQ ID NO：8检测断点的右侧接合点及上下游、左侧接合点及上下游，两侧接合点目标片段均为阳性，则判定为检测到上述核酸变异，并可判定被检测山羊的角型为无角。
在本发明的第四方面，本发明提出了一种探针组。根据本发明的实施例，所述探针组用于确定待测样品基因组1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。
根据本发明的实施例，所述探针组包括第一探针和/或第二探针，其核苷酸序列如下所示：第一探针：
Figure PCTCN2021071422-appb-000006
第二探针：
Figure PCTCN2021071422-appb-000007
其中，所述第一探针用于识别第二断点及其接合点，所述第二探针用于识别第一断点及其接合点，断点接合点处用粗体字表示，所述第一探针和第二探针用于识别无角山羊。
根据本发明的实施例，所述探针组包括第三探针：
Figure PCTCN2021071422-appb-000008
断点接合点处用粗体字表示，所述第三探针用于识别有角山羊。
根据本发明的实施例，所述探针为围绕断点接合点处10bp、15bp或20bp，所述探针可以配合上述引物组使用，也可以单独使用识别断点及其接合位点。根据本发明实施例的探针组，可以携带荧光基团、生物素基团等，便于指示。
在本发明的第五方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒用于确定待测样品基因组1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。根据本发明实施例的试剂盒中包含与检测上述变异的试剂及使用说明，可以快速高效地检测待检测山羊基因组中是否含有上述变异，进而判断山羊的角型。
根据本发明的实施例，所述试剂盒包括在本发明第三方面所提出的引物组和/或在本发明第四方面所提出的探针组。
根据本发明的实施例，所述试剂盒进一步包括PCR扩增酶、PCR扩增缓冲液和dNTPs中的至少一种。
确定山羊角型的方法
在本发明的第六方面，本发明提出了一种确定山羊角型的方法。根据本发明的实施例，所述方法包括：参考图3，S100，获取待测山羊样品的核酸；S200，确定所述核酸1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段；当存在插入突变，是所述待测山羊角型为无角的指示；当不存在插入突变，是所述待测山羊角型为有角的指示。根据本发明实施例的方法可以在早期无创检测山羊的角型，避免山羊因为手术去除山羊角造成的感染等疾病，进而避免对山羊养殖产业造成的损失，也避免山羊因为有角而造成的养殖成本升高，避免山羊养殖的损失。
根据本发明具体的实施例，所述方法包括以下步骤：以待测山羊的基因组DNA为模板，利用本发明的引物对进行PCR扩增，获得PCR产物；对PCR产物进行电泳检测，根据电泳结果判断山羊角型，若得到扩增条带则待测山羊角型为无角，若未得到扩增条带则待测山羊角型为有角。
根据本发明的实施例，从待检测山羊的生物样品中获取核酸样本，生物样品的类型并不受特别限制，只要从该生物样品中能够提取到反映生物样品是否存在突变的核酸分子即可。根据本发明的实施例，生物样品可以为选自待测山羊的血液、皮肤、皮下组织的至少一种，优选外周血。由此，可以方便地进行取样和检测。
在得到核酸样本之后，可以对核酸样本进行分析扩增，检测所述扩增产物片段大小，以便确定山羊角型。其中，使用SEQ ID NO:5、SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO： 8扩增得到的扩增产物为492bp和284bp为待测山羊角型是无角的指示；未得到扩增产物为待测山羊角型是有角的指示；使用SEQ ID NO:9、SEQ ID NO：10、SEQ ID NO：7和SEQ ID NO：8扩增得到的扩增产物为285bp和284bp为待测山羊角型是无角的指示；未得到扩增产物为待测山羊角型是有角的指示。
根据本发明的实施例，所述山羊选自下列至少之一：贵州黑山羊、乌珠穆沁山羊、金堂黑山羊、马头山羊或波尔山羊。根据本发明实施例的方法可以对所有品种的山羊进行角型的鉴定，发明人发现，上述物种山羊可以更好地区分角型。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Illumina公司。
实施例1 山羊角型鉴定(利用序列SEQ ID NO：5～8、11～12进行鉴定)
(1)采集已知角型的山羊耳缘组织，通过组织基因组DNA提取试剂盒(离心柱型)提取DNA。山羊表型信息如表2所示。其中，柴达木绒山羊和马头山羊只有一种角型，即全部是有角个体，而其他品种山羊既有有角个体，也有无角个体。
(2)利用L1和R1正反向引物(SEQ ID NO：5～8)进行PCR扩增：PCR反应体系如表3所示，扩增程序如表4所示。
(3)对PCR产物进行电泳检测，检测结果如图6所示。
表2 山羊品种及表型信息
山羊品种 角型/数量
柴达木绒山羊CDG 有角3/无角0
马头山羊MTG 有角0/无角3
金堂黑山羊JGG 有角3/无角3
巫山白山羊WSG 有角3/无角3
乌珠穆沁山羊UMG 有角3/无角3
贵州黑山羊GBG 有角3/无角3
表3 PCR扩增体系
试剂 体积/μL
rTaq(Takara)mix 10μL
引物F/R 0.2/0.2μL
DNA 20ng
H 2O 加至20μL
表4 PCR反应程序
Figure PCTCN2021071422-appb-000009
图4示出了6个品种山羊断点-接合点PCR电泳结果，其中左侧接合点检测电泳结果，无角个体为284bp条带，有角个体无条带；右侧接合点检测电泳结果，无角个体为492bp条带，有角个体无条带。
对无角个体进行sanger测序，结果如图5所示，结合点处序列的拼接模式与预期一致。
实施例2 山羊角型鉴定(利用序列SEQ ID NO：7～12进行鉴定)
(1)采集已知角型的山羊耳缘组织，通过血液基因组DNA提取试剂盒(离心柱型)提取DNA。山羊表型信息如表5所示。
(2)利用L1和R2正反向引物(SEQ ID NO：7～10)进行PCR扩增：PCR反应体系如表6所示，扩增程序如表7所示。
(3)对PCR产物进行电泳检测，检测结果如图6所示。
表5 山羊品种及表型信息
山羊品种 角型/数量
贵州黑山羊GBG 有角5/无角3
乌珠穆沁山羊UMG 有角4/无角4
金堂黑山羊JGG 有角4/无角4
表6 PCR扩增体系
试剂 体积/μL
rTaq(Takara)mix 10μL
引物F/R 0.2/0.2μL
DNA 20ng
H2O 加至20μL
表7 PCR反应程序
Figure PCTCN2021071422-appb-000010
图6示出了3个品种山羊左右侧接合点PCR电泳结果，其中右侧接合点检测电泳结果，无角个体为285bp条带，有角个体无条带；左侧接合点检测电泳结果，无角个体为284bp条带，有角个体无条带。
综上所述，根据检测在g.129424780bp位点是否存在插入突变，所述插入的核酸片段为 山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段，可以判断山羊的角型；利用本发明所提供的引物，可以简单准确地判断山羊基因组1号染色体是否具有上述突变，进而判断山羊的角型。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (18)

  1. 一种核酸分子，其特征在于，所述核酸分子与山羊基因组1号染色体相比，在g.129424780bp位点存在插入突变，所述插入突变的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。
  2. 根据权利要求1所述的核酸分子，其特征在于，所述山羊选自下列至少之一：贵州黑山羊、乌珠穆沁山羊、金堂黑山羊、马头山羊或波尔山羊。
  3. 检测权利要求1所述核酸分子的试剂在制备试剂盒中的用途，其特征在于，所述试剂盒用于确定山羊角型。
  4. 根据权利要求3所述的用途，其特征在于，所述试剂包括引物，所述引物适用于扩增所述插入突变在所述g.129424780bp位置处形成的断点及其接合点上下游至少100bp；
    任选地，所述引物适用于扩增所述插入突变在所述g.129424780bp位置处形成的断点及其接合点上下游至少200bp。
  5. 根据权利要求3所述的用途，其特征在于，所述试剂包括探针，所述探针适用于识别所述插入突变在所述g.129424780bp位置处形成的断点及其接合点上下游至少10bp；
    任选地，所述探针适用于识别所述插入突变在所述g.129424780bp位置处形成的断点及其接合点上下游至少20bp。
  6. 根据权利要求4所述的用途，其特征在于，所述插入突变在所述g.129424780bp位置处形成的第一断点及其接合点上下游的序列如SEQ ID NO:1和/或SEQ ID NO:3所示。
  7. 根据权利要求6所述的用途，其特征在于，所述插入突变在所述g.129424780bp位置处形成的第二断点及其接合点上下游的序列如SEQ ID NO:2和/或SEQ ID NO:4所示。
  8. 根据权利要求4所述的用途，其特征在于，所述引物具有SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10所示的序列至少之一。
  9. 一种引物组，其特征在于，所述引物组用于确定待测样品基因组1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。
  10. 根据权利要求9所述的引物组，其特征在于，包括第一对引物和第二对引物，其核酸序列如下所示：
    第一对引物上游序列：5’-CTACAGCATAAGATTCCGCATA-3’(SEQ ID NO：5)；
    第一对引物下游序列：5’-CATCAGGGAGTTCACAGTTCAT-3’(SEQ ID NO：6)；
    第二对引物上游序列：5’-GGCCATAGCTAAATGGTACTTAG-3’(SEQ ID NO：7)；
    第二对引物下游序列：5’-CCTGGACTTGCTGATTCTGAG-3’(SEQ ID NO：8)；
    任选地，如SEQ ID NO：5和SEQ ID NO：6所示的引物用于扩增所述插入突变在所述g.129424780bp位置处形成的第二断点及其接合点上下游的序列至少一部分；
    任选地，如SEQ ID NO：7和SEQ ID NO：8所示的引物用于扩增所述插入突变在所述g.129424780bp位置处形成的第一断点及其接合点上下游的序列至少一部分。
  11. 根据权利要求9所述的引物组，其特征在于，包括第一对引物和第二对引物，其 核酸序列如下所示：
    第一对引物上游序列：5’-CCATTTCTTCTACTAACCCATC-3’(SEQ ID NO：9)；
    第一对引物下游序列：5’-TTGAGCACTACTTTACTACCGT-3’(SEQ ID NO：10)；
    第二对引物上游序列：5’-GGCCATAGCTAAATGGTACTTAG-3’(SEQ ID NO：7)；
    第二对引物下游序列：5’-CCTGGACTTGCTGATTCTGAG-3’(SEQ ID NO：8)；
    任选地，如SEQ ID NO：9和SEQ ID NO：10所示的引物用于扩增所述插入突变在所述g.129424780bp位置处形成的第二断点及其接合点上下游的序列至少一部分；
    任选地，如SEQ ID NO：7和SEQ ID NO：8所示的引物用于扩增所述插入突变在所述g.129424780bp位置处形成的第一断点及其接合点上下游的序列至少一部分。
  12. 一种探针组，其特征在于，所述探针组用于确定待测样品基因组1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。
  13. 根据权利要求12所述的探针组，其特征在于，所述探针组包括第一探针和/或第二探针，其核苷酸序列如下所示：
    第一探针：5’-AATAAGGGTATAACTCGCTTCTAGGGACTC-3’(SEQ ID NO:13)；
    第二探针：5’-GTAACTGCCAGTATTGTCACTACGAACAAA-3’(SEQ ID NO:14)，其中，所述第一探针用于识别第二断点及其接合点，所述第二探针用于识别第一断点及其接合点；
    任选地，所述探针组包括第三探针：5’-AATAAGGGTATAACTTCACTACGAACAAAG-3’(SEQ ID NO:15)，所述第三探针用于识别有角山羊。
  14. 一种试剂盒，其特征在于，所述试剂盒用于确定待测样品基因组1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段。
  15. 根据权利要求14所述的试剂盒，其特征在于，所述试剂盒包括权利要求9～11任一项所述的引物组和/或权利要求12～13任一项所述的探针组。
  16. 根据权利要求15所述的试剂盒，其特征在于，所述试剂盒进一步包括PCR扩增酶、PCR扩增缓冲液和dNTPs中的至少一种。
  17. 一种确定山羊角型的方法，其特征在于，包括：
    获取待测山羊样品的核酸；
    确定所述核酸1号染色体g.129424780bp位点是否存在插入突变，所述插入的核酸片段为山羊基因组1号染色体g.150334286-150818095bp区域至少一部分的反向片段；
    当存在插入突变，是所述待测山羊角型为无角的指示；
    当不存在插入突变，是所述待测山羊角型为有角的指示；
    任选地，所述山羊选自下列至少之一：贵州黑山羊、乌珠穆沁山羊、金堂黑山羊、马头山羊或波尔山羊。
  18. 权利要求1～2任一项所述的核酸分子、权利要求9～11任一项所述的引物组、权利要求12～13任一项所述的探针组，权利要求14～16任一项所述的试剂盒在选育山羊无角品种中的应用。
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