CN111378766B

CN111378766B - Molecular marker related to cashmere fineness character and detection primer and application thereof

Info

Publication number: CN111378766B
Application number: CN202010364206.8A
Authority: CN
Inventors: 王泽英; 白文林; 岳畅; 豆兴堂; 郭素平; 郑圆媛; 惠太宇; 王延茹; 李高谦; 岳奎华; 郭素玲; 王占红; 魏春梅; 武师良; 白智贤; 孙家明; 张鑫江; 蔡伟东
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2022-07-08
Anticipated expiration: 2040-04-30
Also published as: CN111378766A

Abstract

The invention is suitable for the technical field of biology, and provides a molecular marker related to fineness properties of cashmere, and a detection primer and application thereof, wherein the molecular marker comprises a G1355A locus of NFKBIA gene of a cashmere goat; the nucleotide sequence of the molecular marker is shown in a sequence table SEQ ID NO 1; the G1355A site is the R position in the nucleotide sequence, and the base is A or G. The G1355A site has the advantages of AG genotypes in cashmere fineness, cashmere yield, cashmere length and cashmere purification rate. Therefore, the AG genotype of the G1355A site can be used for the breeding of fine cashmere goats to improve the problems of poor cashmere quality and the like of the traditional Liaoning cashmere goats.

Description

Molecular marker related to cashmere fineness character and detection primer and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a molecular marker related to cashmere fineness character, and a detection primer and application thereof.

Background

Down producing goats come in a wide variety, but can be essentially divided into two broad categories: one kind of down producing goat in Russian area features long short down and less down; the other is a cashmere goat with long and short wool and more wool than wool, which is represented by China. Cashmere goats, which are important for the daily life of the human society and are known as fine cashmere, are high-grade and expensive textile raw materials, so that the cashmere diameter becomes one of the important factors determining the fiber value. The cashmere is produced in secondary hair follicles of the skin of the cashmere goat, the diameter of the fiber is 13-18 mu m, the total yield is less, and the cashmere only accounts for about two percent of all animal fibers, so the cashmere becomes the most precious animal fiber type raw material. The cashmere fiber is mainly characterized by softness, lightness, thinness, small thermal conductivity, spinnability, plasticity and the like. The fineness and the length of cashmere and wool fibers determine the style and the performance of all textile products, the smaller the fiber diameter is, the more cashmere yarns are in the same count, and the better the uniformity and the strength are, so that the quality of a spinning product is determined by the fiber fineness. Cashmere fineness is a key for evaluating the quality and measuring the price of the cashmere, the essence of phenotypic characteristics of the cashmere fineness is determined by gene expression, and candidate genes of cashmere fineness related characters are gradually screened out through high-throughput data analysis and whole genome correlation analysis in recent years, but key regulatory genes, expression quantities and molecular regulation mechanisms of the cashmere fineness are not clear. At present, through high-throughput sequencing and biological information analysis, the non-coding RNA is found to be differentially expressed and regulate cashmere fineness related target genes in periodic growth and development of different individual skins, secondary hair follicles and secondary hair follicles among and in cashmere goat varieties.

Research proves that the NFKBIA gene has influence on cashmere quality, and NF-kB plays an important role in regulating apoptosis, cell proliferation and differentiation. Activation of NF-. kappa.B under stimulation by several pro-apoptotic mechanisms leads to phosphorylation and degradation of I.kappa.B-. alpha.in the cytoplasm. At the same time, NF-. kappa.B translocates to the nucleus and affects the transcription of the targeted gene. The NFKBIA gene may regulate the development of aneroid ectodermal immunodeficiency (EDA), which is characteristic of the immune deficiency of thin hair, teeth. EDA ID, which is dominant Autosomal (AD), is caused by heterozygous NFKBIA mutations, is a very rare feature of clinical presentation and is largely unknown. Genetic analysis finds that a new heterozygosis missense mutation, namely p.Ser36Tyr, exists in the NFKBIA gene, and can cause degradation defect of the NFKBIA gene and impaired NF-kB activation. The NFKBIA gene is mainly enriched in the NF-kB pathway. Rel/nuclear factor-kappaB (Rel/NF-kappaB) transcription factors have influence on the control of apoptosis, cell proliferation and tumorigenesis, and researches show that the expression of Rel/NF-kappaB signals in the skin of a mouse can be selectively inhibited by targeting over-expression of an over-inhibitory factor IkappaB alpha, so that the increase of the fundamental frequency of apoptotic cells and the spontaneous occurrence of squamous cell carcinoma are caused. The presence of hyperplasia and hair follicle degeneration also well demonstrate the important role of Rel/NF-kappaB signaling in normal epidermal development. In addition, transgenic skin has strong sensitivity to UV-induced apoptosis. This suggests that the rel/nf-kappa pathway may be involved in the process of skin apoptosis and cancer development. The Rel/nf-kappa signal plays an important role in normal epidermal development, particularly in the control of cell growth in hair follicles and keratinocytes. A previous study demonstrated the inhibitory effect of NF-. kappa.B on epidermal growth in mice. Most researches prove that the NFKBIA gene plays a role in cancer and has a regulation effect on cell proliferation and division.

Although research shows that the NFKBIA gene can regulate cashmere growth, the function of cashmere fineness is not found.

Disclosure of Invention

The embodiment of the invention aims to provide a molecular marker related to the fineness character of cashmere, and aims to solve the problems in the background technology.

The embodiment of the invention is realized in such a way that a molecular marker related to the fineness character of cashmere, which comprises a G1355A locus of NFKBIA gene of a cashmere goat; the nucleotide sequence of the molecular marker is shown in a sequence table SEQ ID NO 1; the G1355A site is the R position in the nucleotide sequence, and the base is A or G.

As a preferable scheme of the embodiment of the invention, the G1355A site has the advantages of AG genotypes in cashmere fineness, cashmere yield, cashmere length and cashmere net rate.

Another objective of the embodiments of the present invention is to provide a detection primer, which is used for detecting the above molecular marker; the detection primer is used for detecting the molecular marker in

claim

1 or 2; the detection primer comprises at least one of an NFKBIA-1 primer pair, an NFKBIA-2 primer pair, an NFKBIA-3 primer pair, an NFKBIA-4/5 primer pair and an NFKBIA-6 primer pair; the nucleotide sequence of the NFKBIA-1 primer pair is shown as a sequence table SEQ ID NO. 2-3; the nucleotide sequence of the NFKBIA-2 primer pair is shown as a sequence table SEQ ID NO. 4-5; the nucleotide sequence of the NFKBIA-3 primer pair is shown as a sequence table SEQ ID NO 6-7; the nucleotide sequence of the NFKBIA-4/5 primer pair is shown as a sequence table SEQ ID NO. 8-9; the nucleotide sequence of the NFKBIA-6 primer pair is shown as a sequence table SEQ ID NO. 10-11.

The embodiment of the invention also aims to provide application of the molecular marker in breeding fine down cashmere goats.

The embodiment of the invention also aims to provide application of the detection primer in preparation of a kit for detecting molecular markers related to the fineness character of cashmere.

The embodiment of the invention also aims to provide application of the detection primer in detecting the mutation site in the NFKBIA gene of the cashmere goat.

As another preferable scheme of the embodiment of the invention, the method comprises the following steps:

obtaining DNA of a down producing goat to be detected to obtain a DNA template;

carrying out PCR amplification on the detection primer and the DNA template to obtain a PCR amplification product;

sequencing the PCR amplification product to obtain a sequencing gene sequence;

and comparing the sequencing gene sequence with a standard gene sequence of the down producing goat NFKBIA to obtain a mutation site in the down producing goat NFKBIA gene to be detected.

The molecular marker related to the cashmere fineness character provided by the embodiment of the invention comprises a G1355A locus of a NFKBIA gene of a cashmere goat; because the G1355A locus has the advantages and genotypes in cashmere fineness, cashmere yield, cashmere length and cashmere purification rate: AG > GG > AA. Therefore, the AG genotype of the G1355A site can be used for the breeding of fine cashmere goats to improve the problems of poor cashmere quality and the like of the traditional Liaoning cashmere goats.

Drawings

FIG. 1 is an agarose gel electrophoresis of DNA templates from different cashmere goat samples.

FIG. 2 is an agarose gel electrophoresis image of PCR amplification products obtained by the five sets of primer pairs respectively.

FIG. 3 is a diagram showing the result of comparison between the sequencing gene sequence obtained by sequencing the PCR amplification product and the NFKBIA standard gene sequence.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

Example 1

The embodiment provides a molecular marker related to fineness traits of cashmere, which comprises a G1355A locus of NFKBIA gene of cashmere goats; the nucleotide sequence of the molecular marker is shown as a sequence table SEQ ID NO:1, and specifically comprises the following steps: ACRCCACTCCACTTGGCTG, respectively; wherein the G1355A site is the R position in the nucleotide sequence, and the base thereof is A or G; the AG genotype of G1355A site can be used for the breeding of fine down goat.

In addition, the embodiment also provides a detection primer for detecting the molecular marker, specifically, the detection primer comprises five sets of primer pairs, specifically, the detection primer is used for detecting the molecular marker according to

claim

1 or 2; the detection primer comprises at least one of an NFKBIA-1 primer pair, an NFKBIA-2 primer pair, an NFKBIA-3 primer pair, an NFKBIA-4/5 primer pair and an NFKBIA-6 primer pair; the nucleotide sequence of the NFKBIA-1 primer pair is shown as a sequence table SEQ ID NO. 2-3; the nucleotide sequence of the NFKBIA-2 primer pair is shown as a sequence table SEQ ID NO. 4-5; the nucleotide sequence of the NFKBIA-3 primer pair is shown as a sequence table SEQ ID NO. 6-7; the nucleotide sequence of the NFKBIA-4/5 primer pair is shown as a sequence table SEQ ID NO. 8-9; the nucleotide sequence of the NFKBIA-6 primer pair is shown as a sequence table SEQ ID NO 10-11; the detection primer can also be used for detecting the mutation site in the NFKBIA gene of the cashmere goat.

The five Primer pairs are obtained by designing specific primers for the CDS region of the NFKBIA gene by using Primer 5.0 software according to the NFKBIA gene sequence (accession number is NC-019475.2) of the cashmere goat, and are synthesized by Shanghai. The sequences, annealing temperatures, and amplified fragment sizes of the primer pairs are shown in Table 1 below.

TABLE 1

Example 2

The embodiment provides a method for detecting mutation sites in NFKBIA gene of a cashmere goat by using the detection primer, which comprises the following steps:

s1, obtaining DNA of the down producing goat to be detected to obtain a DNA template;

s2, carrying out PCR amplification on the detection primer and the DNA template to obtain a PCR amplification product;

s3, sequencing the PCR amplification product to obtain a sequencing gene sequence;

s4, comparing the sequencing gene sequence with a standard gene sequence of the down producing goat NFKBIA to obtain a mutation site in the down producing goat NFKBIA gene to be detected.

Specifically, in practical application, the method can be used for an experiment for searching the marker genotype related to economic characters of the fineness of the cashmere of the Liaoning down goats, and the experimental method can comprise the following steps:

(1) in Liaoyang city cashmere goat breeding farm of Liaoning province, 1140 Liaoning cashmere goats and cashmere goat blood samples and cashmere samples with the birth times of 1-4 fetuses are collected (the blood samples and the cashmere samples are stored in a laboratory); wherein the blood sample can be collected from the jugular vein of cashmere goat (ACD: 6:1), and stored at-20 deg.C. In addition, the determination of cashmere growth performance is completed by Liaoning province animal science research institute, and an analysis report is formed by statistics and data management analysis of a cashmere goat precision breeding management system.

(2) 1mL of the blood sample is centrifuged (5000rpm, 5min), the supernatant is discarded, the precipitate is retained, and the washing is repeated 3 times to obtain the precipitate, which is poured to dryness. Then, mixing the precipitate with 450mL of cell lysate and 6mL of proteinase K, and carrying out water bath treatment for 4 hours (the water bath temperature can be controlled to be 55-66 ℃) to obtain a mixed solution; then, extracting the mixture 2 times (12000rpm, 5min) with a mixed solvent of phenol and chloroform of equal volume (the volume ratio of phenol to chloroform is 25:24), and precipitating with 2 times volume of anhydrous ethanol (-20 deg.C, 30 min); when floc precipitation occurred (12000rpm, 5min), the supernatant was discarded, the precipitate was washed with 800mL of 70% ethanol (12000rpm, 3min), the supernatant was discarded, dried, and then the OD value was measured by UV spectrophotometry to calculate OD260/OD 280. And finally adding 50mL of TE buffer solution for dissolving to obtain a DNA template, and storing at-20 ℃ for later use.

(3) The five primer sets provided in example 1 were subjected to PCR reaction with the DNA template obtained above. Specifically, the PCR reaction system is 25 μ L, which comprises: 12.5 μ L of 2 XTaq Master Mix, 1.0 μ L of upstream primer, 1.0 μ L of downstream primer, 1.0 μ L of DNA template, 9.5 μ L of sterile triple distilled water; the PCR antisense conditions are shown in Table 2 below. After amplification, 5. mu.L of the amplification product was mixed with 1. mu.L of the sample addition buffer, and subjected to spotting and electrophoresis (1.0% agarose gel) with 5. mu.L of Marker as a reference, and subjected to 120V electrophoresis for about 20 min. And (4) finishing the electrophoresis to detect a target strip, photographing and marking, sending to Shanghai's chemical company for bidirectional sequencing, and performing sequence comparison, SNP (Single nucleotide polymorphism) map, mutation site base recognition and genotype determination.

TABLE 2

The results of the above experiment are as follows:

1. the agarose gel electrophoresis pattern of the DNA templates of the different cashmere goat samples obtained according to the above method is shown in FIG. 1. 1-6 in FIG. 1 correspond to the DNA templates of the same cashmere goat sample, respectively.

2. The agarose gel electrophoresis pattern of the PCR amplification products obtained by the five primer sets is shown in figure 2. In addition, the sequencing gene sequence obtained by sequencing the PCR amplification product is compared with the NFKBIA standard gene sequence, the comparison result is shown in figure 3, as can be seen from the figure, 15 mutation sites occur in the CDS region of the down producing goats in the batch, and are highly linked, G1355A is a first polymorphic type, G1547 is linked with C952G, A976G, C2053T, C2058, A2063G, C2096T, T2103, A2136C, G2288A, G2337A and A2390G to form a second polymorphic type, and A2060 2420G is linked with T2423C to form a third polymorphic type.

3. The effective sites of the NFKBIA gene are shown in Table 3.

TABLE 3

As can be seen from Table 3, in the Liaoning cashmere goat population, the frequency and genotype frequency differences of the 3 types of polymorphic genes G1355A, G1547A and A2420G are obvious, A and G are dominant alleles, and the frequency is more than 0.5; the alleles C and G are dominant genes in Liaoning down producing goat population and have better homozygosity; GG and AA genotypes are dominant in Liaoning down goats. G1355A and A2420G polymorphic informationThe amount of PIC is less than 0.25, and the polymorphism is low, which indicates that the variation degree of the site is low and the site is conservative; the heterozygosity of the two sites is low, which indicates that the genetic variation is possibly low; at the same time, the effective allele factor is also low, and may be poor in maintaining the allele ability; chi shape²The gene frequency and the genotype frequency are not stably inherited in the inheritance of generations, and the artificial selection intensity is high.

4. The results of the NFKBIA gene replacement effect analysis are shown in Table 4.

TABLE 4

As can be seen from Table 4, the additive effect values of the NFKBIA gene at the positions G1355A and A2420G are negative numbers, which indicates that the natural mutation and substitution of the allele at the position are positive effects, and the down producing performance can be improved by more than 70%. G1547A is negative for down-regulation, and natural mutation and substitution can reduce down-regulation by about 40%.

5. The analysis of the velvet-producing properties of the polymorphic sites of the NFKBIA gene is shown in Table 5.

TABLE 5

As can be seen from Table 5, the difference between the maximum cashmere fineness, the minimum cashmere fineness and the average cashmere fineness of the three polymorphic types of the NFKBIA gene of the Liaoning cashmere goat is not significant, which indicates that the average cashmere fineness in the population is basically consistent. However, the fineness of the G1547A site GG genotype cashmere is about 14.9 minimum, and the yield and net cashmere rate of the G1355A site AG genotype cashmere are the highest (P < 0.01). The difference of different genotypes of different sites on the length of cashmere is not obvious.

6. The results of gene aggregation analysis of the velvet-producing ability of the population of 3 SNP sites are shown in Table 6.

TABLE 6

As shown in Table 6, the Liaoning cashmere goat population result determined by experiments shows that the dominant genotype of G1547A in the wool production, wool length and wool cleaning rate is AA > GG, and the dominant genotype of fineness is AA < GG. The predominant genotypes of G1355A in fineness, lint yield, lint length and lint-free rate are AG > GG > AA. By performing allelic polymerization at 2 sites, ideal polymerization can form 6 genotype combinations, AAAA, AAGG, AAAG, AGGG, AAAA, and AAAA, respectively.

7. Jingchi type²The test data is reliable, so haplotype analysis is carried out. The results of the combined prediction and analysis of the population tapeout performance of the 2 genotypes AA and GG at the G1547A locus and the 3 genotypes AG, AA and GG at the G1355A locus are shown in Table 7.

TABLE 7

As shown in Table 7, it was found that 3 haplotype combinations of H1: AG, H2: GA, and H3: GGHE 9 haplotypes could be formed in 940 sheep populations by SHEsis (http:// analysis. bio-x.cn/myanalysis. php). Wherein the dominant haplotype combination of the comprehensive velvet production is H1H3: AGGG, the dominant haplotype combination of the capacity production, the velvet length and the net velvet rate is H3H3: GGGGGG, the dominant haplotype combination of the fineness is H2H2: GGAA, and the disadvantaged haplotype combination of the velvet production is H1H1: AAGG.

8. The results of the haplotype combination and productivity association analysis are shown in Table 8.

TABLE 8

As shown in Table 8, haplotype combination H1H1: AAGG has the highest cashmere yield; haplotype combination H2H2: the average, maximum and cashmere fineness of GGAA are significantly different from other combinations; haplotype combination H1H3: the net cashmere yield of the AGG is the maximum; H1H3: the minimum lint fineness of AGGG is optimal.

In conclusion, the population genotype analysis shows that the dominant genotype of the G1547A locus in the velvet production quantity, velvet length and velvet purification rate is AA > GG, and the dominant genotype of fineness is AA < GG; and the G1355A locus has the dominant genotypes of AG > GG > AA in fineness, velvet production, velvet length and velvet net rate. Therefore, the AG genotype of the G1355A site can be used for the breeding of the Liaoning down goats.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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Claims

1. A molecular marker related to fineness traits of cashmere is characterized in that the molecular marker comprises a G1355A locus of NFKBIA gene of cashmere goats; the nucleotide sequence of the molecular marker is shown in a sequence table SEQ ID NO. 1; the G1355A site is the R position in the nucleotide sequence, and the base is A or G.

2. The molecular marker related to the fineness traits of cashmere, according to claim 1, characterized in that the predominant genotypes of G1355A at cashmere fineness, cashmere yield, cashmere length and cashmere net rate are all AG genotypes.

3. Use of a molecular marker as claimed in claim 1 or 2 for the selective breeding of fine-down cashmere goats.

4. The application of a detection primer in preparing a kit for detecting molecular markers related to fineness traits of cashmere is characterized in that the detection primer is used for detecting the molecular markers of claim 1 or 2; the detection primer comprises at least one of an NFKBIA-1 primer pair, an NFKBIA-2 primer pair, an NFKBIA-3 primer pair, an NFKBIA-4/5 primer pair and an NFKBIA-6 primer pair; the nucleotide sequence of the NFKBIA-1 primer pair is shown as a sequence table SEQ ID NO. 2-3; the nucleotide sequence of the NFKBIA-2 primer pair is shown as a sequence table SEQ ID NO. 4-5; the nucleotide sequence of the NFKBIA-3 primer pair is shown as a sequence table SEQ ID NO 6-7; the nucleotide sequence of the NFKBIA-4/5 primer pair is shown as a sequence table SEQ ID NO. 8-9; the nucleotide sequence of the NFKBIA-6 primer pair is shown as a sequence table SEQ ID NO. 10-11.