阅读说明：本技术 麦洼牦牛特异性基因、引物组及应用 (Wheat-hollow yak specific gene, primer group and application ) 是由 信金伟 柴志欣 张成福 姬秋梅 钟金城 张强 陈晓英 曹涵文 朱勇 次旦央吉 姜 于 2019-11-07 设计创作，主要内容包括：本发明公开了麦洼牦牛特异性基因、引物组及应用,以解决现有技术中尚未有利用麦洼牦牛特有基因组进行麦洼牦牛品种鉴别、分子育种的问题。本发明的麦洼牦牛特异性基因,包括如SEQ ID NO.1所示的核苷酸序列。本发明引物组包括上引物和下引物,其核苷酸序列分别为：GAGCCCTGAGAACTATCGTGACTT；ATTGGACTCCTGGCGACTTTT。本发明所述的麦洼牦牛特异性基因在鉴别麦洼牦牛品种、分子育种上的应用。利用本发明的特异性基因及引物组,可以从基因水平上进行麦洼牦牛的品种鉴别及分子育种,对于优质牦牛基因资源的保护、优质牦牛资源育种、牦牛近郊品系的亲缘关系鉴定起到极大的推动作用。(The invention discloses a specific gene of a Mailuya yak, a primer group and application thereof, and aims to solve the problem that the unique genome of the Mailuya yak is not utilized to identify the variety of the Mailuya yak and carry out molecular breeding in the prior art. The wheat hollow yak specific gene comprises a nucleotide sequence shown as SEQ ID NO. 1. The primer group comprises an upper primer and a lower primer, and the nucleotide sequences of the primers are respectively as follows: GAGCCCTGAGAACTATCGTGACTT, respectively; ATTGGACTCCTGGCGACTTTT are provided. The wheat-hollow yak specific gene disclosed by the invention is applied to identification of wheat-hollow yak varieties and molecular breeding. By utilizing the specific gene and the primer group, the variety identification and molecular breeding of the wheat-lying yaks can be carried out on the gene level, and the method plays a great promoting role in protecting high-quality yak gene resources, breeding high-quality yak resources and identifying the genetic relationship of suburb strains of the yaks.)

1. The wheat hollow yak specific gene is characterized by comprising a nucleotide sequence shown as SEQ ID NO. 1.

2. The primer set for detecting the nucleotide sequence according to claim 1, wherein the primer set comprises an upper primer and a lower primer, and the nucleotide sequences thereof are respectively: GAGCCCTGAGAACTATCGTGACTT, respectively;

ATTGGACTCCTGGCGACTTTT。

3. a kit comprising the primer set of claim 2.

4. The use of the wheat-hollow yak specific gene of claim 1 for identifying a variety of wheat-hollow yaks.

5. The use of the wheat-hollow yak-specific gene of claim 1 in molecular breeding.

6. A method for identifying a maiken yak variety by using the primer group of claim 2, which comprises the following steps:

step 1, synthesizing a primer;

step 2, preparing a DNA solution;

step 3, preparing a PCR reaction system;

and 4, PCR amplification and electrophoresis detection.

7. The method of claim 6, wherein the primer concentration in step 1 is 10 pmol/. mu.L.

8. The method according to claim 6 or 7, wherein each 20. mu.L of the amplification reaction system comprises the following components:

9. the method of claim 8, wherein the PCR amplification conditions are: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, and extension at 72 ℃ for 5s for 33 cycles.

10. The method of claim 9, wherein the electrophoresis detection condition is that the PCR amplification product is analyzed by gel imaging after electrophoresis for 20min by using 1.0% agarose gel 120V.

Technical Field

The invention belongs to the technical field of livestock raising, and particularly relates to a wheat-spotted yak specific gene, a primer group and application.

Background

The yaks are special cattle species distributed in alpine and subalpine regions which are adjacent to the Qinghai-Tibet plateau, can fully utilize the forage grass resources of the alpine grassland, and have strong adaptability to the ecological environment conditions of the alpine grassland. The domestic animal can live freely under the severe environmental conditions of thin air, short growth period of pasture, cold and long hay-period, and can breed offspring to provide production and living data of meat, milk, wool, fuel, working capacity and the like for local herders, is indispensable in the economy of local animal husbandry, and can be called as 'all-round' domestic animals. Yak is an extremely valuable gene bank in genetic resources. However, the number and the variety group of the Chinese yaks are large, the distribution is wide, only 19 yak varieties are authenticated at present, and a large number of yak varieties which are not authenticated still exist. The current yak variety identification is mainly classified from yak distribution areas and appearance characteristics, and has no exact identification on a gene level. The method is a great limitation on the protection of yak varieties in China, the development of gene resources of different yak varieties and genetic breeding.

Pan-genome (pan-genome) is a concept proposed in 2005 by Tetterin H et al in microbial research, and includes both proper and consensus genomes. Where a unique genome refers to a gene that is present only in a certain population and a consensus genome refers to a gene that is present in all different populations of a species (Tetterin H, et al 2005).

This means that if a consensus sequence of yak species is obtained, it is possible to determine whether or not the target is yak by simple DNA sequencing. If the unique sequences of different varieties of yaks can be obtained, the yaks can be judged by DNA sequence determination, and the assignment is clear. The method plays a great promoting role in protecting high-quality yak gene resources in China, breeding high-quality yak resources, identifying the genetic relationship of suburb yak strains and the like.

The Maidong yaks are mainly produced in southern regions of Hongyuan county and Rulgan county in Sichuan province, are distributed in peripheral counties such as Alba, Pan, Nanping and loam pond, are local yak varieties which are firstly attracted by people to be widely regarded in China and are named because central production regions belong to Maidong tribe. In the prior art, a report of identifying the Mailuya yak variety by using a specific genome is not available, so that the problem that the skilled person needs to solve urgently is solved by providing the specific genome of the Mailuya yak for identifying the Mailuya yak variety and carrying out molecular breeding.

Disclosure of Invention

The invention aims to provide a specific gene of the Mailuya yak, and solves the problem that the unique genome of the Mailuya yak is not utilized to identify the variety of the Mailuyak and carry out molecular breeding in the prior art.

The second purpose of the invention is to provide a primer group for detecting the gene specific to the wheat hollow yak.

The third object of the present invention is to provide a kit comprising the primer set.

The fourth purpose of the invention is to provide the application of the wheat hollow yak specific gene in identifying wheat hollow yak varieties.

The fifth purpose of the invention is to provide the application of the wheat hollow yak specific gene in molecular breeding.

The invention also aims to provide a method for identifying the variety of the maiken yaks by adopting the primer group.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the wheat hollow yak specific gene comprises a nucleotide sequence shown as SEQ ID NO. 1.

The primer group for detecting the nucleotide sequence according to claim 1 comprises an upper primer and a lower primer, and the nucleotide sequences of the primer group are as follows: GAGCCCTGAGAACTATCGTGACTT, respectively;

ATTGGACTCCTGGCGACTTTT。

the kit of the invention comprises the primer group.

The wheat-hollow yak specific gene disclosed by the invention is applied to identification of wheat-hollow yak varieties.

The invention relates to application of a wheat hollow yak specific gene in molecular breeding.

The method for identifying the variety of the Mailuya yaks by adopting the primer group comprises the following steps:

step 1, synthesizing a primer;

step 2, preparing a DNA solution;

step 3, preparing a PCR reaction system;

and 4, PCR amplification and electrophoresis detection.

As an embodiment of the present invention, the primer concentrations in step 1 are all 10 pmol/. mu.L.

In one embodiment of the present invention, each 20. mu.L of the amplification reaction system contains the following components:

as an embodiment of the present invention, the PCR amplification conditions are: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, and extension at 72 ℃ for 5s for 33 cycles.

As an embodiment of the invention, the condition of the electrophoresis detection is that the PCR amplification product is electrophoresed for 20min by using 1.0% agarose gel 120V and then analyzed by gel imaging.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a specific gene and a primer group of the wheat hollow yak for the first time through creative labor. By utilizing the specific gene and the primer group, the variety identification of the wheat-lying yak can be carried out on the gene level, and molecular breeding is carried out, so that the method plays a great promoting role in protecting high-quality yak gene resources, breeding high-quality yak resources and identifying the genetic relationship of suburb strains of the yak.

Drawings

FIG. 1 is a schematic diagram of library construction according to the present invention.

FIG. 2 is a diagram showing the results of PCR electrophoresis in example 2 of the present invention, wherein M is: d2000 DNA Ladder, 1-3 respectively represent Maidong yak, Pariyu yak and Spumak yak.

FIG. 3 is a diagram showing the results of PCR electrophoresis in example 3 of the present invention.

Detailed Description

The present invention is further illustrated by the following examples, which include, but are not limited to, the following examples.

The idea of the invention is that 3 important yak species individuals of the palygorskite, the malpigeon and the sibu yak are subjected to genome-wide sequencing, the specific gene sequence of the malpigeon is found through comparative analysis, a primer group is designed according to specific genes, and the malpigeon of the confirmed species is adopted for verification.