CN116601306A

CN116601306A - Goat horn-related nucleic acid molecules and uses thereof

Info

Publication number: CN116601306A
Application number: CN202180075073.5A
Authority: CN
Inventors: 杨漫漫; 李勇; 魏强; 苗泽圃; 王然
Original assignee: Shenzhen Huada Agricultural Application Research Institute; BGI Shenzhen Co Ltd
Current assignee: Shenzhen Huada Agricultural Application Research Institute; BGI Shenzhen Co Ltd
Priority date: 2021-01-13
Filing date: 2021-01-13
Publication date: 2023-08-15
Also published as: WO2022151020A1

Abstract

Compared with the goat genome No. 1 chromosome, the nucleic acid molecule has insertion mutation at g.129424780bp site, and the insertion mutation nucleic acid fragment is a reverse fragment of at least one part of the g.15034286-150818095 bp region of the goat genome No. 1 chromosome. Also provides the application of the nucleic acid molecule, and the primer group, the probe group and the kit thereof in breeding goat pollenless varieties.

Description

Goat horn-related nucleic acid molecules and uses thereof

PRIORITY INFORMATION

Without any means for

Technical Field

The application relates to the biotechnology field and the agricultural field, in particular to a nucleic acid molecule related to goat horn types and application thereof, and more particularly relates to a nucleic acid typing method, a primer group, a kit, a method for determining goat horn types, a device for determining goat horn types and a goat breeding system.

Background

The sheep is a country with the largest goat breeds in the world, and the sheep horns are easy to cause frequent and serious bruise to the sheep in the goat raising process, so that the quality of the sheep and the quality of skin are influenced. The hornless sheep has a plurality of advantages: the management is convenient, the loss caused by the damage of the ram horned hopper is reduced, and the fence is not easy to be damaged by the hornless ram; when in barn feeding, the number of the hornless sheep is more than that of the horned sheep in the same sheep barn; the sheep horn also consumes a lot of nutrition in terms of feed conversion efficiency. For horned sheep, the burning-in method and caustic alkali method are generally adopted for removing the horns. Both methods are easy to cause infection, cause various infectious diseases of goats, do not accord with animal welfare principles, and cause certain economic loss.

Therefore, developing a noninvasive detection method capable of effectively and accurately judging the goat horn is necessary for guiding the breeding of the variety of the hornless goat and the production of animal husbandry.

Disclosure of Invention

The present application has been made based on the findings and knowledge of the inventors regarding the following facts and problems:

the current research on sheep horn type is mainly focused on sheep, and the Relaxin/insulin-like peptide receptor (RXFP 2) gene has been found to have a regulating effect on sheep horn size. Montgomery et al found for the first time that microsatellite marker OarVH58 was linked to the horn of sheep and that the non-horn gene of sheep was locked again on chromosome 10; studies by Xiaolong Wang et al found that one synonymous mutation c.1125A/G of exon 14 of the RXFP2 gene was significantly associated with sheep horn type. However, no report on the accurate prediction and determination of the goat horn type is known.

In a first aspect of the application, the application provides a nucleic acid molecule. According to an embodiment of the application, the nucleic acid molecule has an insertion mutation at the g.129424780bp site compared to the goat genome chromosome 1, and the nucleic acid fragment of the insertion mutation is an inverted fragment of at least a part of the g.15043286-150818095 bp region of the goat genome chromosome 1. The inventor finds that the non-horned goat has a copy number variation at the g.129424780bp position of the goat genome No. 1 chromosome, wherein the copy number variation is that the sequence of the g.15034286-150818095 bp region of the goat genome No. 1 chromosome is reversely inserted at the g.129424780bp position.

In a second aspect of the application, the application provides the use of a reagent for detecting a nucleic acid molecule as described above in the preparation of a kit. According to an embodiment of the application, the kit is used for determining goat horn type. The reagent according to the embodiment of the application can effectively detect whether the nucleic acid molecules exist or not or detect the copy number of the nucleic acid molecules, thereby rapidly and accurately judging the horn shape of the detected goat.

In a third aspect of the present application, the present application provides a primer set. According to the embodiment of the application, the primer group is used for determining whether insertion mutation exists at the g.129424780bp locus of the genome No. 1 of the sample to be detected, and the inserted nucleic acid fragment is a reverse fragment of at least one part of the g.15034286-150818095 bp region of the genome No. 1 of the goat. According to the primer group provided by the embodiment of the application, whether the genome of the goat to be detected contains the variation can be accurately and effectively detected by using simple biotechnology means such as PCR and the like, and the existence of the variation can be accurately judged based on the band information according to the PCR and gel electrophoresis results, so that the detection is more convenient and direct.

In a fourth aspect of the application, the application provides a probe set. According to the embodiment of the application, the probe set is used for determining whether insertion mutation exists at the g.129424780bp locus of the genome No. 1 of the sample to be detected, and the inserted nucleic acid fragment is a reverse fragment of at least one part of the g.15034286-150818095 bp region of the genome No. 1 of the goat. According to the probe set provided by the embodiment of the application, the breakpoint formed in the goat genome with the insertion mutation and the sequence upstream and downstream of the junction point of the breakpoint can be specifically identified, so that goats with the mutation can be accurately and effectively detected and screened.

In a fifth aspect of the application, the application provides a kit. According to the embodiment of the application, the kit is used for determining whether the insertion mutation exists at the g.129424780bp locus of the genome No. 1 of the sample to be detected, and the inserted nucleic acid fragment is a reverse fragment of at least one part of the g.15034286-150818095 bp region of the genome No. 1 of the goat. The kit provided by the embodiment of the application comprises the reagent for detecting the variation and the use instruction, so that whether the genome of the goat to be detected contains the variation can be detected rapidly and efficiently, and the horn type of the goat to be detected can be judged.

In a sixth aspect of the application, the application provides a method of determining a goat horn pattern. According to an embodiment of the application, the method comprises: obtaining nucleic acid of a goat sample to be detected; determining whether an insertion mutation exists at a g.129424780bp site of the nucleic acid 1 chromosome, wherein the inserted nucleic acid fragment is a reverse fragment of at least a part of a g.15034286-150818095 bp region of the goat genome 1 chromosome; when the insertion mutation exists, the insertion mutation is an indication that the goat horn type to be detected is non-horn; and when no insertion mutation exists, indicating that the horn type of the goat to be detected is horned. The method provided by the embodiment of the application is based on the nucleic acid of the goat to be detected, acquires the information of the goat to be detected from the gene level by using a biotechnology means, and judges the horn type of the goat by judging whether the specific site on the chromosome 1 of the goat to be detected has the variation of the copy number of the gene. The method can realize noninvasive detection of the goat horns, and can accurately judge the goat horns when the goat is not horned only by collecting a very small amount of tissues, peripheral blood, excrement and the like of the goat to be detected, so as to realize the differentiation and screening of the horned goat and the hornless goat.

In a seventh aspect of the application, the application provides application of the nucleic acid molecule, the primer group, the probe group and the kit in breeding of goat polled varieties.

Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.

Drawings

The foregoing and/or additional aspects and advantages of the application will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a diagram showing a pattern of CNV insertion at 129.4Mb on chromosome 1 (chr 1) of goat according to an embodiment of the present application;

FIG. 2 shows the electrophoresis results of the left and right side engagement of a horned/polled individual of a blue goat in the embodiment of the present application, wherein lanes 1 to 3 and 7 to 9 in FIG. 2A show that the left and right side engagement of the horned individual is amplified, and no amplified band can be obtained; lanes 4-6 and 10-12 are the amplification of the left and right side engagement of the non-angle individual, and the target bands are 284bp and 492bp respectively; FIG. 2B shows the result of electrophoresis of reference genes, wherein all individuals in lanes 1-12 can amplify 197bp of target band;

FIG. 3 is a flow chart of a method of determining a goat horn pattern in accordance with an embodiment of the present application;

FIG. 4 shows the results of PCR electrophoresis of left and right side engagement of 6 breeds of goats, wherein L represents the results of detection electrophoresis of left side engagement, and wherein a 284bp band was amplified from a non-angular (poled) individual, and a non-angular (horned) individual was amplified; r represents the electrophoresis result of the right side joint detection, wherein the individuals without corners (poled) are 492bp bands, and the individuals with corners (horned) are free of bands; HSP90 is an internal reference, and the amplification length is 197bp. The goats comprise a Qidamu goat (CDG), a horse goat (MTG), a Jintang black goat (JGG), a wushan white goat (WSG), a Wu Zhumu Qin goat (UMG) and a Guizhou Black Goat (GBG) from top to bottom in sequence;

FIG. 5 shows the sequence of the left and right side junctions according to the sequence of the corner-free individual sanger according to an embodiment of the application, consistent with expectations: the unshaded part is a 129M region, the shaded part represents a g.15034286-150818095 bp region, and the intermediate sequence is omitted;

FIG. 6 shows PCR electrophoresis results of the left and right side engagement of 3 breeds of goats according to an embodiment of the present application.

Detailed Description

Reference will now be made in detail to embodiments of the present application, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present application and should not be construed as limiting the application.

Interpretation of the terms

Unless specifically stated otherwise, as used herein, the terms "first," "second," "third," and the like are used for convenience of description and are not intended to imply or indicate any difference in order or importance between them, nor are they intended to be construed as being limited to only one element.

Copy number variation ((Copy number variation, CNV)) as used herein refers to an increase or decrease in copy number of large fragments of a genome due to rearrangement of the genome, and is mainly represented by deletion and duplication at a sub-microscopic level. Specifically, the "copy number variation" of the present application includes the reverse insertion of the sequence of the g.15034286-150818095 bp region of the goat genome chromosome 1 into the g.129424780bp position of the goat genome chromosome 1.

In this context, the goats are defined as no angle from birth to year, and the goats are defined as angle from birth to adult, regardless of sickle angle, plate angle, right angle, etc.

For purposes of the present specification and claims, reference to a nucleic acid will be understood by those skilled in the art to include virtually any one, or both, of the complementary double strands. For convenience, in the present description and claims, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. For example, reference to SEQ ID NO. 1, actually includes its complement, and one skilled in the art will also appreciate that one strand may be used to detect the other strand and vice versa.

It should be noted that the goat genome referred to herein is Capra hircus ARS1 (NCBI genome sequence number: GCA_001704415.1, https:// www.ncbi.nlm.nih.gov/genome/.

It should be noted that, herein, "the first breakpoint and the junction thereof" and "the left breakpoint and the junction thereof" have the same meaning; the "second breakpoint and junction thereof" has the same meaning as the "right breakpoint and junction thereof".

Nucleic acid molecules

In a first aspect of the application, the application provides a nucleic acid molecule. According to an embodiment of the application, the nucleic acid molecule has an insertion mutation at the g.129424780bp site compared to the goat genome chromosome 1, and the inserted nucleic acid fragment is an inverted fragment of at least a part of the g.15043286-150818095 bp region of the goat genome chromosome 1.

The inventors found that the 129.4Mb region on chromosome 1 (chr 1) had a CNV (copy number variation ) in the hornless Qinghai sheep by performing simplified genome sequencing on 477 goats (398 horns, 79 hornless), simultaneously performing deep whole genome re-sequencing on the other 10 goats (6 hornless, 4 hornless), including three-generation, two-generation and one-generation sequencing, by performing comparative analysis, and further performing three-generation sequencing on 1 hornless goats, performing local assembly on chromosome 1 of the goats genome, and then performing fine positioning on 129.4Mb regions, determining the exact position of a breakpoint, and deducing that the sequence of the g.15043286-150818095 bp region on chromosome 1 of the hornless goats is reversely inserted into the g.1294780 bp position of chromosome 1 of the genome, thereby destroying the formation of goats.

According to an embodiment of the application, the goat is selected from at least one of the following: guizhou black goats, wu Zhumu Qin goats, jintang black goats, horse goats or Boer goats. The nucleic acid molecules according to the examples of the present application have universality in goat populations, and the inventors have confirmed that the correlation between the nucleic acid molecules and the horns of goats is based on the results of 477 goats 'simplified genome sequencing and 10 goats' deep whole genome resequencing, the nucleic acid molecules are specifically present in the genome of the hornless goats, the horned goats are not present, and the variety of goats is not particularly limited, and the inventors have confirmed that the CNV markers of the present application are conserved among various goat varieties.

The CNV marker provided by the application can be used for identifying the phenotype characteristics of goat horns, so as to guide livestock production and local variety protection. Meanwhile, the CNV marker locus has relatively large scientific research value, and a plurality of genetic evolution rules are researched through the obvious angle model.

Application of reagent in preparation of kit

In a second aspect of the application, the application provides the use of a reagent for detecting a nucleic acid molecule as set forth in the first aspect of the application in the preparation of a kit. According to an embodiment of the application, the kit is used for determining goat horn type. According to the embodiment of the application, the reagent for detecting the nucleic acid molecule can specifically detect the nucleic acid molecule or the copy number thereof or the mutation existing in the nucleic acid molecule, and further judge the horn type of the detected goat according to the detection result.

According to the embodiment of the application, a breakpoint is formed at the g.129424780bp position of the goat genome 2 chromosome, and the sequences of the g.15034286-150818095 bp regions on the goat genome 1 chromosome are reversely inserted, so that left and right joints are formed, the immediately upstream sequence of the left joint is the normal sequence of the goat genome 1 chromosome (the upstream sequence at the breakpoint when the reverse sequence is not inserted), the immediately downstream sequence of the left joint is the sequence of the g.15034286-150818095 bp region on the reversely inserted goat genome 1 chromosome, the immediately upstream sequence of the right joint is the sequence of the g.15034286-150818095 bp region on the reversely inserted goat genome 1 chromosome, and the immediately downstream sequence of the right joint is the normal sequence of the goat genome 1 chromosome (the downstream sequence at the breakpoint when the reverse sequence is not inserted).

The positions g150334286bp, g150818095bp and g129424780bp of the chromosome 1 of the goat genome referred to in the application all refer to physical coordinates (Accession number: NC_ 030808.1) on the chromosome. In addition, the reverse insertion of the sequence of the g150334286-150818095bp region of chromosome 1 forms two junctions, i.e., L (left junction) and R (right junction), at the g129424780bp position, and the primer pair is designed upstream and downstream (i.e., on both sides) of either junction, so that the junction can be amplified, and the goat horn type can be judged accordingly. In a preferred embodiment, primers are designed on both sides of the two junctions, and the two junctions are amplified separately, enabling a more accurate determination of the goat horn shape. The nucleic acid sequence and the detection primer thereof provide a basis for early and accurate identification for the cultivation of the hornless goats.

In the application, the sequence of the g150334286-150818095bp region of chromosome 1 is reversely inserted to have certain conservation at the g129424780bp position, so that the identification of the goat horn type can be realized by only designing proper primers on two sides of a breakpoint-junction point. There are many primers that can be designed at the junction or near or far positions, for example, forward primer and reverse primer are 20bp, 50bp, 80bp, 100bp, 120bp, 150bp, 160bp, 180bp, 200bp, 250bp, 300bp, 450bp, 500bp, 600bp, 800bp, 1000bp, 1200bp, 1500bp, 1800bp, 2000bp, 3000bp, 5000bp, or 50-500bp apart, respectively.

According to an embodiment of the application, the reagent comprises a primer adapted to amplify the breakpoint of the insertion mutation at the g.129424780bp position and at least 100bp upstream and downstream of its junction point.

According to an embodiment of the application, the primer is adapted to amplify the insertion mutation at least 200bp upstream and downstream of the breakpoint at the g.129424780bp position and its junction point.

According to an embodiment of the application, the reagent comprises a probe adapted to recognize the breakpoint of the insertion mutation at the g.129424780bp position and at least 10bp upstream and downstream of its junction point.

According to an embodiment of the application, the probe is adapted to recognize the breakpoint of the insertion mutation at the g.129424780bp position and at least 20bp upstream and downstream of its junction point.

According to an embodiment of the application, the probe may comprise a label of a fluorescent group, a biotin group or the like for the purpose of tracing or characterizing the detected nucleic acid molecule, wherein the fluorescent group may further comprise a luminescent group and a quenching group for the purpose of characterizing whether the nucleic acid molecule is detected or not. The probe may be one of DNA, RNA or a combination thereof, and the probe may be designed at a position on both sides of the two-sided junction or near or far, for example, the probe may be designed at a position of 20bp, 50bp, 80bp, 100bp, 120bp, 150bp, 160bp, 180bp, 200bp, 250bp, 300bp, 450bp, 500bp, 600bp, 800bp, 1000bp, 1200bp, 1500bp, 1800bp, 2000bp, 3000bp, 5000bp, or 50-500bp from the specific recognition breakpoint.

The inventors designed primers based on the sequences flanking the breakpoint after insertion of the reverse sequence, as shown in table 1, the primer sequences SEQ ID NOs: 5-10, wherein the amino acid sequence as shown in SEQ ID NO:5 and SEQ ID NO:6 is used for amplifying a second breakpoint (namely a right breakpoint) formed at the g.129424780bp position of the insertion mutation and sequences upstream and downstream of a joint of the second breakpoint, so as to obtain a 492bp target strip; as set forth in SEQ ID NO:7 and SEQ ID NO:8 is used for amplifying a first breakpoint (namely a left breakpoint) formed at the g.129424780bp position of the insertion mutation and sequences upstream and downstream of a junction point of the first breakpoint, so that 284bp of a target band is obtained by amplification, wherein the target band is represented by SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8 are used simultaneously, and the left breakpoint and the joint site and the right breakpoint thereof are detected respectively. Furthermore, 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 to amplify the sequence of the insertion mutation upstream and downstream of the second breakpoint (i.e.the right breakpoint) formed at the g.129424780bp position and its junction point, yielding a 285bp band of interest. The primer sequence SEQ ID NO:11-12 are internal reference gene amplified sequences, and the estimation of the CNV is verified through PCR amplification and electrophoresis detection. When the target fragments of L (left side junction) and R (right side junction) were detected using the above primers, it was shown that CNV was present in the 129.4Mb region and the goat phenotype was non-angular; when L and R electrophoresis were striped, it was shown that the 129.4Mb region was free of CNV, the goat phenotype was angular, and specifically, the fragment lengths were 284bp and 492bp, respectively. Lanes 1-3 and 7-9 in FIG. 2A show that amplified bands could not be obtained by amplifying the side-by-side junctions of the horned individuals; lanes 4-6 and 10-12 are the amplification of the left and right side engagement of the non-angle individual, and the target bands are 284bp and 492bp respectively; FIG. 2B shows the result of electrophoresis of reference genes, and all individuals in lanes 1-12 can amplify 197bp of the target band.

Table 1: angle identification sequence

According to an embodiment of the application, the sequence of the insertion mutation upstream and downstream of the first breakpoint at the g.129424780bp position and its junction is shown in SEQ ID NO. 1 or SEQ ID NO. 3.

Wherein the underline indicates the break point and the junction point.

According to an embodiment of the application, the sequence of the second breakpoint at the position of g.129424780bp and upstream and downstream of its junction point is shown in SEQ ID NO. 2 or SEQ ID NO. 4.

Wherein the underline indicates the break point and the junction point.

According to an embodiment of the application, SEQ ID NOS.1 to 4 are sequences upstream and downstream of the breakpoint and the junction point of the breakpoint formed by the insertion mutation at the position of g.129424780bp, wherein SEQ ID NOS.1 and SEQ ID NO. 3 are left-hand breakpoint-junction sequences, SEQ ID NO. 3 comprises the entire SEQ ID NO. 1 sequence, SEQ ID NO. 2 and SEQ ID NO. 4 are right-hand breakpoint-junction sequences, and SEQ ID NO. 4 comprises the entire SEQ ID NO. 2 sequence.

According to an embodiment of the application, the primer has the sequence of SEQ ID NO:5SEQ ID NO:6SEQ ID NO:7SEQ ID NO:8SEQ ID NO:9SEQ ID NO:10, and at least one of the sequences shown in fig. 10.

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)。

According to an embodiment of the application, SEQ ID NO:5 and SEQ ID NO:6 right side junction and upstream and downstream sequences for detecting breakpoints, SEQ ID NO:9 and SEQ ID NO:10 can also be used to detect the right-hand side junction of a breakpoint and the upstream and downstream sequences, SEQ ID NO:7 and SEQ ID NO:8 are used to detect the left-hand side junction of the breakpoint and the upstream and downstream sequences. For detection, it is necessary to use the sequence of SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8 detecting the right side and upstream and downstream and left side and upstream and downstream of the breakpoint, or using SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO:7 and SEQ ID NO:8 detecting the right side contact and upstream and downstream, the left side contact and upstream and downstream of the breakpoint, and the sequence of SEQ ID NO:11 and SEQ ID NO:12 are used to detect gDNA (genomic DNA) quality. If the target fragments of both the two binding sites are positive, it is determined that the nucleic acid molecule is detected, and it is determined that the horn shape of the goat to be detected is no horn.

Primer set, probe set and kit

According to an embodiment of the present application, the primer set includes a first pair of primers and a second pair of primers, the nucleic acid sequences of which are as follows:

the first primer pair upstream sequence: 5'-CTACAGCATAAGATTCCGCATA-3' (SEQ ID NO: 5);

first primer pair downstream sequence: 5'-CATCAGGGAGTTCACAGTTCAT-3' (SEQ ID NO: 6);

second pair of primer upstream sequences: 5'-GGCCATAGCTAAATGGTACTTAG-3' (SEQ ID NO: 7);

second primer pair downstream sequence: 5'-CCTGGACTTGCTGATTCTGAG-3' (SEQ ID NO: 8).

According to an embodiment of the present application, the nucleic acid sequences of the first pair of primers are as follows:

the first primer pair upstream sequence: 5'-CCATTTCTTCTACTAACCCATC-3' (SEQ ID NO: 9);

first primer pair downstream sequence: 5'-TTGAGCACTACTTTACTACCGT-3' (SEQ ID NO: 10).

According to an embodiment of the application, SEQ ID NO:5 and SEQ ID NO:6 right side junction and upstream and downstream sequences for detecting breakpoints, SEQ ID NO:9 and SEQ ID NO:10 can also be used to detect the right-hand side junction of a breakpoint and the upstream and downstream sequences, SEQ ID NO:7 and SEQ ID NO:8 are used to detect the left-hand side junction of the breakpoint and the upstream and downstream sequences. For detection, it is necessary to use the sequence of SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8 detecting the right side and upstream and downstream and left side and upstream and downstream of the breakpoint, or using SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO:7 and SEQ ID NO:8 detecting the right side junction, the upstream and downstream side junction, the left side junction and the upstream and downstream side junction of the breakpoint, and judging that the nucleic acid variation is detected and the detected goat is corner-free if target fragments of the two side junctions are positive.

In a fourth aspect of the application, the application provides a probe set. According to the embodiment of the application, the probe set is used for determining whether insertion mutation exists at the g.129424780bp locus of the genome No. 1 of the sample to be detected, and the inserted nucleic acid fragment is a reverse fragment of at least one part of the g.15034286-150818095 bp region of the genome No. 1 of the goat.

According to an embodiment of the present application, the probe set includes a first probe and/or a second probe, the nucleotide sequences of which are as follows: a first probe:a second probe:the first probe is used for identifying a second breakpoint and a junction point thereof, the second probe is used for identifying a first breakpoint and a junction point thereof, the junction point of the breakpoint is indicated by bold characters, and the first probe and the second probe are used for identifying the hornless goats.

According to an embodiment of the application, the probe set comprises a third probe:the breakpoint junction is indicated in bold, and the third probe is used to identify the horny goat.

According to the embodiment of the application, the probe is 10bp, 15bp or 20bp around the breakpoint joint, and the probe can be matched with the primer set for use, or can be used for identifying the breakpoint and the joint site thereof independently. The probe group according to the embodiment of the application can carry a fluorescent group, a biotin group and the like, and is convenient for indication.

In a fifth aspect of the application, the application provides a kit. According to the embodiment of the application, the kit is used for determining whether the insertion mutation exists at the g.129424780bp locus of the genome No. 1 of the sample to be detected, and the inserted nucleic acid fragment is a reverse fragment of at least one part of the g.15034286-150818095 bp region of the genome No. 1 of the goat. The kit provided by the embodiment of the application comprises the reagent for detecting the variation and the use instruction, so that whether the genome of the goat to be detected contains the variation can be detected rapidly and efficiently, and the horn type of the goat can be judged.

According to an embodiment of the application, the kit comprises a primer set as set forth in the third aspect of the application and/or a probe set as set forth in the fourth aspect of the application.

According to an embodiment of the application, the kit further comprises at least one of a PCR amplification enzyme, a PCR amplification buffer and dNTPs.

Method for determining goat horn

In a sixth aspect of the application, the application provides a method of determining a goat horn pattern. According to an embodiment of the application, the method comprises: referring to fig. 3, S100, nucleic acid of a goat sample to be tested is obtained; s200, determining whether an insertion mutation exists at the g.129424780bp locus of the nucleic acid 1, wherein the inserted nucleic acid fragment is a reverse fragment of at least one part of the g.15034286-150818095 bp region of the goat genome 1; when the insertion mutation exists, the insertion mutation is an indication that the goat horn type to be detected is non-horn; and when no insertion mutation exists, indicating that the horn type of the goat to be detected is horned. According to the method provided by the embodiment of the application, the horn shape of the goat can be detected in an early stage in a noninvasive manner, diseases such as infection and the like caused by the removal of the goat horn by operation are avoided, further, the loss to the goat breeding industry is avoided, the raising cost of the goat caused by the horn is also avoided, and the loss of goat breeding is avoided.

According to a specific embodiment of the application, the method comprises the steps of: taking the genome DNA of the goat to be detected as a template, and carrying out PCR amplification by using the primer pair to obtain a PCR product; and (3) performing electrophoresis detection on the PCR product, judging the goat horn type according to the electrophoresis result, if the amplification strip is obtained, determining that the goat horn type to be detected is non-horn, and if the amplification strip is not obtained, determining that the goat horn type to be detected is horn.

According to an embodiment of the present application, 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 the presence or absence of mutation of the biological sample can be extracted from the biological sample. According to an embodiment of the present application, the biological sample may be at least one selected from blood, skin, subcutaneous tissue of the goat to be tested, preferably peripheral blood. Thus, sampling and detection can be performed conveniently.

After obtaining the nucleic acid sample, the nucleic acid sample may be subjected to analytical amplification, and the amplified product fragment size detected, so as to determine the goat horn type. Wherein, SEQ ID NO. 5, SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8, amplifying to obtain amplification products of 492bp and 284bp, wherein the amplification products are indicative of the horn type of the goat to be detected as no horn; the amplification product is not obtained and is an indication that the goat horn to be detected is angular; SEQ ID NO 9, SEQ ID NO: 10. SEQ ID NO:7 and SEQ ID NO:8, amplifying to obtain an amplification product of 285bp and 284bp, wherein the amplification product is an indication that the goat horn type to be detected is non-horn; the amplification product was not obtained as an indication that the goat horn type to be tested was angular.

According to an embodiment of the application, the goat is selected from at least one of the following: guizhou black goats, wu Zhumu Qin goats, jintang black goats, horse goats or Boer goats. According to the method provided by the embodiment of the application, the horns of goats of all varieties can be identified, and the inventor finds that the goats of the species can better distinguish the horns.

The scheme of the present application will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present application and should not be construed as limiting the scope of the application. The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, code Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used are conventional products available commercially, such as those available from Illumina corporation, without the manufacturer's knowledge.

Example 1 identification of goat horn type (identification Using sequences SEQ ID NOs: 5 to 8, 11 to 12)

(1) The goat ear margin tissue of known horn type was collected, and DNA was extracted by a tissue genomic DNA extraction kit (centrifugal column). Goat phenotype information is shown in table 2. Wherein, the chassises goat has only one angle type, namely all the goats are angular individuals, and other goats have both angular individuals and non-angular individuals.

(2) PCR amplification was performed using the L1 and R1 forward and reverse primers (SEQ ID NOS: 5-8): the PCR reaction system is shown in Table 3, and the amplification procedure is shown in Table 4.

(3) The PCR products were subjected to electrophoresis, and the detection results are shown in FIG. 6.

TABLE 2 goat breeds and phenotype information

Goat breeds	Angle/number
Czochralski down goat CDG	Angular 3/no angular 0
Horse head goat MTG	Angular 0/no angular 3
Jintang black goat JGG	Angular 3/non-angular 3
Wushan white goat WSG	Angular 3/non-angular 3

Wu Zhumu goat UMG	Angular 3/non-angular 3
Guizhou black goat GBG	Angular 3/non-angular 3

TABLE 3 PCR amplification System

Reagent(s)	Volume/. Mu.L
rTaq(Takara)mix	10μL
Primer F/R	0.2/0.2μL
DNA	20ng
H ₂ O	To 20. Mu.L

TABLE 4 PCR reaction procedure

FIG. 4 shows the results of 6 goat breakpoint-juncture PCR electrophoresis of the species, wherein the left side junction detected the electrophoresis results, the individuals without angles were 284bp bands, and the individuals with angles were not bands; the right side is connected with the electrophoresis result, the non-angular individual is 492bp band, and the angular individual is non-band.

The splice sequencing was performed on the non-corner individuals and the results are shown in FIG. 5, where the splice pattern of the sequences at the junction is consistent with expectations.

Example 2 identification of goat horn type (identification Using the sequences SEQ ID NO: 7-12)

(1) The goat ear margin tissue of known horn type was collected, and DNA was extracted by a blood genomic DNA extraction kit (centrifugal column). Goat phenotype information is shown in table 5.

(2) PCR amplification was performed using the L1 and R2 forward and reverse primers (SEQ ID NOS: 7-10): the PCR reaction system is shown in Table 6, and the amplification procedure is shown in Table 7.

TABLE 5 goat breeds and phenotype information

Goat breeds	Angle/number
Guizhou black goat GBG	Angular 5/no angular 3
Wu Zhumu goat UMG	Angular 4/non-angular 4
Jintang black goat JGG	Angular 4/non-angular 4

TABLE 6 PCR amplification System

Reagent(s)	Volume/. Mu.L
rTaq(Takara)mix	10μL
Primer F/R	0.2/0.2μL
DNA	20ng
H2O	To 20. Mu.L

TABLE 7 PCR reaction procedure

FIG. 6 shows the PCR electrophoresis results of the left and right side engagement of 3 goat breeds, wherein the right side engagement detects the electrophoresis results, the individuals without angles are 285bp bands, and the individuals with angles are not bands; the left side is jointed with the electrophoresis result, the non-angular individual is 284bp band, and the angular individual is non-band.

In summary, according to the detection of the presence or absence of an insertion mutation at the g.129424780bp locus, the inserted nucleic acid fragment is a reverse fragment of at least a part of the g.15034286-150818095 bp region of the genome 1 of the goat, so that the horn type of the goat can be judged; the primer provided by the application can simply and accurately judge whether the chromosome 1 of the goat genome has the mutation or not, thereby judging the horn type of the goat.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application.

Claims

A nucleic acid molecule, wherein said nucleic acid molecule has an insertion mutation at the g.129424780bp locus compared to chromosome 1 of the goat genome, and wherein said insertion mutated nucleic acid fragment is an inverted fragment of at least a portion of the g.15043286-150818095 bp region of chromosome 1 of the goat genome.
The nucleic acid molecule of claim 1, wherein the goat is selected from at least one of the following: guizhou black goats, wu Zhumu Qin goats, jintang black goats, horse goats or Boer goats.
Use of a reagent for detecting a nucleic acid molecule according to claim 1 for the preparation of a kit for determining the goat horn type.
The use according to claim 3, wherein the reagent comprises a primer adapted to amplify the breakpoint of the insertion mutation at the g.129424780bp position and at least 100bp upstream and downstream of its junction;

optionally, the primer is adapted to amplify the insertion mutation at least 200bp upstream and downstream of the breakpoint at the g.129424780bp position and its junction point.
The use according to claim 3, wherein the reagent comprises a probe adapted to recognize the breakpoint of the insertion mutation at the g.129424780bp position and at least 10bp upstream and downstream of its junction;

optionally, the probe is adapted to recognize at least 20bp upstream and downstream of the breakpoint and its junction point of the insertion mutation at the g.129424780bp position.
The method according to claim 4, wherein the sequence of the insertion mutation upstream and downstream of the first breakpoint and the junction thereof at the position of g.129424780bp is shown in SEQ ID NO. 1 and/or SEQ ID NO. 3.
The use according to claim 6, characterized in that the sequence of the insertion mutation upstream and downstream of the second breakpoint at the g.129424780bp position and its junction point is shown in SEQ ID NO. 2 and/or SEQ ID NO. 4.
The use according to claim 4, wherein the primer has the sequence of SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO:10, and at least one of the sequences shown in fig. 10.
The primer group is used for determining whether insertion mutation exists at the g.129424780bp locus of the genome No. 1 of a sample to be detected, and the inserted nucleic acid fragment is a reverse fragment of at least one part of the g.15043286-150818095 bp region of the genome No. 1 of a goat.
The primer set of claim 9, comprising a first primer pair and a second primer pair having a nucleic acid sequence as set forth in seq id no:

the first primer pair upstream sequence: 5'-CTACAGCATAAGATTCCGCATA-3' (SEQ ID NO: 5);

first primer pair downstream sequence: 5'-CATCAGGGAGTTCACAGTTCAT-3' (SEQ ID NO: 6);

second pair of primer upstream sequences: 5'-GGCCATAGCTAAATGGTACTTAG-3' (SEQ ID NO: 7);

second primer pair downstream sequence: 5'-CCTGGACTTGCTGATTCTGAG-3' (SEQ ID NO: 8);

optionally, the sequence as set forth in SEQ ID NO:5 and SEQ ID NO:6 for amplifying at least a portion of the sequence of the insertion mutation upstream and downstream of the second breakpoint at the g.129424780bp position and its junction;

optionally, the sequence as set forth in SEQ ID NO:7 and SEQ ID NO:8 is used to amplify at least a portion of the sequence of the insertion mutation upstream and downstream of the first breakpoint at the g.129424780bp position and its junction point.
The primer set of claim 9, comprising a first primer pair and a second primer pair having a nucleic acid sequence as set forth in seq id no:

the first primer pair upstream sequence: 5'-CCATTTCTTCTACTAACCCATC-3' (SEQ ID NO: 9);

first primer pair downstream sequence: 5'-TTGAGCACTACTTTACTACCGT-3' (SEQ ID NO: 10);

second pair of primer upstream sequences: 5'-GGCCATAGCTAAATGGTACTTAG-3' (SEQ ID NO: 7);

second primer pair downstream sequence: 5'-CCTGGACTTGCTGATTCTGAG-3' (SEQ ID NO: 8);

optionally, the sequence as set forth in SEQ ID NO:9 and SEQ ID NO:10 for amplifying at least a portion of the sequence of said insertional mutation upstream and downstream of the second breakpoint at said g.129424780bp position and its junction;

optionally, the sequence as set forth in SEQ ID NO:7 and SEQ ID NO:8 is used to amplify at least a portion of the sequence of the insertion mutation upstream and downstream of the first breakpoint at the g.129424780bp position and its junction point.
A probe set for determining whether an insertion mutation exists at a g.129424780bp locus of a genome 1 of a sample to be tested, wherein the inserted nucleic acid fragment is a reverse fragment of at least a part of a g.15043286-150818095 bp region of the genome 1 of a goat.
The probe set of claim 12, wherein the probe set comprises a first probe and/or a second probe having a nucleotide sequence as follows:

a first probe: 5'-AATAAGGGTATAACTCGCTTCTAGGGACTC-3' (SEQ ID NO: 13);

a second probe: 5'-GTAACTGCCAGTATTGTCACTACGAACAAA-3' (SEQ ID NO: 14), wherein the first probe is used to identify a second breakpoint and its junction, and the second probe is used to identify a first breakpoint and its junction;

optionally, the set of probes comprises a third probe: 5'-AATAAGGGTATAACTTCACTACGAACAAAG-3' (SEQ ID NO: 15) and the third probe is used for identifying a horned goat.
A kit for determining whether an insertion mutation exists at a g.129424780bp locus of a genome 1 of a sample to be detected, wherein the inserted nucleic acid fragment is a reverse fragment of at least a part of a g.15043286-150818095 bp region of the genome 1 of a goat.
Kit according to claim 14, characterized in that it comprises a primer set according to any one of claims 9 to 11 and/or a probe set according to any one of claims 12 to 13.
The kit of claim 15, further comprising at least one of PCR amplification enzymes, PCR amplification buffers, and dNTPs.
A method of determining a goat horn pattern, comprising:

obtaining nucleic acid of a goat sample to be detected;

determining whether an insertion mutation exists at a g.129424780bp site of the nucleic acid 1 chromosome, wherein the inserted nucleic acid fragment is a reverse fragment of at least a part of a g.15034286-150818095 bp region of the goat genome 1 chromosome;

when the insertion mutation exists, the insertion mutation is an indication that the goat horn type to be detected is non-horn;

when no insertion mutation exists, the insertion mutation is an indication that the goat horn type to be detected is angular;

optionally, the goat is selected from at least one of the following: guizhou black goats, wu Zhumu Qin goats, jintang black goats, horse goats or Boer goats.
Use of the nucleic acid molecule according to any one of claims 1 to 2, the primer set according to any one of claims 9 to 11, the probe set according to any one of claims 12 to 13, the kit according to any one of claims 14 to 16 for breeding a non-horned variety of goat.