CN112538533B - Molecular marker related to long-hair rabbit hair length and application thereof - Google Patents
Molecular marker related to long-hair rabbit hair length and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of rabbit molecular marker breeding, in particular to a molecular marker related to long-hair rabbit down hair length and application thereof. The molecular marker comprises a KRT26 gene mutant, wherein the KRT26 gene is shown as SEQ ID NO:1, the KRT26 gene mutant is formed by mutating a base G at the 41844263 th site of KRT26 into A, and comprises three genotypes of GG, GA and AA, wherein the lengths of GG-type villi and AG-type villi are remarkably higher than that of AA-type villi. The detection of the KRT26 gene polymorphic site provides a marker auxiliary means for the selection of long-hair rabbit hair length characters.
Description
Technical Field
The invention relates to the field of rabbit molecular marker breeding, in particular to a molecular marker related to long-hair rabbit down hair length and application thereof.
Background
The long-hair rabbit quilt hair is mainly composed of coarse hair and down hair, wherein the down hair is the main part of the long-hair rabbit quilt hair, is densely distributed on the surface of the long-hair rabbit hair, is waved and bent, is slender and soft, has good physicochemical property and spinnability, has extremely high textile value in the textile industry, and is called as 'gold fiber' comparable to cashmere. In recent years, the demand of high-quality worsted rabbit hair is increasing at home and abroad, but at present, a high-quality hair type long hair rabbit group is lacked in production, and the developed hair type long hair rabbit breeding has a wide market prospect.
The textile value of long-hair rabbit wool fibers depends mainly on the length, fineness and homogeneity of the wool. For a long time, the breeding and production of long-hair rabbits excessively pursue the hair yield, the improvement on the fiber quality is ignored, and the hair of rabbit hair textiles is easy to fall off due to the insufficient length of the produced rabbit hair, so the textile value is seriously influenced. The research on the villus length breeding method is very little, the conventional breeding method is influenced by the hair-raising period, the determination level, the population scale, the genetic evaluation technology and the like, and the selection accuracy is relatively low.
Keratin is a protein widely present in the epidermis of humans and animals, is a major component of hair, feathers, hooves, shells, horns, belongs to a fibrous, non-nutritive protein, and is an important structural protein of connective tissues (guanfeng, 2007). Keratins are divided into two classes, the keratin intermediate filament (KRT), which is an important member of the keratin family, and the Keratin Associated Protein (KAP). KRT has two types of I type and II type, and KRT26, KRT27, KRT31 and KRT38 belong to type I KRT genes and are mainly expressed in inner root sheath, medulla and inner layer of hair follicle to regulate and control the growth and development of the whole hair follicle; KRT85 belongs to type II KRT gene, and is mainly expressed in matrix and stratum corneum of hair follicle to regulate and control micro and macro structure of hair fiber. The KRT26(keratin 26) gene can encode a protein containing 468 amino acids, is specifically expressed in the inner root sheath of hair follicle as a special type I keratin, mainly regulates the growth and development of the whole hair follicle, and the gene structure and function variation can affect the hair fiber characteristics of animals.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a molecular marker related to the length of long-hair rabbit hair, the molecular marker has obvious genetic effect on the length of the long-hair rabbit hair, and can be applied to molecular breeding of the length of the long-hair rabbit hair.
In order to realize the purpose, the invention adopts the technical scheme that:
a molecular marker related to the length of long-hair rabbit hair comprises a KRT26 gene mutant, wherein the KRT26 gene is shown as SEQ ID NO:1, and the KRT26 gene mutant is formed by mutating a base G at the 41844263 th position of a KRT26 gene site to A.
In the technical scheme of the invention, the base G at 41844263 of KRT26 gene locus of the long-hair rabbit is mutated into A to form a mutant, and the lengths of GG-type and AG-type villi are extremely longer than those of AA-type villi. In actual breeding, a group homozygous for allele G or a group with genotype AG obtained by crossing can be selected respectively to increase the villus length of filial generation.
The invention also provides an amplification primer for detecting the molecular marker. The amplification primers comprise primers for amplifying KRT26 gene, and the primers for amplifying KRT26 gene are shown as SEQ ID NO. 2 and SEQ ID NO. 3.
The invention also provides application of the molecular marker in long-hair rabbit hair length character selection.
In addition, the invention also provides a method for screening the molecular marker related to the length of the long-hair rabbit hair, which comprises the following specific steps: constructing a genome DNA mixed pool, respectively adopting the primer sequences to carry out PCR amplification on the genome DNA of the individual sample of the long-hair rabbit to be detected to obtain a PCR product, screening out a gene SNP locus according to the PCR product, then adopting a flight mass spectrometry detection method to carry out SNP typing, and determining whether the base at 41844263 of the KRT26 gene locus is G or mutated into A.
As a preferred embodiment of the screening method, the flight mass spectrometry detection method comprises the following steps of:
s1, designing a PCR (polymerase chain reaction) and a single-base amplification primer according to SNP (Single nucleotide polymorphism) site information, and performing quality inspection on a genome DNA sample to obtain a qualified genome DNA sample;
s2, carrying out PCR reaction on the qualified genome DNA sample, and then carrying out SAP digestion and extension reaction to finally obtain a reaction product;
s3, diluting the reaction product, desalting by using resin, spotting the sample subjected to desalting on a sample target, naturally crystallizing, then loading the sample on a machine for mass spectrum detection, and collecting data.
In a preferred embodiment of the screening method of the present invention, the length of the PCR product is 250 bp.
The invention adopts a flight mass spectrometry detection method to carry out SNP typing on an SNP site existing in KRT26 gene, sequence comparison shows that the basic group G at the 41844263 th position of KRT26 gene site of long-hair rabbit is mutated into A, the long-hair rabbit hair length and KRT26 gene are subjected to correlation analysis and group verification, the result shows that three genotypes obtained by SNP typing are closely related to the hair length property of long-hair rabbit, the basic group G at the 41844263 th position of KRT26 gene site of long-hair rabbit is mutated into A, and GG, AG and AA three genotypes are formed, wherein the hair lengths of GG type and AG type are both obviously longer than the hair length of AA type (P is less than 0.01), and the rest hair producing properties have no obvious influence at the site (P is more than 0.05). The detection of the KRT26 gene polymorphic site provides a new material for molecular breeding, and also provides scientific basis and auxiliary means for marker-assisted selection of long-hair rabbit traits.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a molecular marker related to the length of long-hair rabbit hair for the first time, wherein the molecular marker is KRT26 gene, the 41844263 th basic group G of the KRT26 gene site is mutated into A, and the lengths of GG type and AG type villi are obviously longer than those of AA type villi. In the actual breeding process, the groups homozygous for the allele G can be respectively selected, or the groups with the genotype of AG can be obtained through hybridization combination, so that the villus length of the long-hair rabbit offspring can be increased.
Drawings
FIG. 1 is a schematic diagram showing the result of agarose gel electrophoresis detection of a PCR amplification product;
FIG. 2 is a schematic diagram showing the sequencing result of the PCR amplification product clone of KRT26 gene;
FIG. 3 is a gene sequencing peak diagram of the PCR amplification product of KRT26 gene.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Examples
1. Collecting samples: the invention takes the long-hair rabbits jointly cultivated by Shandong Mengyin Yida rabbit industry Co Ltd and Shandong agriculture university as test materials, the total effective samples are 957, and the long-hair rabbits are bred under the same condition. The indexes of hair yield, hair length and the like of each individual are respectively measured on the 283 th day after birth (fourth shearing) and the 73 th day during the hair-nourishing period. The hair sample at the lower side center part of the skin is cut by using scissors, a piece of ear tissue with the size of soybean grains is cut by using surgical scissors, the cut ear tissue is put into a 1.5mL centrifuge tube filled with 75% alcohol and is taken back to a laboratory for preservation at-20 ℃ for extracting genome DNA.
2. The experimental method comprises the following steps: the genome DNA of the collected ear tissue sample is extracted by a high-salt method, and the concentration and the quality of the DNA are detected by a spectrophotometer. The extracted DNA was stored at-20 ℃.
3. Mixed pool sequencing and screening gene SNP locus
(1) Constructing a DNA mixed pool: 100 genomic DNA samples were randomly drawn and mixed into one DNA pool in equal volume for each 20 genomic DNA samples.
(2) Design of candidate gene primers: the gene sequence of rabbit KRT26 in the ensemble database is obtained, primers of 5 'and 3' regulatory regions and exon coding regions of the genes are designed by using Primer5.0, the primers are synthesized by Shanghai Biotech service company, and the information of the primers is shown in Table 1.
TABLE 1 amplification of genomic DNA primers
PCR amplification:
and performing gradient PCR according to the synthesized primer information, searching for the optimal annealing temperature, and amplifying the target fragment by using the optimal annealing temperature. The PCR amplification reaction system is shown in Table 2 below:
TABLE 2 PCR amplification System
Setting of PCR amplification temperature:
pre-denaturation at 95 ℃ for 10 min;
35 cycles of: denaturation at 95 ℃ for 45 sec;
the annealing temperature is 53 ℃;
extension 72 ℃ for 30 sec;
extending at 72 ℃ for 10 min;
after the reaction is finished, the PCR product is stored in a refrigerator at 4 ℃ and is used for sample loading detection and subsequent analysis.
And (3) detecting a PCR product: the PCR product obtained by the method of agarose gel electrophoresis is detected, the result is shown in figure 1, the specificity of the PCR product is better, and the size of the amplified fragment is consistent with that of the target fragment.
Sequencing a product: sending the qualified PCR product to Shanghai Biotech service company for sequencing, and screening out a gene SNP locus; SNP typing is carried out by adopting a flight mass spectrometry detection method, and sequencing results are subjected to sequence comparison by using DNAMAN and Chromas software to search SNPs (figure 1, figure 2 and figure 3).
SNP typing: the SNP typing test is completed by Beijing Congpson Biotechnology GmbH, and comprises the following steps:
(1) designing a primer: according to SNP locus information, a PCR reaction and a single base extension primer are designed by utilizing MassARRAY design software AssayDesignSuitev2.0, and the specificity of the primer is checked on line through UCSC.
(2) And (3) genome DNA quality inspection: detecting the concentration, purity and degradation degree of the DNA by agarose gel electrophoresis, judging the standard of the detection result: in the electrophoresis detection gel image, the DNA band is single and clear, has no impurities, and has no dispersion and trailing phenomena.
(3) Electrophoresis conditions:
(ii) 0.8% agarose gel, 170V, 25min
Sample loading quantity: 500ng sample + 3. mu.l Loading Buffer
③Marker：Trans2000 Plus3μl
(4) And (3) PCR reaction:
the PCR reaction system is shown in the following Table 3:
TABLE 3 PCR reaction System
② the PCR reaction cycle parameters are shown in the following table 4:
TABLE 4 cycling parameters for PCR reactions
(5) SAP digestion
Reaction system for SAP digestion is shown in table 5 below:
TABLE 5 reaction System for SAP digestion
② circulation parameters for SAP digestion are shown in table 6 below:
TABLE 6 cycle parameters for SAP digestion
(6) Extension reaction
Extension reaction system is shown in the following table 7:
TABLE 7 elongation reaction System
② the cycle parameters of the extension reaction are shown in the following table 8:
TABLE 8 circulation parameters for the extension reaction
(7) And (3) computer detection:
firstly, diluting a reaction product (9 ul in total) by 3 times, and desalting by using resin;
secondly, the sample after desalination treatment is spotted on a sample target, and natural crystallization is carried out;
and thirdly, performing mass spectrum detection on the computer and collecting data.
And according to the typing result, performing SNPs population genetic analysis and correlation analysis of the SNPs population genetic analysis and the long-hair rabbit hair fiber diameter character by using R software and SAS software.
By analyzing fig. 2, the results show that: through sequence alignment, the base G at 41844263 of the KRT26 gene site is mutated into A; the base mutation can also be shown by the appearance of different peaks on the same sequence site shown in the peak figure 3, the double peaks indicate heterozygote, and the single peaks indicate homozygote.
Experimental example, correlation analysis of long-hair rabbit hair length character and KRT26 gene
And performing variance analysis by using a GLM program of SAS9.2, and performing correlation analysis on each genotype of the polymorphic sites and the wool production traits. The BLUP model is:
Y＝Xb+Za+e
y: producing a wool trait phenotype value; x: a matrix of individual numbers related to the fixation effect; b: fixation effects (SNP sites, sex effects); z: an individual number matrix of individual additive genetic effects; a: an individual additive genetic effect; e: random error.
The character of the length of the villus is measured according to GBT13835 rabbit hair fiber test method, allele and genotype frequency are calculated according to KRT26 gene SNPs, Hayes Weinberg balance test is carried out on the calculated allele and genotype frequency, genotypes AA, AG and GG are respectively obtained, the result of the length of the villus corresponding to each genotype is shown in a table 9, and the unit of the length of the villus is cm.
TABLE 9 analysis result table relating KRT26 gene and long-hair rabbit hair length traits
As shown in the data in table 9, the mutation of the base G at 41844263 of KRT26 gene site to a has a significant effect on the length of the villus (P <0.01), wherein AA is 0.37cm lower than GG and 0.40cm lower than AG; the lengths of the fuzz of GG type and AG type are significantly longer than those of AA type. In the actual breeding process, the groups homozygous for allele G can be selected separately, or the hybrid groups of genotype AG can be obtained by hybrid combination, so as to increase the villus length of the offspring.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Shandong agricultural university, New Cone technology Limited, Shandong
<120> molecular marker related to long-hair rabbit hair length and application thereof
<130> 2020.10.10
<160> 3
<170> PatentIn version 3.5
<210> KRT26 Gene
<211> 600
<212> DNA
<213> Artificial Synthesis
<400> 1
cttcccagcc cacgagtagg acgctggatg ggaagaggag cagccagaac ttgaacaaca 60
actcggacat agggtttggg agaagcaaac cgcagcttag ctccctggga cagatgaaag 120
catacaccgc cattgcactt tgcatattca cagccttcct tggttcctct cacagaaaga 180
gtcctacgag agctccttgg cggagatgga aggaaattac tgcctccagc tccagcaaat 240
ccaggatcag atcggggcca tggaggggca gctgcagcag atccggacgg aaaccgccgg 300
ccagaagctg gagcacgagc agctgctaga catcaaagtc ttcttagaga aggagatcga 360
gacgtactgc aacttactcg acggccaaga caggtgagcc acccgccctc acgtcagcca 420
ggctttctcc gcgtgtttcc cgagagcgcg tcctagaatt tcctgctagg aggatcacgc 480
cccagaggtc ctcactcacg tactgcggcg cgtcgcagtc aaagcagagc acagtctaga 540
aagtacccgt atgcatccta atataaagga ggcttgttta gaaacaggat gtctgccggt 600
<210> primer sequences
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 2
aaagagtcct acgagagctc 20
<210> primer sequences
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 3
ctgtcttggc cgtcgagtaa 20
Claims (2)
1. A molecular marker related to the length of long-hair rabbit hair is characterized in that the molecular marker is a KRT26 gene mutant, and the KRT26 gene mutant is formed by mutating a 289 th basic group G in a sequence shown in SEQ ID NO. 1 into A.
2. Use of a molecular marker according to claim 1 for marker-assisted selection of long-hair rabbit hair length traits.
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| WO2019051103A1 (en) * | 2017-09-06 | 2019-03-14 | Lawrence Livermore National Security, Llc | Methods and systems to perform genetically variant protein analysis, and related marker genetic protein variations and databases |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019051103A1 (en) * | 2017-09-06 | 2019-03-14 | Lawrence Livermore National Security, Llc | Methods and systems to perform genetically variant protein analysis, and related marker genetic protein variations and databases |
Non-Patent Citations (5)
| Title |
|---|
| 6个家兔群体KAP6.1基因的遗传多样性分析;吴添文等;《湖南农业大学学报(自然科学版)》;20101215;第36卷(第06期);第666-671页 * |
| Ensembl.ENSOCUG00000008999.《Ensembl》.2020,第1-3页. * |
| ENSOCUG00000008999;Ensembl;《Ensembl》;20201130;第1-3页 * |
| 中国美利奴羊(新疆型)KRT27基因的多态性及其与毛细度性状关联分析;石晓雷等;《中国草食动物科学》;20150401;第35卷(第02期);第1-4页 * |
| 长毛兔候选基因5’调控区多态性及其与产毛性状的关联分析;张露;《中国优秀硕士学位论文全文数据库 农业科技辑》;20180815(第8期);第6-7页1.5产毛性状相关候选基因研究进展，第14-18页2.5.4混池测序筛选基因SNP位点、2.5.5SNP分型 * |
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