CN107217091A - A kind of detection method of milch goat Fecundity Trait related gene SNP - Google Patents

A kind of detection method of milch goat Fecundity Trait related gene SNP Download PDF

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CN107217091A
CN107217091A CN201710130614.5A CN201710130614A CN107217091A CN 107217091 A CN107217091 A CN 107217091A CN 201710130614 A CN201710130614 A CN 201710130614A CN 107217091 A CN107217091 A CN 107217091A
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snp
snps
milch goat
goat
milch
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沈伟
张瑞乾
赖方秾
李兰
闵令江
赵勇
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Qingdao Agricultural University
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Abstract

The present invention relates to the fields such as high-flux sequence, genomics and bioinformatics technique, specifically based on milch goat SNP, eliminate the method for analysis to analyze the detection method of the SNP associated with milch goat Fecundity Trait and candidate gene using selectivity.Comprise the following steps：1) by by the comparing of high-flux sequence to reference gene group, then using the SNP site on free ANNOVAR software annotation milch goats.2) colony's sequencing sequence that two lambings are different is evaluated with heterozygosity fraction (Hp) using genetic differentiation coefficient (Fst), so as to filter out the SNPs in each sliding window.3) SNPs of homozygosis is filtered out in low yield colony and yielding Populations, identify in Liang Ge colonies distinctive single base nonsense replace and it is non-synonymous replace because both variations by by directly affect protein translation and amino acid sequence so as to influenceing protein structure and function.

Description

A kind of detection method of milch goat Fecundity Trait related gene SNP
Technical field：
Present invention is mainly applied to molecular genetic assistant breeding field, it is related to high-flux sequence, genomics and biological letter Cease the fields such as technology, and in particular to the related SNPs molecular labelings of milch goat Fecundity Trait and application.
Background technology：
Milch goat occupies critical role in China's husbandry sector, and it can provide fur, meat and dairy produce etc., good to its It is one of major issue of livestock hair to plant seed selection and expand numerous.China's milch goat industry is in recent years in a high speed development During, however, still locating to collection prolificacy and the genetic background research of the excellent milch goat kind of the high output of milk at present In the starting stage, therefore, unique hereditary information that the complicated quantitative character in high litter size colony behind is included is understood in depth, will The basis worked as genetic marker assist-breeding.
The Fecundity Trait of milch goat is an important indicator of female livestock breed performance, in the consistent bar of raising, managerial skills Under part, the ewe more than litter size can produce bigger economic benefit.Fecundity Trait is as one by answering that multiple genes are controlled Miscellaneous quantitative character, with relatively low genetic force, and is vulnerable to the environmental factor residing for parent and individual factors influence.Conventional Cross breeding method can not meet this controlled by multiple genes of reproductive capacity, and the individual seed selection demand of complex character of breeding cycle length. And conventional genetic marker assistant breeding can only carry out seed selection work to known a small amount of molecular labeling.Positioned currently with QTL Method has determined that candidate gene much related to Fecundity Trait.But the limitation of QTL localization methods is larger, it is impossible to differentiate new The gene related to Fecundity Trait.Development and first goat full-length genome collection of illustrative plates in the world with high throughput sequencing technologies Successful drafting, whole-genome association (the Genome-wide Association based on high throughput sequencing technologies Study, GWAS) turn into the identification candidate gene relevant with Important Economic property or the specifically new strategy of genome area. This is for understanding associated gene and distinctive SNP (Single nucleotial with litter size character Polymorphisms, SNPs) etc. genetic background there is powerful technical advantage.
SNP is that genetic polymorphism is hereditary variation kind content at most most common type.It refers to genome In DNA sequence dna caused by single nucleotide acid (A/T/C/G) change polymorphism, can be largely classified into the conversion of base with Transversion and the insertion of base and missing two types.SNP has the characteristics of having a very wide distribution quantity more, such as A SNP just occurs in general every 1000 bases in the genome of people.By nature or the differentiable tool of artificial selection There is quantity variance character subgroup, if wherein including the SNPs associated with the character, analysis can be eliminated by selectivity Statistical method carries out genetic statistics, because the SNP associated with purpose character is often that cluster occurs, if do not considered in this site Neighbouring chiasma is exchanged or the new hair mutation of individual, then this crowd of SNPs may occur in a certain adjacent domain of target group Enrichment is concentrated, the phenomenon of this region heterozygosity reduction is in turn resulted in.Based on this, we are carried out using to the extreme colony of high yield goat Full-length genome resurveys sequence, and then the SNPs that the method searching for eliminating analysis using selectivity may be associated with high-yield character, with And the candidate gene that these SNPs are nearby included, the SNPs filtered out is applied to genetic molecule breeding field, and then help Realize that the high individual of litter size character carries out quick molecular marking supplementary breeding work.
The content of the invention：
The problem of present invention is solved is to provide mononucleotide related to litter size character in a kind of screening milch goat genome The method of polymorphism mark, to high yield, low yield colony, using full-length genome weight sequencing technologies, initial data mistake is produced by sequencing Compare onto the reference gene group of Yunnan black goat, then annotated the SNPs files of each group after filter, with reference to genome note Information is released, the SNPs in special near transcriptional start sites 1KB regions between group is filtered out using the python local scripts write, So as to accelerate the process of milch goat stock breeding.
In order to realize foregoing invention purpose, the present invention is adopted the following technical scheme that：
1. find the different milch goat of litter size marks low yield colony and yielding Populations respectively, from the muscle groups of every goat Knit middle extraction DNA and carry out mixed pond.Sequence is resurveyed by genome and obtains sample genome reads information；
2. being compared using some sequence alignment programs with black goat reference gene group, the positional information of sequence is obtained, Then SNPs information is annotated.High-quality SNPs data messages are filtered out by the setting of some software parameters；
3. based on high-quality SNPs data, calculated by the method for programming and obtain genetic differentiation coefficient and heterozygosis property coefficient Data, the method for eliminating analysis using selectivity filters out group difference SNPs.
In order to realize foregoing invention purpose, the inventive method is operated in accordance with the following steps：
Family colony is found, many generations are preferably found, heredity and significant difference yielding Populations and low yield group can be stablized Body, if finding less than such family colony, can also use the extreme colony of high yield and low yield, specific colony's selection is according to reality Test depending on purpose, it is necessary to note the interference for avoiding environmental factor to bring as far as possible.
Each milch goat genes of individuals group DNA is extracted first, then according to packet mixed in equal amounts DNA, through electrophoresis and nucleic acid Band is clear without obvious degradation and pollution-free after analyzer detection, and 350bp fragments are broken at random, and end is repaired, plus sequencing Joint, purifying, amplification prepare sequencing library, are diluted to sequencing and require concentration, treat library after the assay was approved, carry out Illumina HiSeq full-length genomes resurvey sequence.High-quality sequence after filtering is compared onto goat reference gene group using BWA softwares, led to Cross using SamTools softwares, calculate and obtain SNPs, SNPs is then annotated by ANNOVAR come identify nonsense replace, it is non-synonymous Replace the candidate gene type with synonymous replacement.Use genetic differentiation coefficient (genetic differentiation Coefficient, Fst) and heterozygosity fraction (Heterozygosity scores, Hp) the two parameters progress full-length genome Statistics, then filters out heterozygosity reduction and the high genomic segment of group difference in group, and then filter out and milch goat production The SNPs and high-yield character candidate gene of lamb trait associations.
The present invention detects milch goat genome based on high throughput sequencing technologies using genome-wide screening analysis method SNPs, find Laoshan dairy goat gene polynorphisms, and probe into itself and milch goat lambing using these genes as candidate gene Relation between character.The present invention is that goat yeaning traits functional genome research and genetic marker assistant breeding are provided newly Thinking.
Brief description of the drawings：
Fig. 1 goat chromosome SNP distribution situations Circos schemes.An outermost circle is the numbering of every item chromosome, it Length and position of the reflection per item chromosome.A middle circle is represented per the detected SNPs quantity of 150KB genes window, model Enclose is that between 0-4000, green line represents SNPs quantity in each window<=1500, red line is represented in each window SNP quantity>1500.An innermost circle represents the average heterozygosis rate of SNPs in each window, and scope is in 0-100%.Green Triangle represent heterozygosis rate>=40%, red triangle represents heterozygosis rate<40%.
The Manhattan figure of heterozygosis rate and the differentiation degree analysis of the extreme colonies of Fig. 2.Red title is represented in 150KB windows most Small D-ZHp Gene Name.(B) the full-length genome genetic differentiation coefficient (Z-Fst) between low yield colony and yielding Populations is graceful Hatton schemes.Red title is represented in Gene Name maximum 150KB size windows Z-Fst.
Distribution situation between Fig. 3 Fst and ZHp.The point of blueness is represented in candidate window to be had more compared with yielding Populations Many homozygosis SNPs.(B) in yielding Populations each window Z-Fst and Z-Hp distribution situation, blue point represents candidate's window There are more homozygosis SNPs compared with low yield colony in mouthful.(C) blue title represents the exclusive candidate gene of low yield colony, (D) red title represents the exclusive candidate gene of yielding Populations.(E) monopolize and common time in low yield colony and yielding Populations Select gene window distribution situation.(F) yellow title represents the title of the candidate gene mark in candidate window.
Embodiment：
Material used and experimental method in the present invention：
1. experiment material
Laoshan dairy goat 37, the Laoshan milk that sample collection differs greatly in two groups of lambings of Qingdao Ao Tete sheep studs Goat, the litter size of 20 milch goats is 1 in low yield colony, and it is 3, one to have 13 individual litter sizes in yielding Populations The litter size of milch goat is 4.Sample collection is milch goat ear musculature.
2. milch goat ear musculature DNA is extracted and is sequenced
37 milk are extracted using QIAamp DNA Mini Kit (QIAGEN, 51304, Hilden, Germany) kit The DNA of goat.The DNA equivalent of extraction is mixed into pond and carries out library construction, is then sequenced using Illumina HiSeq 2000 flat Platform, to high yield low yield, colony carries out high-flux sequence respectively.
3. data analysis
3.1 sequencing datas filter and compare reference gene group
Quality control is carried out to sequencing data, contains sequence measuring joints sequence using free cutadapt softwares shearing Reads, for example：
cutadapt-a GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTG CTTG-A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCA TT-m 50-o both-ends One end filename 1.fq.gz both-ends sequencing other end filename is sequenced
2.fq.gz>＆log.txt, represents that one end file 1.fq.gz for both-end being sequenced generation is carried out
GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTT CTGCTTG joint sequences The shearing of row, for other end file 2.fq.gz, progress joint sequence is AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTG TAGATCTCGGTGGTCGCCGTATCATT shearing, reads length after shearing is discarded less than 50.
Then, referred to using free BWA softwares by the fastq sequence alignments after filtering to goat reference gene group Genome is Yunnan black goat genome (http://www.ncbi.nlm.nih.gov/genome/Term=Capra_ Hircus), the genome sequence fasta files of download are indexed using BWA foundation first, ordered as bwa indexGCF_ 000317765.1_CHIR_1.0_genomic.fasta-p GCF_000317765.1_CHIR_1.0_genomic are then right Two end datas are compared using aln algorithms respectively, are ordered and are：bwa aln-e 10-l 32-i 15-q 10-t 11 GCF_ 000317765.1_CHIR_1.0_genomic high_1.fq>File is sequenced in high yield group one end by this order of high_1.sai High_1.fq is compared to just setting up in the reference gene group of index, subsequent bwa aln-e 10-l 32-i 15-q 10-t 11 GCF_000317765.1_CHIR_1.0_genomic high_2.fq>High_2.sai this orders the high yield group other end File high_2.fq is sequenced to compare to just setting up in the reference gene group of index, afterwards input order bwa sampe-f pair-end.samGCF_000317765.1_CHIR_1.0_genomic high_1.sai high_2.sai high_1.fq High_2.fq generates the sam files after being compared with reference gene group, and file carries out deduplication, ginseng using SamTools after comparison Number is rmdup.
3.2 SNP are detected and annotated
After comparing deduplication, the SNPs of Liang Ge colonies, ' mpileup ' order are detected using freeware SamTools Parameter be set to the '-F0.002-d 1000 ' of-m 2.Then the SNPs of acquisition is filtered using python scripts, filtered Principle be：1. mass value >=20；2. depth >=4 are sequenced；3. the SNP site of multiple gene type is filtered out；It can so obtain Obtain high-quality SNPs.Then SNP is annotated using ANNOVAR to identify nonsense replacement, non-synonymous replacement and the time of synonymous replacement Select genotype.
3.3 selectivity eliminate analysis SNPs selection region
Genetic differentiation coefficient (Fixation index, Fst) and heterozygosity fraction (Heterozygosity are used respectively Scores, Hp) the two parameters evaluate the otherness and single intragroup heterozygosity of Liang Ge colonies sequencing sequence.Heredity Coefficient of differentiation (Fst) be for estimate between population and in population genetic similarity index.Fst calculation formula is： It is average value, σ x2The difference of the gene frequency of an allele in subgroup is represented, Fst calculating makes Completed with software PoPoolation2.Heterozygosity fraction (Hp) is for assessing gene pleiomorphism and research Population Genetics Parameter, Hp calculation formula is：
nmajorAnd nminorIt is the minimum and maximum equipotential in given special sliding window Genotype frequency summation, Hp calculating is completed using the python scripts write.After orthogonal conversion, we select with Then 150KB length select the 5% and ZFst fraction highests that ZHp fractions are minimum in each window as a selection window 5% intersection area carries out statistical analysis.Count SNPs quantity and calculate per the flat of 150KB sliding window SNPs heterozygosis rates Average, between low yield colony and yielding Populations per 150KB windows in SNPs quantity in No. 8 chromosomes, No. 12 chromosomes, No. 18 dyes There is larger difference in some regions on colour solid, X chromosome, the artificial selection between low yield colony and yielding Populations is probably to lead Cause the reason for some regions of above-mentioned chromosome produce greatest differences.
The higher genetic region of homozygosis rate representative in screening yielding Populations, calculates the gene window of different length first Window size is worked as in the SNPs quantity (50,100,150 and 200KB) of mouth and then discovery>The SNPs quantity that window is counted during=150KB Less than 20.This shows that 150KB sliding windows are adapted to selectivity and eliminate analysis.Calculate the sliding window of 50% (75KB) length scale Heterozygosis rate.Except the kinetochore do not analyzed and a total of 33,643 chromosome windows of mitochondrial DNA (mtDNA) pass through ZHp Fraction is estimated.Find that most representational annotation differential gene region is in No. 8 chromosomes after calculating and sequence 54.15-54.90MB regions, 15.23-15.30MB regions of No. 10 chromosome, the 26.70-26.77MB in No. 25 chromosome Region, the 58.50-58.57MB regions in X chromosome.
After the fraction for comparing ZHp and ZFst, the 5% of the minimum 5% and ZFst fractions maximum of ZHp fractions is selected, it Be homozygosis and the strong candidate region of difference in low yield colony and yielding Populations.Next selection is in low yield colony and height Produce selection region highly pure and mild in difference maximum (5% before Fst fractions) between colony and each colony (in low yield colony and In yielding Populations ZHp it is minimum 5%).Then 137 candidate genes filtered out in yielding Populations determine 73 special bases Cause.113 candidate genes filtered out in low yield colony determine 68 specific genes.

Claims (4)

1. a kind of detection method of milch goat Fecundity Trait related gene SNP, it is characterised in that：By based on High throughput sequencing technologies eliminate SNP of the analysis method to milch goat Fecundity Trait related gene using selectivity Analyzed.
2. a kind of detection method of milch goat Fecundity Trait related gene SNP according to claim 1, It is characterized in that screening milch goat gene monokaryon glycosides using two parameters of genetic differentiation coefficient (Fst) and heterozygosity fraction (Hp) Sour polymorphism.
3. according to the method described in claim 1, it is characterised in that the SNP site information that selectivity elimination Analysis and Screening goes out is to close In milch goat Fecundity Trait related gene.
4. according to the method described in claim 1, it is characterised in that each window is found using sliding window with reference to ergodic algorithm SNP site in mouthful.
CN201710130614.5A 2017-03-07 2017-03-07 A kind of detection method of milch goat Fecundity Trait related gene SNP Pending CN107217091A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107868843A (en) * 2017-12-07 2018-04-03 江汉大学 A kind of method for screening the high polymorphic molecular marker site of mung bean
CN108913760A (en) * 2018-07-26 2018-11-30 武汉生命之美科技有限公司 The method that a kind of pair of single nucleotide polymorphism and specific trait relevance are assessed and quantified
CN110093406A (en) * 2019-05-27 2019-08-06 新疆农业大学 A kind of argali and its filial generation gene research method
CN115851964A (en) * 2022-07-27 2023-03-28 西北农林科技大学 SNP molecular marker related to milk production traits and lamb production traits of milk goats, liquid chip detection kit and application

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CN102690890A (en) * 2012-06-19 2012-09-26 扬州大学 Molecular marker-assisted selection primer and method for Hu-sheep maternal behaviors

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107868843A (en) * 2017-12-07 2018-04-03 江汉大学 A kind of method for screening the high polymorphic molecular marker site of mung bean
CN108913760A (en) * 2018-07-26 2018-11-30 武汉生命之美科技有限公司 The method that a kind of pair of single nucleotide polymorphism and specific trait relevance are assessed and quantified
CN108913760B (en) * 2018-07-26 2022-06-07 武汉生命之美科技有限公司 Method for evaluating and quantifying relevance between single nucleotide polymorphism and specific traits
CN110093406A (en) * 2019-05-27 2019-08-06 新疆农业大学 A kind of argali and its filial generation gene research method
CN115851964A (en) * 2022-07-27 2023-03-28 西北农林科技大学 SNP molecular marker related to milk production traits and lamb production traits of milk goats, liquid chip detection kit and application

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Application publication date: 20170929