CN112226520B

CN112226520B - Method for detecting molecular marker in goat circular RNA and application thereof

Info

Publication number: CN112226520B
Application number: CN202011222071.8A
Authority: CN
Inventors: 陶虎; 陈明新; 刘洋; 张年; 杨娟; 熊琪; 杨前平; 李晓锋; 索效军; 张凤; �田宏; 张鹤山; 熊军波
Original assignee: Institute of Animal Science and Veterinary of Hubei Academy of Agricultural Sciences
Current assignee: Institute of Animal Science and Veterinary of Hubei Academy of Agricultural Sciences
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2022-10-28
Anticipated expiration: 2040-11-05
Also published as: CN112226520A

Abstract

The invention provides a method for detecting a molecular marker in goat circular RNA and application thereof, wherein the molecular marker is positioned in a DNA sequence of the goat circular RNA circ _ ZCCHC24, a nucleotide sequence of the molecular marker is shown as a sequence table SEQ ID NO.1, the length of the sequence is 2045bp, a G > A base mutation (named g.651G > A) exists at the 651bp position in the sequence, a G > T base mutation (named g.1082G > T) exists at the 1082bp position in the sequence, and an A > G base mutation (named g.1310A > G) exists at the 1310bp position in the sequence. The invention also provides a detection kit for detecting the molecular marker related to the number of lambs born or/and the growth traits in the goat circular RNA, which comprises the following components: primer pairs shown as SEQ ID NO. 4-7; the single-base extension primer of SNaPshot shown as SEQ ID NO. 8-10. The invention explores a new molecular marker, is simultaneously associated with the number of lambs born by the goats or/and the growth traits, realizes the early selection of the number of lambs born by the goats or/and the growth traits, has a quick and accurate detection method, and is not influenced by the culture environment condition factors.

Description

Method for detecting molecular marker in goat circular RNA and application thereof

Technical Field

The invention relates to the technical field of goat marker-assisted selection, and relates to a method for detecting molecular markers in goat circular RNA and application thereof.

Background

The goat breeding history is long in China and is a common meat source for residents. In recent years, the goat breeding scale is continuously enlarged in the world, and particularly, the goat breeding world is in China. With the increasing demand of consumers for healthy diet, the specific gravity of mutton in meat consumption is promoted to increase continuously due to the healthy and safe nutritional characteristics of mutton. The growth traits including body weight and body size are the most important economic traits in goat production. Therefore, the research on how to improve the growth traits of the goats through breeding is of great significance to the goat industry and the goat breeding research.

Single Nucleotide Polymorphism (SNP) refers to a polymorphism of a DNA sequence caused by variation of a Single nucleotide (A, T, C and G) at the same position in a genome between individuals, and mainly includes four forms of base conversion, transversion, insertion or deletion. The SNP has the characteristics of large quantity, wide distribution, low heterozygosis rate, good genetic stability, suitability for high-throughput automatic detection and the like. Therefore, SNP can be used as a first choice tool for research such as molecular breeding, gene mapping, population evolution and the like. Molecular Marker-assisted selection (MAS) utilizes molecular markers associated with specific traits as an aid for selective breeding. The method has the advantages of rapidness, accuracy and no environmental influence, thereby accelerating the breeding speed, and particularly having great advantages for low heritability and properties which are difficult to measure. With the continuous development of the applied genomics, molecular biology and molecular genetics theoretical technology, the MAS technology can effectively accelerate the selection progress of important economic traits of the goats, and can bring great economic benefits to the sustainable development of the goat industry in China.

Circular RNA (Circular RNA) is a novel endogenous non-coding RNA, which is a covalent closed Circular structure formed by connecting the 5 'end and the 3' end of an exon and/or an intron in a reverse splicing mode in the pre-mRNA splicing process. The CircRNA is widely existed in eukaryotic cells and has the characteristics of species conservation, tissue specificity, stability and the like. There are limited studies of CircRNA regulation of follicular development. Research by Capel et al found that mouse sperm determinant gene SRY can encode circRNA; in the process of aging of pregnant and lying-in women, circRNA in human ovarian granulosa cells may play a role in glucose metabolism, mitotic cell cycle, ovarian hormone production and the like; the CircRNA-9119 can participate in the expression regulation of PTGS2 genes in the endometrial epithelial cells of goats through miRNA-mediated signal pathways. So far, no research report about the goat circular RNA circ _ ZCCHC24 as a molecular marker of lambing number and growth traits exists.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for detecting molecular markers in goat circular RNA and application thereof.

One of the purposes of the invention is to provide three molecular markers related to the goat lambing number or/and growth traits, wherein the molecular markers are positioned in a DNA sequence of a goat circular RNA circ _ ZCCHC24, the nucleotide sequence of the molecular markers is shown in a sequence table SEQ ID NO.1, the length of the sequence is 2045bp, and the three molecular markers comprise: in the sequence, there are one G > A base mutation at 651bp (named g.651G > A), one G > T base mutation at 1082bp (named g.1082G > T) and one A > G base mutation at 1310bp (named g.1310A > G).

Preferably, the base polymorphism site of G or A at 651bp in the sequence is represented by GG, GA or AA three genotypes, wherein G is dominant allele.

Preferably, the G or T base polymorphism site at 1082bp in the sequence is represented by GG, GT or TT three genotypes, wherein G is dominant allele.

Preferably, the site of the nucleotide polymorphism of A or G at the 1310bp in the sequence shows three genotypes of AA, GA or GG, wherein the A is dominant allele.

The second purpose of the invention is to provide the application of the molecular marker in the auxiliary selection of the goat lambing number or/and growth character marker. The application of the molecular marker in at least one of the characters of the goat lambing number character and the goat growth character marker is within the protection scope of the invention.

The invention also aims to provide a detection kit for molecular markers related to the number of lambs born or/and the growth traits in goat circular RNA, which comprises the following components:

a primer pair for amplifying a sequence shown in SEQ ID NO.2 containing g.651G > A sites: the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5; the single-base extension primer of the SNaPshot for detecting the g.651G > A site is shown as SEQ ID NO. 8;

a primer pair for amplifying a sequence shown in SEQ ID No.3 comprising g.1082G > T and g.1310A > G sites: the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.6 and SEQ ID NO. 7; the single-base extension primer of the SNaPshot for detecting the g.1082G > T locus is shown as SEQ ID NO. 9; the single-base extension primer of SNaPshot for detecting g.1310A > G sites is shown as SEQ ID NO. 10.

The fourth purpose of the invention is to provide a method for detecting the molecular marker related to the number of lambs born or/and the growth traits in the goat circular RNA by using the detection kit, which comprises the following steps:

step 1, using genome DNA extracted from a goat blood sample to be detected as a template, constructing a PCR amplification system through a PCR primer pair selected from SEQ ID NO. 4-5 and SEQ ID NO. 6-7, performing PCR amplification on sequences shown as SEQ ID NO.2 and SEQ ID NO.3, and purifying a PCR product;

step 2, with the purified PCR product as a template, constructing a SNaPshot reaction system through a SNaPshot single-base extension primer selected from the group consisting of the snaPshot single-base extension primers shown in SEQ ID NO. 8-10 to perform single-base extension reaction, and purifying the single-base extension product prepared by the reaction;

and 3, detecting the purified single-base extension product by using a genetic analyzer, and analyzing the result by using gene analysis software.

Preferably, the PCR amplification system in step 1 comprises: PCR Mix is 10 muL, 0.5 mumol/L upstream primer, 0.5 mumol/L downstream primer, template DNA2 is 50ng, and deionized water is added to make up to 20 muL; the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 4min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 10s, and 35 cycles; extension at 72 ℃ for 5min.

Preferably, the purified PCR product in the step 2 is a PCR product after SAP and ExoI enzyme treatment; the SNaPshot Reaction system also comprises a Reaction Mix reagent; the PCR amplification conditions were 96 ℃ denaturation 10s,50 ℃ annealing 5s,60 ℃ extension 30s, and 25 cycles.

The fifth purpose of the invention is to provide the application of the detection kit in the auxiliary selection of the goat lambing number or/and growth character marker.

The invention has the beneficial effects that:

the invention discovers a molecular marker related to the goat lambing number or/and growth traits, which is positioned in a DNA sequence of a goat circular RNA circ _ ZCCHC24, wherein the nucleotide sequence of the molecular marker is shown as a sequence table SEQ ID NO.1, the length of the sequence is 2045bp, and a G & gtA base mutation exists at the 113bp position in the sequence; the invention discovers the molecular marker for the first time, is simultaneously associated with two characters (the number of lambs born by the goats and the growth character), realizes the early selection of the number of lambs born by the goats and the growth character, has quick and accurate detection method, and is not influenced by the culture environment condition factors.

Drawings

FIG. 1 is the agarose gel electrophoresis pattern of the SEQ ID NO.2 and SEQ ID NO.3 sequence fragments in goat circular RNA circ _ ZCCHC24, 1: the sequence shown as SEQ ID NO.2, 2: a sequence shown as SEQ ID NO. 3;

FIG. 2 is a graph showing the results of the GeneMapper V4.0 software reading of the g.651G > A site in the goat circular RNA circ _ ZCCHC24 of the present invention; a, picture A: a GG genotype; and B, drawing: GA genotype; and (C) diagram: an AA genotype;

FIG. 3 is a graph showing the results of the GeneMapper V4.0 software reading of g.1082G > T sites in goat circular RNA circ _ ZCCHC24 according to the present invention; a, drawing: a GG genotype; and B, drawing: a GT genotype; and (C) diagram: the TT genotype;

FIG. 4 is a graph showing the results of the GeneMapper V4.0 software reading of the g.1310A > G sites in the goat circular RNA circ _ ZCCHC24 of the present invention; a, picture A: an AA genotype; and B, drawing: GA genotype; and (C) diagram: GG genotype.

Detailed Description

Example 1 establishment of SNP detection method in goat circular RNA circ _ ZCCHC 24.

1. Extracting goat genome DNA:

the test goat varieties are Boer goats, macheng black goats and black-headed sheep (hybrid Boer goats and Macheng black goats), and the samples are from sheep farms at animal husbandry and veterinary research institute of agricultural academy of sciences in Hubei province. The goat genome DNA is extracted by adopting a blood genome DNA extraction kit (produced by Beijing Tiangen Biotechnology Co., ltd.), and the specific steps refer to the kit specification. And (4) detecting the concentration and quality of the extracted DNA, and storing at-40 ℃ for later use.

2. Obtaining SNP genetic marker detection fragments:

(1) PCR amplification

Two pairs of primers are designed according to the SNP genetic marker detection sequence (shown as SEQ ID NO. 1) in the genome sequence of the goat circular RNA circ _ ZCCHC24, and a fragment (shown in figure 1) of two polymorphic sites is amplified, wherein the fragment 1 is a sequence shown as SEQ ID NO.2 containing g.651G > A sites, and the fragment 2 is a sequence shown as SEQ ID NO.3 containing g.1082G > T and g.1310A > G sites. The primers are as follows:

upstream primer for amplification of fragment 1: 5'-AGGCATACATGTGTGAGGTG-3' (nucleotide sequence shown in SEQ ID NO. 4), downstream primer: 5'-GCCTGGCACTGGCGAAGTAC-3' (nucleotide sequence shown in SEQ ID NO. 5); upstream primer for amplification of fragment 2: 5'-ACTGGATGGGGTCACTGGGG-3' (nucleotide sequence shown in SEQ ID NO. 6), downstream primer: 5'-CACGCCTCCAGGTTGATACAG-3' (nucleotide sequence shown in SEQ ID NO. 7).

The genomic DNA of Boer goats, macheng black goats and black head goats are respectively used as templates, the primers are used for PCR amplification, and the PCR reaction system is as follows: PCR Mix was 10. Mu.L, 0.5. Mu. Mol/L forward primer, 0.5. Mu. Mol/L reverse primer, 50ng template DNA, and made up to 20. Mu.L with deionized water. The PCR reaction program is: pre-denaturation at 95 ℃ for 4min; then, 35 cycles of denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s and extension at 72 ℃ for 15s are carried out; extension at 72 ℃ for 5min and storage at 16 ℃.

(2) PCR product purification

The PCR product was purified using Gel Extraction Kit (Shanghai Biotechnology, ltd.), and the detailed procedures were as described in the specification.

3. Detection of molecular markers by the SNaPshot method:

designing a single-base extension primer (the nucleotide sequence is shown as SEQ ID NO. 8) for detecting the g.651G > A site according to the genome sequence of the goat circular RNA circ _ ZCCHC 24; a single-base extension primer of SNaPshot (nucleotide sequence shown as SEQ ID NO. 9) for detecting g.1082G > T sites; the single-base extension primer of SNaPshot (nucleotide sequence shown in SEQ ID NO. 10) for detecting g.1310A > G sites.

Adding 5U SAP and 2U Exo I into 15 μ L of purified PCR product, shaking, mixing, keeping the temperature at 37 ℃ for 1h, and keeping the temperature at 75 ℃ for 15min to inactivate SAP and Exo I enzyme; using a SNaPshot Multiplex Kit (Applied Biosystems) to suck 3 microliter of the treated 15 microliter of PCR product for performing the SNaPshot detection, wherein 10 microliter of PCR Reaction system, 5 microliter of Reaction Mix reagent, 3 microliter of PCR product treated by SAP and ExoI enzyme, 0.5 microliter of extension primer and 1 microliter of deionized water respectively are used, and the PCR amplification program comprises 96 ℃ denaturation 10s,50 ℃ annealing 5s,60 ℃ extension 30s,25 cycles and 4 ℃ storage; diluting the SNaPshot product by 20 times, wherein the dilution system is Hi-Di Formamide 9.25 mu L, GS-120LIZ 0.25 mu L and the reaction system is modification at 95 ℃ for 5min and ice bath for 4min; preparing a mixed solution containing 350 mu L of Hi-Di formamide and 50 mu L of Matrix standard substance, denaturing at 95 ℃ for 5min, rapidly cooling with ice for 5min, bisecting 2 tubes, and performing spectrum correction on a 3730XL DNA Analyzer after subpackaging the mixture to an upper machine plate; performing capillary electrophoresis on the prepared sample by using a 3730XL DNA Analyzer and collecting a signal; the results of the experiment were finally analyzed using GeneMapper V4.0 software (as shown in fig. 2-4).

Example 2 detection of polymorphism distribution of molecular markers prepared according to the present invention in a goat population.

In the present example, polymorphisms of g.651G > A, g.1082G > T and g.1310A > G sites in the genomic sequence of goat circular RNA circ _ ZCCHC24 were detected in the Boer goat, hei-ceps and Macheng black goat populations, respectively, and the detection results are shown in Table 1.

Table 1-genotype frequency and allele frequency of SNP sites in goat circ _ ZCCHC24 in the population:

from the results of table 1, it can be seen that: the three polymorphic loci of g.651G > A, g.1082G > T and g.1310A > G, except that the locus of g.1082G > T does not have TT genotype in Boer goat population, other loci are expressed into three genotypes in Boer goat population, blackhead goat population and Macheng black goat population, wherein the genotypes of the g.651G > A and the g.1082G > T loci are homozygously occupied, and the locus of g.1310A > G loci is heterozygously occupied. The allele G of the g.651G > A and the g.1082G > T loci in the three goat populations is a dominant allele, the frequency of the allele A and the frequency of the allele G in the g.1310A > G loci are not different greatly, and the allele A is slightly dominant and is a dominant allele.

Example 3 correlation analysis and application of the molecular marker prepared by the invention and goat lambing number traits.

In order to determine whether the three polymorphic loci of g.651G > A, g.1082G > T and g.1310A > G in the genome sequence of the goat circular RNA circ _ ZCCHC24 are related to the difference of the lambing number traits of Boer goats, blackhead goats and Macheng black goats, the method established in example 1 is adopted for carrying out polymorphism detection, and the correlation between the three genotypes of the three loci of the goat and the lambing number traits is analyzed. The SAS statistical software (SAS Institute Inc, version 9.1) GLM program was used for analysis of variance of different SNP genotype combinations and for significance testing, the models used were:

Yijklm＝μ+Pi+Sj+Fk+Gl+Mm+eijklm；

yijklm is the phenotypic value of the trait, μ is the mean value, pi is the influence of the ith birth (i =1, 2, 3, 4), sj is the genotype effect, fk is the influence of the kth sheep field (k =1, 2), gl is the influence of the ith genotype (l = 1-3), mm is the dam effect, eijklm is the residual effect. The results of the statistical analysis are shown in table 2:

table 2-association analysis of goat g.651g > a locus with lambing number trait:

note: in peer-to-peer comparison, different lower case letters represent significant differences (P < 0.05); * Representing significant differences (P < 0.05).

As can be seen from Table 2, the total number of lambs born by the AA genotype of the g.651G > A site in the blackhead sheep population is significantly higher than that of the GA genotype and the GG genotype (P < 0.05). The total lambing number of AA genotype individuals is higher, the total lambing number of GA and GG genotype individuals is lower, and the additive effect and the dominant effect reach a significant level (P < 0.05).

Table 3-association analysis of goat g.1082g > T locus with lambing number trait:

note: in the same row comparison, different capital letters represent significant differences (P < 0.01); * Represents very significant difference (P < 0.01).

As can be seen from Table 3, in the Boer goat population, the number of first-born lambs of GT genotype at the g.1082G > T locus was significantly higher than that of GG genotype (P < 0.01). The GT genotype individual has a higher number of head born lambs, and the GG genotype individual has a lower number of lambs. No lambing was recorded for the TT genotype, and the additive effect reached a very significant level (P < 0.01). The g.1082G > T locus has no significant difference in the number of the head fetus lambs and the total lambs of each genotype in the black head goat and the Macheng black goat population.

In conclusion, the g.651G > A locus has better lambing number trait of the AA genotype individual in the total lambing number trait of the black head goat, and the g.1082G > T locus has better lambing number trait of the GT genotype individual in the head fetus lambing number trait of the Boer goat. Therefore, in the early detection, when the black-headed goat individual has an AA genotype at the g.651G > A locus in the genome sequence of the goat circular RNA circ _ ZCCHC24 or the Boer goat individual has a GT genotype at the g.1082G > T locus, the individual is indicated to have better lambing number trait potential.

Example 4 correlation analysis and application of the molecular marker prepared by the invention and growth traits of the year.

In order to determine whether the differences of the three polymorphic loci of g.651G > A, g.1082G > T and g.1310A > G in the genomic sequence of the goat circular RNA circ _ ZCCHC24 and the growth traits of the Weekly black-headed sheep are related or not, the method established in example 1 is adopted for carrying out polymorphism detection, and the correlations of the three genotypes of the three loci of the goat with the growth traits of the Weekly black-headed sheep, such as body weight, body size and the like are analyzed. SAS statistical software (SAS Institute Inc, version 9.1) GLM program is adopted to perform correlation analysis of locus genotype and growth traits of the year, and significance test is performed, wherein the adopted model is as follows:

Yikjlm＝μ+Gi+Fk+Aj+Sl+Pm+eikjlm

y is the phenotypic value of the trait, mu is the average value of the individual traits, gi is the genotype effect, fk is the field fixation effect, aj is the age fixation effect, sl is the gender fixation effect, pm is the fetal number fixation effect, and e is the random error.

Correlation analysis between three genotypes of g.651G > A, g.1082G > T and g.1310A > G loci and growth traits of the year of the week is carried out in 100 black-head sheep groups, and the results of statistical analysis are shown in tables 4, 5 and 6.

Table 4-association analysis of goat g.651g > a locus with growth trait of the third year of life of black-headed sheep:

note: in the same row comparison, different lower case letters represent significant differences (P < 0.05), different upper case letters represent very significant differences (P < 0.01), and "x" represents very significant differences (P < 0.01).

As can be seen from Table 4, in the black-head sheep population, the GA genotype of the g.651G > A site is significantly higher than the AA genotype (P < 0.05) and significantly higher than the GG genotype (P < 0.01) in the body height traits of the goats, while the AA genotype and the GG genotype have no significant difference in the body height traits of the goats, and the dominant effect of the body height traits of the goats reaches a significant level (P < 0.01).

Table 5-association analysis of goat g.1082g > T locus with growth trait of the third year of life of black head sheep:

note: in the same row comparison, different lower case letters represent significant differences (P < 0.05), different upper case letters represent very significant differences (P < 0.01), and a-letter represents very significant differences (P < 0.01).

As can be seen from table 5, in the body weight trait of the year of the black head sheep, the TT genotype of the g.1082g > T site is significantly higher than the GG genotype (P < 0.01), significantly higher than the GT genotype (P < 0.05), significantly higher than the GT genotype (P < 0.01), and the additive effect is a very significant level (P < 0.01); in the yearly somatic height trait, the GT genotype is significantly higher than the GG genotype (P < 0.05); in the body oblique growth trait of the year, the TT genotype is very significantly higher than the GG genotype and the GT genotype (P < 0.01), the GT genotype is very significantly higher than the GG genotype (P < 0.01), and the additive effect is very significant level (P < 0.01); in the mid-year-old bust and tube traits, the GT genotype was significantly higher than the GG genotype (P < 0.05).

Table 6-association analysis of goat g.1310a > G locus with growth trait of the third year of black-headed sheep:

note: in the same row comparison, different lower case letters represent significant differences (P < 0.05) and different upper case letters represent very significant differences (P < 0.01).

As can be seen from Table 5, in the high trait of the third year of life of the blackhead sheep population, the GA genotype at the g.1310A > G locus was significantly higher than the AA genotype (P < 0.05), and was significantly higher than the GG genotype (P < 0.01).

In conclusion, three polymorphic sites of g.651G > A, g.1082G > T and g.1310A > G in the genomic sequence of the goat circular RNA circ _ ZCCHC24 are obviously or extremely obviously different from one or more indexes of body weight, body height, oblique body length, chest circumference and tube circumference in the growth trait of the Weekly-old black-headed sheep. In early detection, when the G.651G > A locus of an individual has GA genotype, the G.1082G > T locus has TT genotype or GT genotype, and the G.1310A > G locus has GA genotype, the individual has better growth trait potential.

The invention is not to be considered as limited to the particular embodiments shown, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

SEQ ID NO.1

ATCCCAGGCTTTGGTCCAAGCCTACACGAGAGAGAAAAGAGGCCCAGAGAGGGAAAGGGCTGGGCCAGAGTCACAGAGCAGGGTGTCCAGGGAGCCAAGAAGAGTGAGGGCTGTATCCTCCAGGGAGGAGTGCCGGACCTCCAGAGGCATACATGTGTGAGGTGTGGGCTCTGTCCTGGTTGGAGGAGGGGTGTCTTGATTCCCTGGGACCAGCTCTCAGCTTACTACCCTCCCTTCCTACACTGAGGGGACACAGAAAAAGAACTGGTATGCCCAAGAACTGGAGCCTAGTAACCTGAGCCCCGTGCCCAGGTTTGTCATCAGCCCCTGGGAAGCTACCTCTTTCTAGTCTTGGCCTGGCCCCGGGAGAAGGGTCTCATGGCCAGCCTTACTGCAGGCCCCACCTTTGCTGAATTCCAGTGTGCCTGGCCGCCCAGAGCCCGTGACTCCTATAAATAGAGGCTGTATACACCAGGAGGGGGCCTGGGCCACGTCTGCTTTGAAGCCAGGCAAGACATCTGGTTCCTGTCAACTGGAGGTGGGAGAGGTGAGAGAGGTGGGCCGTTTCCTCCCCGGGCTTCCAGAGAGCTGGCCCAGACTGGGCTGAGGGCTCCCCGGACCCACTGTGCACAGGCGTCGGCCTGCAGGAGRCCAGGCAAAGTGCCGAACTTCAGTGTCCTCTTCTGTACAGTGGAAACGAGGCTGCGAGCTGGTCACCCCACCAGCACACGGCAGAGATGCGGCTCTCGTAGGTGGCTTTCTGCTGCTTGTCTTACCTCCTGGCAGCGTGTAAAGAACTAATGATCACCACCGTCACCATCATCATGTACTGAGTACTTCGCCAGTGCCAGGCAGGAAATAAAGGGCTATGTTAGCCTCCTAGAAACCCTTGGAGGTAGCTGTCATTACTCCTGTTTACTGGATGGGGTCACTGGGGTCCCGAGTGGTTCACTGGCTTGTCCATGGATGCACAACTCGAGCAGGGATTTGAACATGGGCTCCAGGAAGCTGGAGCCCACAGGTGACTAAAGAGTCAGTATGAAACTAGGAATGAAACCACCATATGACCCAGAAATCCCACKCCTAGGCATATACCCTGAGGAAACGAAAATTGAAAAAGACACACGTATCCCATTGTTCATTGCAACACTATTTGAACTATAGAACATGGAAGCAACCTAGATGTCCGTCAACAGATGAATGAATAAAAAGTTGTGTACATATACACAATGGAATATTATCAGCCATAAAAAGAAACACATTTGAGTCAGTTCTAATGAGGTGGATGAACTTAGAACCTATTATACAGRGTGAAGTAAGTCAGAAAGAGAAAAATAAACATTATATTCTAATGCATATATACAGAATCTAGAAAAATGGTGCTGAAGAATTTATTTGCAGGGCAGCAATGGAGAAACAGACTTAGGGAATAGACTTATGGACAGGGGAGAGGGGAGGAGAGGGTGAGATGTATGGAAAGAGTAACATGGAAACTTATATTACCATATGTAAAATAGATAGCCAACGGGAATTTGCTGTATGGCTCAGGAAACTCAAACGGGCTCTGTATCAACCTGGAGGCGTGGGATGGGGAGGGAGATGGGAGGGGGGTTCAGAAGAGAGGGGATATATGTATACCTATGGCTGATTCGTGTTGAGGTTTGACAGAAAGCAACAAAATTCTGTAAAGCAATTATCCTTCAATAAAAAATAAGAAAAAAGAAAAATCAGGGAAGACAAAAAACAAAGACTGGGTATGAATGCGCGTGTGTGTAGGAGCCTGTGGAAGCACGGCTGCACCTTGGGCCGGGCCCCCAGGCCTCACAGCCTCCGTCCCCCTCAGGCGCTGGGCAGCAGCGTGTACAAGAGCGCCTCACCCTACGGCTCCCTCAGCAACATCGCCGACGGCCTCAGCTCCCTCACCGAGCACTTCTCCGACCTGACCCTCGCCTCCGAGACCCGCAAGCCCAGCAAGCGGCCCCCACCCAACTACCTGTGCCACCTGTGCTTCAACAAAGGGCACTACATCAAGGACTGCCCCCAGG

R represents G/A, K represents G/T

SEQ ID NO.2：

AGGCATACATGTGTGAGGTGTGGGCTCTGTCCTGGTTGGAGGAGGGGTGTCTTGATTCCCTGGGACCAGCTCTCAGCTTACTACCCTCCCTTCCTACACTGAGGGGACACAGAAAAAGAACTGGTATGCCCAAGAACTGGAGCCTAGTAACCTGAGCCCCGTGCCCAGGTTTGTCATCAGCCCCTGGGAAGCTACCTCTTTCTAGTCTTGGCCTGGCCCCGGGAGAAGGGTCTCATGGCCAGCCTTACTGCAGGCCCCACCTTTGCTGAATTCCAGTGTGCCTGGCCGCCCAGAGCCCGTGACTCCTATAAATAGAGGCTGTATACACCAGGAGGGGGCCTGGGCCACGTCTGCTTTGAAGCCAGGCAAGACATCTGGTTCCTGTCAACTGGAGGTGGGAGAGGTGAGAGAGGTGGGCCGTTTCCTCCCCGGGCTTCCAGAGAGCTGGCCCAGACTGGGCTGAGGGCTCCCCGGACCCACTGTGCACAGGCGTCGGCCTGCAGGAGRCCAGGCAAAGTGCCGAACTTCAGTGTCCTCTTCTGTACAGTGGAAACGAGGCTGCGAGCTGGTCACCCCACCAGCACACGGCAGAGATGCGGCTCTCGTAGGTGGCTTTCTGCTGCTTGTCTTACCTCCTGGCAGCGTGTAAAGAACTAATGATCACCACCGTCACCATCATCATGTACTGAGTACTTCGCCAGTGCCAG GC

R represents G/A

SEQ ID NO.3：

ACTGGATGGGGTCACTGGGGTCCCGAGTGGTTCACTGGCTTGTCCATGGATGCACAACTCGAGCAGGGATTTGAACATGGGCTCCAGGAAGCTGGAGCCCACAGGTGACTAAAGAGTCAGTATGAAACTAGGAATGAAACCACCATATGACCCAGAAATCCCACKCCTAGGCATATACCCTGAGGAAACGAAAATTGAAAAAGACACACGTATCCCATTGTTCATTGCAACACTATTTGAACTATAGAACATGGAAGCAACCTAGATGTCCGTCAACAGATGAATGAATAAAAAGTTGTGTACATATACACAATGGAATATTATCAGCCATAAAAAGAAACACATTTGAGTCAGTTCTAATGAGGTGGATGAACTTAGAACCTATTATACAGRGTGAAGTAAGTCAGAAAGAGAAAAATAAACATTATATTCTAATGCATATATACAGAATCTAGAAAAATGGTGCTGAAGAATTTATTTGCAGGGCAGCAATGGAGAAACAGACTTAGGGAATAGACTTATGGACAGGGGAGAGGGGAGGAGAGGGTGAGATGTATGGAAAGAGTAACATGGAAACTTATATTACCATATGTAAAATAGATAGCCAACGGGAATTTGCTGTATGGCTCAGGAAACTCAAACGGGCTCTGTATCAACCTGGAGGCGTG

K represents G/T, R represents G/A

SEQ ID NO.4：

AGGCATACATGTGTGAGGTG

SEQ ID NO.5：

GCCTGGCACTGGCGAAGTAC

SEQ ID NO.6：

ACTGGATGGGGTCACTGGGG

SEQ ID NO.7：

CACGCCTCCAGGTTGATACAG

SEQ ID NO.8：

TTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTCGGCCTGCAGGAG

SEQ ID NO.9：

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATGACCCAGAAATCCCAC

SEQ ID NO.10：

TTTTTTTTTTTTTTTTTTTTTTATTTTTCTCTTTCTGACTTACTTCAC

Sequence listing

<110> institute of zootechnics of academy of agricultural sciences of Hubei province

<120> method for detecting molecular marker in goat circular RNA and application thereof

<141> 2020-10-12

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2045

<212> DNA

<213> Capra hircus

<400> 1

atcccaggct ttggtccaag cctacacgag agagaaaaga ggcccagaga gggaaagggc 60

tgggccagag tcacagagca gggtgtccag ggagccaaga agagtgaggg ctgtatcctc 120

cagggaggag tgccggacct ccagaggcat acatgtgtga ggtgtgggct ctgtcctggt 180

tggaggaggg gtgtcttgat tccctgggac cagctctcag cttactaccc tcccttccta 240

cactgagggg acacagaaaa agaactggta tgcccaagaa ctggagccta gtaacctgag 300

ccccgtgccc aggtttgtca tcagcccctg ggaagctacc tctttctagt cttggcctgg 360

ccccgggaga agggtctcat ggccagcctt actgcaggcc ccacctttgc tgaattccag 420

tgtgcctggc cgcccagagc ccgtgactcc tataaataga ggctgtatac accaggaggg 480

ggcctgggcc acgtctgctt tgaagccagg caagacatct ggttcctgtc aactggaggt 540

gggagaggtg agagaggtgg gccgtttcct ccccgggctt ccagagagct ggcccagact 600

gggctgaggg ctccccggac ccactgtgca caggcgtcgg cctgcaggag rccaggcaaa 660

gtgccgaact tcagtgtcct cttctgtaca gtggaaacga ggctgcgagc tggtcacccc 720

accagcacac ggcagagatg cggctctcgt aggtggcttt ctgctgcttg tcttacctcc 780

tggcagcgtg taaagaacta atgatcacca ccgtcaccat catcatgtac tgagtacttc 840

gccagtgcca ggcaggaaat aaagggctat gttagcctcc tagaaaccct tggaggtagc 900

tgtcattact cctgtttact ggatggggtc actggggtcc cgagtggttc actggcttgt 960

ccatggatgc acaactcgag cagggatttg aacatgggct ccaggaagct ggagcccaca 1020

ggtgactaaa gagtcagtat gaaactagga atgaaaccac catatgaccc agaaatccca 1080

ckcctaggca tataccctga ggaaacgaaa attgaaaaag acacacgtat cccattgttc 1140

attgcaacac tatttgaact atagaacatg gaagcaacct agatgtccgt caacagatga 1200

atgaataaaa agttgtgtac atatacacaa tggaatatta tcagccataa aaagaaacac 1260

atttgagtca gttctaatga ggtggatgaa cttagaacct attatacagr gtgaagtaag 1320

tcagaaagag aaaaataaac attatattct aatgcatata tacagaatct agaaaaatgg 1380

tgctgaagaa tttatttgca gggcagcaat ggagaaacag acttagggaa tagacttatg 1440

gacaggggag aggggaggag agggtgagat gtatggaaag agtaacatgg aaacttatat 1500

taccatatgt aaaatagata gccaacggga atttgctgta tggctcagga aactcaaacg 1560

ggctctgtat caacctggag gcgtgggatg gggagggaga tgggaggggg gttcagaaga 1620

gaggggatat atgtatacct atggctgatt cgtgttgagg tttgacagaa agcaacaaaa 1680

ttctgtaaag caattatcct tcaataaaaa ataagaaaaa agaaaaatca gggaagacaa 1740

aaaacaaaga ctgggtatga atgcgcgtgt gtgtaggagc ctgtggaagc acggctgcac 1800

cttgggccgg gcccccaggc ctcacagcct ccgtccccct caggcgctgg gcagcagcgt 1860

gtacaagagc gcctcaccct acggctccct cagcaacatc gccgacggcc tcagctccct 1920

caccgagcac ttctccgacc tgaccctcgc ctccgagacc cgcaagccca gcaagcggcc 1980

cccacccaac tacctgtgcc acctgtgctt caacaaaggg cactacatca aggactgccc 2040

ccagg 2045

<210> 2

<211> 709

<212> DNA

<213> Capra hircus

<400> 2

aggcatacat gtgtgaggtg tgggctctgt cctggttgga ggaggggtgt cttgattccc 60

tgggaccagc tctcagctta ctaccctccc ttcctacact gaggggacac agaaaaagaa 120

ctggtatgcc caagaactgg agcctagtaa cctgagcccc gtgcccaggt ttgtcatcag 180

cccctgggaa gctacctctt tctagtcttg gcctggcccc gggagaaggg tctcatggcc 240

agccttactg caggccccac ctttgctgaa ttccagtgtg cctggccgcc cagagcccgt 300

gactcctata aatagaggct gtatacacca ggagggggcc tgggccacgt ctgctttgaa 360

gccaggcaag acatctggtt cctgtcaact ggaggtggga gaggtgagag aggtgggccg 420

tttcctcccc gggcttccag agagctggcc cagactgggc tgagggctcc ccggacccac 480

tgtgcacagg cgtcggcctg caggagrcca ggcaaagtgc cgaacttcag tgtcctcttc 540

tgtacagtgg aaacgaggct gcgagctggt caccccacca gcacacggca gagatgcggc 600

tctcgtaggt ggctttctgc tgcttgtctt acctcctggc agcgtgtaaa gaactaatga 660

tcaccaccgt caccatcatc atgtactgag tacttcgcca gtgccaggc 709

<210> 3

<211> 668

<212> DNA

<213> Capra hircus

<400> 3

actggatggg gtcactgggg tcccgagtgg ttcactggct tgtccatgga tgcacaactc 60

gagcagggat ttgaacatgg gctccaggaa gctggagccc acaggtgact aaagagtcag 120

tatgaaacta ggaatgaaac caccatatga cccagaaatc ccackcctag gcatataccc 180

tgaggaaacg aaaattgaaa aagacacacg tatcccattg ttcattgcaa cactatttga 240

actatagaac atggaagcaa cctagatgtc cgtcaacaga tgaatgaata aaaagttgtg 300

tacatataca caatggaata ttatcagcca taaaaagaaa cacatttgag tcagttctaa 360

tgaggtggat gaacttagaa cctattatac agrgtgaagt aagtcagaaa gagaaaaata 420

aacattatat tctaatgcat atatacagaa tctagaaaaa tggtgctgaa gaatttattt 480

gcagggcagc aatggagaaa cagacttagg gaatagactt atggacaggg gagaggggag 540

gagagggtga gatgtatgga aagagtaaca tggaaactta tattaccata tgtaaaatag 600

atagccaacg ggaatttgct gtatggctca ggaaactcaa acgggctctg tatcaacctg 660

gaggcgtg 668

<210> 4

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 4

aggcatacat gtgtgaggtg 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 5

gcctggcact ggcgaagtac 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 6

actggatggg gtcactgggg 20

<210> 7

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 7

cacgcctcca ggttgataca g 21

<210> 8

<211> 44

<212> DNA

<213> Artificial Synthesis

<400> 8

tttttttttt tttttttttt ttttttttcg tcggcctgca ggag 44

<210> 9

<211> 60

<212> DNA

<213> Artificial Synthesis

<400> 9

tttttttttt tttttttttt tttttttttt tttttttttt atatgaccca gaaatcccac 60

<210> 10

<211> 48

<212> DNA

<213> Artificial Synthesis

<400> 10

tttttttttt tttttttttt ttatttttct ctttctgact tacttcac 48

Claims

1. A molecular marker in goat circular RNA is characterized in that: the molecular markers are respectively positioned in DNA sequences of goat circular RNA circ _ ZCCHC24, the nucleotide sequences of the molecular markers are shown in a sequence table SEQ ID NO.1, the length of the sequences is 2045bp, and the molecular markers comprise a G > A base mutation (named as g.651G > A) at the 651bp position, a G > T base mutation (named as g.1082G > T) at the 1082bp position and an A > G base mutation (named as g.1310A > G) at the 108bp position in the sequences.

2. The molecular marker of claim 1, wherein: the base polymorphism sites of G or A at the 651bp position in the sequence are represented by GG, GA or AA three genotypes, wherein G is a dominant allele.

3. The molecular marker of claim 1, wherein: the G or T base polymorphism site at 1082bp in the sequence is represented by GG, GT or TT three genotypes, wherein G is a dominant allele.

4. The molecular marker of claim 1, wherein: the nucleotide polymorphism sites of A or G at the 1310bp in the sequence are expressed as three genotypes of AA, GA or GG, wherein A is a dominant allele.

5. Use of the molecular marker of any one of claims 1-4 in marker-assisted selection of goat lambs, said goats are Boer, black-headed and Macheng goats, said goat lambs are head-born and total lambs, and said growth traits are body height, body weight, oblique body length, chest circumference and tube circumference at age.

6. A detection kit of a molecular marker related to the number of lambs and/or growth traits in goat circular RNA is characterized by comprising:

a primer pair for amplifying a sequence shown in SEQ ID No.3 comprising g.1082g > T and g.1310a > G sites: the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.6 and SEQ ID NO. 7; the single-base extension primer of the SNaPshot for detecting the g.1082G > T locus is shown as SEQ ID NO. 9; the single-base extension primer of SNaPshot for detecting g.1310A > G sites is shown as SEQ ID NO. 10;

the goats are Boer goats, black-head goats and Macheng goats, the goat lambing number is the number of head fetus lambing and the total lambing number, and the growth traits are body height, body weight, oblique body length, chest circumference and tube circumference of the whole year.

7. A method for using the kit for detecting the molecular marker related to the number of lambs or/and the growth trait in the goat circular RNA as claimed in claim 6, which comprises the following steps:

8. The method of use of claim 7, wherein the PCR amplification system of step 1 comprises: PCR Mix is 10 muL, 0.5 mumol/L upstream primer, 0.5 mumol/L downstream primer, template DNA is 50ng, deionized water is added to make up to 20 muL; the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 4min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 10s, and 35 cycles; extension at 72 ℃ for 5min.

9. The method of claim 7, wherein the purified PCR product of step 2 is a PCR product after SAP and ExoI enzyme treatment; the SNaPshot Reaction system also comprises a Reaction Mix reagent; the PCR amplification conditions were 96 ℃ denaturation 10s,50 ℃ annealing 5s,60 ℃ extension 30s, and 25 cycles.

10. The use of the test kit of claim 6 in marker-assisted selection of goat lambs, said goats are Boer, black-headed and Macheng goats, said goat lambs are head-born and total lambs, and said growth traits are yearly height, body weight, oblique body length, chest circumference and tube circumference.