CN109680043B - MSTN gene molecular marker selection method for Chinese softshell turtle growth traits - Google Patents
MSTN gene molecular marker selection method for Chinese softshell turtle growth traits Download PDFInfo
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- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a method for the growth character of Chinese soft-shelled turtlesMSTNA gene molecular marker selection method, namely, the method is used for the trionyx sinensisMSTNOne SNP site on the first intron sequence in the gene, starting with the start codon, is T2194C. Screening out the ones closely related to the growth traits from the Chinese softshell turtle populationMSTNBreeding marker of gene, and screening and constructing breeding population of Chinese soft-shelled turtle with the breeding marker. The method can improve the accuracy of the selection of the growth traits of the Chinese softshell turtles, and lays a foundation for developing molecular marker-assisted breeding of the Chinese softshell turtles and provides a reference.
Description
Technical Field
The invention relates to a Chinese soft-shelled turtleMSTNA detection method and an application range of gene sequence SNP loci belong to the field of molecular biology.
Background
The growth character is one of the important economic characters of the Chinese softshell turtle and is influenced by a plurality of genetic and environmental factors such as muscle growth and development. The growth traits are selected through the phenotype, the genetic progress is slow, the major gene for controlling the growth traits of the Chinese soft-shelled turtle and the genetic marker thereof are searched, the breeding process can be accelerated by using the molecular marker for auxiliary selection, and the molecular marker has important significance for improving the growth performance of the Chinese soft-shelled turtle.
The myostatin gene (myostatin,MSTN) It is a member of the transforming growth factor beta (TGF-beta) superfamily, and plays a role in negative regulation on the growth and development of animal muscles. It is reported thatMSTNPolymorphism of gene and motility of waterThere is a significant correlation between growth traits of plants. For example, Zhang Meng et al (2016) uses direct sequencing method for grass carpMSTNThe gene polymorphism is analyzed, and the 2 nd intron base mutation is detected to have correlation with growth traits such as body length, body mass and the like. Meng et al (2017) on 3 Argopecten irradiansMSTNThe polymorphism of the gene is screened, and 2 SNP loci related to growth traits such as body mass, muscle mass and the like are found. Identification of Penaeus vannamei by Zhou et al (2017)MSTNA plurality of SNP sites of the gene are obviously related to growth traits such as body length, body mass and the like. Chenyi culture and the like (2018) analyze 3 groups of Eriocheir sinensisMSTNThe genetic variation of the 1 st exon of the gene shows that 1 polymorphic site has obvious influence on the growth traits of body weight, shell length and the like. But relates to the Chinese soft-shelled turtleMSTNThe association study of gene base mutation and growth traits is not reported.
The Chinese soft-shelled turtle is an extremely important aquatic economic animal in China, and the yield of the Chinese soft-shelled turtle in 2017 is over 30 million tons. However, the quality of the Chinese soft-shelled turtles in China is uneven, germplasm is mixed and degenerated, the quantity of improved Chinese soft-shelled turtles is small, coverage rate is low, and industrial development is severely restricted. In the face of strong demand of improved variety of Chinese soft-shelled turtles, the traditional breeding means can not be met, and the development of molecular marker-assisted breeding work of the Chinese soft-shelled turtles is necessary to improve the selection efficiency and accelerate the breeding process.
Disclosure of Invention
The invention aims to find the compound with important regulation and control function in the growth of the Chinese softshell turtleMSTNSNP loci on the gene (SEQ ID NO. 1) carry out association analysis on the locus genotype and the growth traits, and an effective molecular marker is provided for marker-assisted selection of the growth traits of the Chinese softshell turtles.
The invention is realized by the following technical scheme:
a method for improving growth characteristics of Chinese soft shell turtleMSTNThe gene molecular marker selecting method includes the following steps:
(1) amplifying from the genome DNA of the Chinese softshell turtle by using an amplification primer;
(2) sequencing amplified fragments obtained from different Chinese softshell turtles, and screening out base mutation sites;
(3) carrying out genotype detection on individuals in the Chinese softshell turtle population, and carrying out least square method estimation on the relationship between the growth character value and the genotype of the Chinese softshell turtle by utilizing a generalized linear model GLM;
(4) and analyzing the difference of the growth traits among different genotypes of the Chinese softshell turtle population by adopting a least square method and multiple comparison, and finally determining SNP (single nucleotide polymorphism) sites which are obviously related to the growth trait values of the Chinese softshell turtles.
Preferably, the nucleotide sequences of the amplification primers in step (1) are respectively P1: SEQ ID NO.2 and P2: SEQ ID NO.3, the specific primer information is as follows:
P1：TCTGTTTATATGATATGCTGGC （SEQ ID NO.2）
P2：TTTTCCAACCAAATCTAGTAGG（SEQ ID NO.3）
preferably, the growth trait values of the trionyx sinensis in the step (3) comprise body mass, length of dorsal concha, width of dorsal concha, length of ventral concha, width of rear side of skirt and body height.
The growth characteristics of Chinese soft-shelled turtleMSTNThe application of the gene molecular marker selection method in marker-assisted selection of the growth traits of the Chinese softshell turtles.
Preferably, when the growth traits of the Chinese softshell turtle are body mass and width of the abdominal shell, the marker is a T2194C site (starting from an initiation codon).
The invention has the beneficial effects that:
the selection method is simple and effective, the body mass and the width of the screened Chinese soft-shelled turtle with the T2194C locus as TT type are respectively and obviously higher than those of TC type individuals, and in the process of growth and breeding of the Chinese soft-shelled turtle, individuals with dominant genotypes can be preferentially selected as parents according to the locus, so that the seed selection efficiency is improved.
Drawings
FIG. 1 shows the growth characteristics of Chinese soft-shelled turtleMSTNSelecting one SNP locus by a gene molecular marker selection method; wherein a is TT type, b is TC type, and c is CC type.
Detailed Description
In order that the invention may be better understood, the invention is further described in connection with the following examples which are intended to be illustrative of the invention and are not intended to be limiting.
Chinese soft-shelled turtleMSTNGene DNA sequence PCR amplification and polymorphism detection
1. Chinese soft-shelled turtleMSTNPCR amplification of gene DNA sequences
Chinese softshell turtle downloaded according to NCBIMSTNSpecific amplification primers P1 and P2 were designed for the full-length sequence of the gene (GenBank No: NW-005852453.1), using 30 genomic DNAs of Chinese soft-shelled turtles as templatesTaqAnd amplifying by using primers P1 and P2 under PCR conditions in a buffer environment of a PCR master mix, purifying and sequencing a product to obtain a target fragment with the length of 417 bp.
The primer sequences are as follows:
P1：TCTGTTTATATGATATGCTGGC（SEQ ID NO.2）
P2：TTTTCCAACCAAATCTAGTAGG（SEQ ID NO.3）
the PCR amplification conditions are as follows: 20.0 mu L of PCR reaction system comprising 10.0 mu L of 2TaqPCR master mix, 0.5. mu.L each of upstream and downstream primers (20. mu. mol/L), 0.5. mu.L of template DNA, and dd H2O to 20.0. mu.L.
The PCR amplification reaction program is as follows:
pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s, and extension at 72 ℃ for 30 s for 35 cycles; extending for 10 min at 72 ℃, and storing at 4 ℃.
2. Chinese soft-shelled turtleMSTNGene base mutation site detection
Sequencing a target fragment with the length of 417 bp by a biological company to obtain a gene sequence, and performing sequence comparison analysis by using BioEdit software to find outMSTNOne SNP site in the first intron of the gene sequence (SEQ ID NO. 1), starting with the start codon, is T2194C (FIG. 1).
Second, Chinese soft-shelled turtleMSTNGenetic structure analysis of gene mutation site in Chinese soft-shelled turtle population
To analyzeMSTNGenetic structure analysis of the gene T2194C locus in the Chinese soft-shelled turtle population selects 150 Chinese soft-shelled turtle populations of 2 winter ages of different strains, same batch breeding and same paddy field breeding. To pair150 Chinese soft-shelled turtlesMSTNThe T2194C locus of the gene was typed, and its genetic parameters were counted (Table 1). Wherein the polymorphic information content (PIC) Desired heterozygosity: (He) Effective allelic factor (a)Ne) The calculation method of (2) is as follows:
1. polymorphic information content (PIC) Is calculated by
The calculation formula is as follows:
2. Degree of heterozygosity: (He) Is calculated by
The calculation formula is as follows:
He 
。
in the formula (I), the compound is shown in the specification,H e : the heterozygosity at a certain position is shown,P i : at a certain positioniThe frequency of the individual alleles is determined,n: allelic factors for a locus.
3. Effective allelic factor (Ne) Is calculated by
The calculation formula is as follows:
Ne=1/∑P i 2
in the formula (I), the compound is shown in the specification,N e : the effective allelic factor for a locus,P i : at a certain positioniIndividual allele frequency.
The results show that the polymorphic information content (PIC) At 0.3146, the desired heterozygosity: (He) 0.3924, effective allele factor (Ne) In the order of 1.6423, is,PICandHethe concentration is between 0.25 and 0.50, which indicates that most SNP sites have moderate polymorphism. Through the checking of the suitability of the chi-square,MSTNthe site of the gene T2194C is in ChinaThe turtle population is in Hardy-Weinberg equilibrium state (P＞0.05）。
TABLE 1 Chinese Soft shelled turtleMSTNGenetic diversity parameters of SNP loci of genes
| SNP Locus as SNP Locus | Polymorphic Information Content (PIC) | Observation of heterozygosity (Ho) | Desired heterozygosity (He) | Effective allelic factor (Ne) | Harvard equilibrium constant (HWE) |
| T2194C | 0.3146 | 0.3467 | 0.3924 | 1.6423 | 0.1512 |
Note: deviation from Hardy-Weinberg law: (P＜0.05）
Three, Chinese soft-shelled turtleMSTNGrowth correlation analysis of genetic SNP markers in Large populations
Collecting growth character values of the 150 Chinese soft-shelled turtles, measuring the body mass of each Chinese soft-shelled turtle by using an electronic balance (accurate to 0.01 g), and measuring by using a vernier caliper (accurate to 0.01 cm)And measuring growth trait values such as the length of the dorsal concha, the width of the dorsal concha, the length of the abdominal concha, the width of the rear side of the skirt, the height of the body and the like of each sample. Then, the skirt tissue of each Chinese softshell turtle sample is sheared and DNA is extracted. Carrying out PCR amplification and sequencing on each Chinese softshell turtle DNA sample by using amplification primers P1 and P2 to obtain the DNA sequence of each Chinese softshell turtle sampleMSTNThe genotype of the gene. The general linear model GLM of the SPSS 19.0 software is used for carrying out correlation analysis of SNP locus genotype and growth traits (body mass, dorsal concha length, dorsal concha width, ventral concha length, ventral concha width, skirt rear side width, body height and the like).
Analysis of molecular breeding markers
The correlation analysis result shows that the T2194C locus has significant difference with the measured growth trait values such as body mass, width of the turbinate and the like (P<0.05) (Table 2), among the measured growth trait values, the body mass and the width phenotype value of the turbinate of TT genotype were the largest, and the phenotype values of the dorsal turbinate width, the ventral turbinate length, the posterolateral skirt width, etc. were also higher than those of TC and CC genotypes. Therefore, comparative analysis shows that the TT genotype of the T2194C locus is most closely related to the growth of the Chinese soft-shelled turtle, and the TT genotype is primarily determined as the Chinese soft-shelled turtleMSTNA breeding marker of the gene.
TABLE 2 Chinese Soft shelled turtleMSTNCorrelation analysis of genes and growth traits
| SNP site SNP site | Genotype (sample) Amount) genotype (number) | Body Mass/g body weight | Length/cm of dorsal concha carapace length | Width/cm of dorsal concha carapace width | Length/cm of abdominal shell plastron length | Width/cm of abdominal shell plastron width | Width/cm of rear side of skirt calipash lateral width | Height/cm of body height |
| T2194C | TT(84) TC (52) CC(14) | 882.99±159.70b 823.54±125.98a 849.01±172.04ab | 17.86±1.39 17.58±1.36 17.99±1.28 | 14.46±2.17 14.01±0.98 13.94±0.77 | 13.73±0.83 13.49 ±0.80 13.51± 0.75 | 12.82±0.81b 12.41±0.81a 12.65±0.85ab | 3.40±0.35 3.31±0.37 3.37±0.36 | 5.06±0.44 5.12±0.36 5.11±0.32 |
Note: the difference between the upper and lower letters in the same column indicates significant difference (P＜0.05）。
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
SEQUENCE LISTING
<110> aquatic research institute of agricultural science institute of Anhui province
<120> MSTN gene molecular marker selection method for Chinese softshell turtle growth traits
<130> 2019
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 4393
<212> DNA
<213> unknown
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atgcaaaagc tacaaatcta tgtttatatt tacctgtttg tgctgattat acttggtcca 60
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gcatgtactt ggaggcaaaa tacgaaatct tccaggatag aagccataaa aattcaaatc 180
ctcagcaaac ttcgtctgga gcaagctcct aatattagca gggatactat aaaacagctt 240
ttacccaaag ctcctccatt gcaagaactg attgatcaat atgacatcca gagagatgac 300
agcagtgatg gttctttgga agacgatgat tatcatgcta caactgagac tattattaca 360
atgcctacag agtgtaagta gtcctgctac tttaatatca tactgttcta ctgataaaca 420
gttgtcctac tgcttaagag caacaaatct tgtctcagtc tcttctaaca ggctgctggc 480
agaagtatgt aagagggagg gagaagagca tatttttaaa tagtttgagt catcatccaa 540
tgacctcatt ataatatttt attttattgt attatttttc tgcttcctta gaatatgcac 600
agtaatagct tataaaatcg tgccaattgc ttaagattgc tatttaagat tcacagttta 660
ctgacctcgg tattttcagt attcaaggta tttcctacag taaatataga gatgtaaaaa 720
gaaataagtg caaataagga ggtaattata attctcttaa caaggatttt atgggccaca 780
actttttaaa acactgcact agaaactgac aaaaatgtca tttagaacat tctaactata 840
atacactata acaagtgcaa ataaagggtt aaatagttgt ttcctatgct gatcttataa 900
gcttgcacca cacaggtaac agttaacagt gaatgtaaac tatatattta aagtaagtgt 960
aagaatgata ggttttagat gttgaagcat ccatgtatat ctatcaactc cagtagttgt 1020
gtcagcaaag aatgaaggca tttactgaat gagcagattc ttagttatct tctgagcgct 1080
aaacatctgc catgactcct atcaagtatc ttttgcacag ctaatagtat agtatcttat 1140
gttcttaatc agctaaagta tttggctatg gcagctgttt ttcaaaataa cacttatgaa 1200
tatgatgaca aaagttgaaa agaagacagg ggacataagg catagatatc ttactagcaa 1260
cagagagcta cagtacacac acctaacaga atgtcagcag ctgtagctac tagagaatat 1320
acatatttat agaagttaat tgactataaa atatataggc taaatgaata gtgtaggata 1380
ctttatagca acattgaatg cattatagat gtttgatcat ggtgatgtag gtaaaggcca 1440
actaatcaac atttagccca ctatttacaa taacctacta ttatctaatt cttctttgcc 1500
aagtgaagaa aggtgatcta tattttgttt atctgtcact tcacaattcc actcttttca 1560
taaaaaacac ccaaccacca ttttagtgta ttacacaagt aaatatctaa agagatgagg 1620
ttatgaatat tgtaactctg caccatcaga aattattact ttgctctcat acccccttac 1680
catggagact gtcacctggg tctaatagat tttgcacctg cactgcatac caaggatagt 1740
tagcttacta cactgaaaac actttaccat aacataattc aaataaaaag aaatatacac 1800
ccgtagatta aaacaagtat attaacagac agcttaatca taaaacaaag gaagggaatt 1860
tagaagatac tgtagcttga acacagctgc ttctgtttat atgatatgct ggcactaatg 1920
ataatgtata agtagaaaca tccataacat cacagaagtg tctaatctga aagtataggt 1980
ttgctgttca atgtgttttt tgtaagatac tgaaggaggg aacgtcagaa aggtgttact 2040
cctttaacca ggctgtacac tgtgttcagt cctcaggtaa tttttggctg aaggaagtgt 2100
tcctttccta gactggctgg tacagctgct cagttagtac aagtactcag attctgaaag 2160
aattctaagg aaatgttcct ctgcagcctc ttttgatctc attattcccc atcattttaa 2220
aatttccgca tgtgttcacc ccttagctaa agaaatcaaa gtgaatagat aacaaggaat 2280
aacgatccta ctagatttgg ttggaaaatg gtaaatctag ctttactgaa ttgatgttca 2340
gaaaatggtt gttcagaaaa tgaccattgg tttcttgtaa taacatatta cttgtagatt 2400
gcttaaaaat tgaggtacaa cgattcttaa tgttagttaa ataatgccat aaactctcaa 2460
ttctgaattt tgtagtgtat ttgataataa attaagttac atgccatctt tgctactaat 2520
attccactga taacatatct tattaagatg tactatacag gttggatttt ttttctgatt 2580
tgtttatgcg agttcctttc tgctcctagg tcaataatct attctttcca ttcatttata 2640
gctgattttc ttgtgcaaat ggaagggaaa ccaaaatgtt gcttctttaa gtttagttct 2700
aaaatacaat ataacaaagt agtaaaggcc caactgtgga tatacttgag gcaagtccag 2760
aagcctacaa cagtattcgt gcagatcctg agactcatca gacccatgaa agacggtaca 2820
agatatactg gaattcgatc tctgaaactt gacatgaacc caggcactgg tatttggcag 2880
agtattgatg tgaagacagt gttacaaaat tggctcaaac aacctgaatc caacttaggc 2940
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ggtgaagatg gactggtaag ttctattgaa aaatttccac agaaacgtct cctattttat 3060
agacccattt acaaaagttg gaattggaaa gcaggtttta aattatattt tgaagacagg 3120
aagtggaagc attttctagg ggttaaaata tagaagtggg atttaagaca agggttttat 3180
atgacacgac aaacttgttt tctgatcttg tacaaatcgc tgtctctctc tgtgtctcag 3240
tttcccattc tgtaaaatag aggtatttac tactacttta gtactaattc aaagagatgc 3300
tgggagactt gcattactct gattgtaaag ggttttatat gcaatggaag ctgctagaaa 3360
agggcaaaat atttgttctc aaatccagga gtaaagaaaa gatactaaaa cgctacttaa 3420
tgaaaagtta ttttttaatg tatagcacaa aatgaaaagt gctgggttcc tgcacttgag 3480
aagaagttgc ctcaaatctt gtaaccttaa ctaagctctt gaattcaaaa caagagggaa 3540
cttctacaca ctctgaataa tgggagttac acgtgcatat aaaggagaca aacagagaac 3600
ctcacaatgg gtcttaaatt aaggacatct tttccttcct tgttgttaat ctttgaaaag 3660
caataatcac ctacagtctg cataagtata tttatgaacc tattacctat accagtaaag 3720
caatacttag cgctaccaaa attcatagtt gtgaaaaaat agtcatgggc catgaaattg 3780
ggtctcccta atgaaatctg gctattgcag gggtcaccgt atcgccagtc ttatttctgc 3840
actgctttca gagcagggtg gccaaagaat ggtgactatt ggctgggagc ccactctgaa 3900
ggcaacactg acaccagcag ctctgcagga gtgagggtgg catggaacgg cattaccact 3960
cttctgtgcc cccaccagca gcaaagcaag ggtgacaaca cagtagcatg ccatactatg 4020
ccaccattac ttctgtgctg ctgcaggtgg cagctctgcc ttcagagctg agattccagc 4080
cagcagttgc tgttctctaa ttgcccagct ctgaagctgc tatccaccag tagcacagaa 4140
ataagggtag cagtacctca acccctattg caataaactt gtggccccct ctagcactcc 4200
tttttgggtc aagactgcta cacttacaac acccccctcc ccccccccgg ggatgacacg 4260
gacctatatt ttaatcccta cagaaatcag tgcaactttt ctaccattca aaaaaagata 4320
atagactact gtttttctac attttgttcc tctggttttc tgagcaatgc caggtaactc 4380
ccgctagtag gaa 4393
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Claims (3)
1. A method for improving growth characteristics of Chinese soft shell turtleMSTNThe application of the gene molecular marker in marker-assisted selection of the growth traits of the Chinese softshell turtle is characterized in that: the growth traits of the Chinese soft-shelled turtle are body mass and width of the abdominal shell, the marker is a T2194C site which takes an initiation codon as a starting point on SEQ ID NO.1, and the screened Chinese soft-shelled turtle is TT type.
2. The method for improving the growth of Chinese softshell turtle according to claim 1MSTNThe application of the gene molecular marker in marker-assisted selection of the growth traits of the Chinese softshell turtle is characterized in that the selection method of the molecular marker comprises the following steps:
(1) amplifying from the genome DNA of the Chinese softshell turtle by using an amplification primer;
(2) sequencing amplified fragments obtained from different Chinese softshell turtles, and screening out base mutation sites;
(3) carrying out genotype detection on individuals in the Chinese softshell turtle population, and carrying out least square method estimation on the relationship between the growth character value and the genotype of the Chinese softshell turtle by utilizing a generalized linear model GLM;
(4) and analyzing the difference of the growth traits among different genotypes of the Chinese softshell turtle population by adopting a least square method and multiple comparison, and finally determining SNP (single nucleotide polymorphism) sites which are obviously related to the growth trait values of the Chinese softshell turtles.
3. The method for improving the growth of Chinese softshell turtle according to claim 2MSTNThe gene molecule is marked inThe application of the marker-assisted selection of the growth traits of the Chinese softshell turtles is characterized in that the nucleotide sequences of the amplification primers in the step (1) are respectively P1: SEQ ID NO.2 and P2: SEQ ID NO. 3.
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| CN112029875B (en) * | 2020-10-19 | 2022-05-10 | 中国水产科学研究院黄海水产研究所 | SNP (Single nucleotide polymorphism) marker related to growth of palaemon carinicauda, detection primer and application |
| CN112899282A (en) * | 2021-03-03 | 2021-06-04 | 中国水产科学研究院南海水产研究所 | SNP (single nucleotide polymorphism) site related to growth traits of yellow fin acanthopagrus latus, and screening method and application thereof |
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| CN107475407A (en) * | 2017-09-19 | 2017-12-15 | 湖南农业大学 | It is a kind of to detect the kit in mononucleotide site related to growth and application in Shelled Turtle Trionyx Sinensis IGFBP1 genetic fragments |
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| CN107475407A (en) * | 2017-09-19 | 2017-12-15 | 湖南农业大学 | It is a kind of to detect the kit in mononucleotide site related to growth and application in Shelled Turtle Trionyx Sinensis IGFBP1 genetic fragments |
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| 中华鳖GHRL基因SNPs的筛选及生长性状的关联分析;李纯;《水生生物学报》;20180328;第42卷(第2期);第307-312页 * |
| 中华鳖IGF2基因SNP标记与生长性状的关联分析;彭娜;《湖南农业大学学报》;20180825;第44卷(第1期);第88-94页 * |
| 基于转录组测序的中华鳖微卫星标记的开发及MSTN基因的克隆表达;张群英;《中国优秀硕士学位论文全文数据库 农业科技辑》;20140915(第9期);第51页 * |
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| CN109680043A (en) | 2019-04-26 |
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