CN113667761A

CN113667761A - Grass carp insulin receptor alpha subtype SNP molecular marker combination and application thereof

Info

Publication number: CN113667761A
Application number: CN202110948917.4A
Authority: CN
Inventors: 樊佳佳; 马冬梅; 朱华平; 白俊杰; 田园园; 苏换换; 陈在佳
Original assignee: Pearl River Fisheries Research Institute CAFS
Current assignee: Pearl River Fisheries Research Institute CAFS
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2021-11-19
Anticipated expiration: 2041-08-18
Also published as: CN113667761B

Abstract

The invention discloses a grass carp insulin receptor alpha subtype SNP molecular marker combination and application thereof, wherein the grass carp insulin receptor alpha subtype molecular marker combination is covered in a grass carp insulin receptor alpha subtype genome, and the relation between the grass carp insulin receptor alpha subtype molecular marker combination and grass carp growth traits is obtained through analysis of a general linear model. The inventor also finds that the H4 double type constructed by the method is significantly higher than other genotype individuals in body weight, body length, body height and body width (P <0.05), the average value of the fullness is 1.59, and the H4 double type is not high enough to serve as a candidate marker for the fast-growing high-quality grass carps, so that the H4 double type can be effectively applied to aspects such as molecular marker assisted breeding of the grass carps.

Description

Grass carp insulin receptor alpha subtype SNP molecular marker combination and application thereof

Technical Field

The invention belongs to the field of gene detection, and particularly relates to a grass carp insulin receptor alpha subtype SNP molecular marker combination and application thereof.

Background

Grass carp (Ctenophagogon idellus) belongs to the subfamily Atlantic (Leuciscinae) and grass carp (Ctenophagogon), and is popular with consumers because of high growth speed and delicious meat quality. The annual output of the grass carps is always stable at the first of the output of the freshwater fishes, and the total output of the grass carps is up to 552 ten thousand tons in the world of 2016. The feeding of the compound pellet feed is a main breeding mode for grass carp breeding, and the feed investment accounts for about 70% of the total input cost, so that how to effectively reduce the feed cost becomes the focus of attention of the breeding industry in order to obtain better economic benefit.

The sugar belongs to the cheapest energy supply substance in three nutrient substances (protein, fat and sugar), the sugar content is required to be increased frequently to reduce the feed cost in the actual culture process of the grass carps, and the grass carps have higher sugar utilization capacity compared with omnivorous fishes and carnivorous fishes although the grass carps belong to the herbivorous fishes. However, when the sugar level in the feed exceeds a certain limit, the problems of reduced immunity, slow growth, high mortality and the like of the fishes are easily caused, so that the breeding of the new grass carp variety capable of efficiently utilizing the high-sugar feed has extremely important significance for the breeding of the grass carps and is also beneficial to greatly reducing the breeding cost of the grass carps.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a grass carp insulin receptor alpha subtype SNP molecular marker combination which comprises SNP, insertion and deletion and can be used for multiple aspects of grass carp variety screening, grass carp variety identification, grass carp breeding, germplasm resource protection, germplasm resource improvement and the like. The grass carp insulin receptor alpha subtype SNP molecular marker combination can quickly screen out grass carp high-quality seeds, and the screened grass carp high-quality seeds are uniform in individual body type, heavy in mass, high in actual breeding benefit and high in application value.

In a first aspect of the invention, a grass carp insulin receptor alpha subtype molecular marker combination is provided, and the grass carp insulin receptor alpha subtype molecular marker combination is covered in a grass carp insulin receptor alpha subtype genome.

The insulin receptor (insulin receptor) plays a mediating role in the biological action of insulin, and is located on the surface of target cells. Insulin receptor alpha is a subtype of the insulin receptor family. The gene of the insulin receptor alpha subtype has a full length of 4068bp, and comprises 21 exons and 20 introns. In teleost fish, the insulin receptor alpha subtype is involved in embryogenesis, food intake regulation and glucose metabolism in some fish species, and the inventors believe that the insulin receptor alpha subtype based on the hindgut of grass carps has a higher level of expression and therefore, can be hypothesized to have a great significance in the digestive metabolism of grass carps.

Insulin signaling is considered to be highly conserved and critical for homeostasis. The polymorphism sites related by the invention are mainly distributed in the intron region of the grass carp insulin receptor alpha subtype, and compared with the intron region, the intron region is easier to generate gene variation, which is probably related to that the intron region has longer intron sequence, does not have coding protein, is relatively less subjected to selection pressure, has higher mutation probability and is easy to accumulate more variation.

In some embodiments of the invention, the genome of the grass carp insulin receptor alpha subtype is obtained in a manner that: the mRNA sequence of the grass carp insulin receptor alpha subtype is obtained from a grass carp EST database (https:// www.ncbi.nlm.nih.gov/SRA/.

According to the first aspect of the present invention, in some embodiments of the present invention, the genome of the grass carp insulin receptor alpha subtype preferably comprises the sequence shown in SEQ ID No.1 and/or SEQ ID No.2 and/or SEQ ID No. 3.

According to a first aspect of the present invention, in some embodiments of the present invention, the molecular marker combination of grass carp insulin receptor alpha subtype comprises: a SNP and at least one of an insertion and a deletion.

In some preferred embodiments of the present invention, the SNP includes at least one of (1) to (10):

(1) the alpha subtype 5 intron s.588G > A of the grass carp insulin receptor;

(2) the s.64C > T intron 6 of grass carp insulin receptor alpha subtype;

(3) the s.114A > T intron 6 of grass carp insulin receptor alpha subtype;

(4) located in intron s.158C > A of grass carp insulin receptor alpha subtype 8;

(5) the s.170G > A intron 8 of grass carp insulin receptor alpha subtype;

(6) the 14 th intron s.520C > T located in grass carp insulin receptor alpha subtype;

(7) the alpha-subtype 14 intron of the grass carp insulin receptor is s.658T > C;

(8) s.752T > C located in the 14 th intron of grass carp insulin receptor alpha subtype;

(9) is located in the 14 th intron s.753G > A of grass carp insulin receptor alpha subtype;

(10) is located in the 15 th intron s.42T > C of grass carp insulin receptor alpha subtype.

Wherein (9) and (10) belong to the complete linkage of SNPs. That is, the base type of either one can be determined by detecting the other. In the present invention, (9) and (10) may be combined into SNP9(s.752A > B, wherein A denotes TG and B denotes CA).

Introns (i.e., non-coding sequences), while not coding for proteins, play important regulatory roles in mRNA splicing, gene transcription and expression.

In some preferred embodiments of the invention, the insertions and deletions are: the s.209 located in the 10 th intron of grass carp insulin receptor alpha subtype is mutated into TCAGAATTGTGTGATTAAAAAC (SEQ ID NO.37) or the base is deleted.

In the present example, the insertions and deletions are indicated by SNP 9.

In the present invention, both SNPs and insertions and deletions according to the first aspect of the invention occur in the sequences shown in SEQ ID NO.1 and/or SEQ ID NO.2 and/or SEQ ID NO. 3. The positions of the SNPs and the mutations of the insertions and deletions can be referred to the labels in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in the detailed description.

According to the first aspect of the invention, in some embodiments of the invention, the molecular marker combination of grass carp insulin receptor alpha subtype is a combination of SNP 1-SNP 4 in the examples of the invention.

Sugar is the cheapest of the three feed nutrients. The fish has weak sugar utilization capability, the sugar level in the feed exceeds a certain limit, the fish can cause symptoms of reduced immunity, slow growth, high mortality and the like, and the inventor finds that the low sugar-resistant function of the fish is caused by insufficient secretion of insulin or blocking of insulin receptor alpha signal transmission. Therefore, the invention takes the grass carp insulin receptor alpha gene as a candidate gene to screen the SNPs sites related to the growth of grass carp, and lays a foundation for the molecular marker-assisted breeding of grass carp.

In a second aspect of the present invention, there is provided a use of the molecular marker combination for grass carp insulin receptor alpha subtype according to the first aspect of the present invention in the following (1) to (5):

(1) the application in grass carp variety screening;

(2) the application in grass carp variety identification;

(3) the application in grass carp breeding;

(4) the application in germplasm resource protection;

(5) application in germplasm resource improvement.

The grass carp insulin receptor alpha subtype molecular marker combinations are obtained by screening, and the molecular marker combinations obtained by screening are subjected to correlation analysis with the body weight, body length, body height, body width and fullness of grass carp breeding populations, so that new species of fast-growing high-quality grass carps can be screened by utilizing the molecular marker combinations, or technical support is provided for auxiliary breeding.

In a third aspect of the present invention, there is provided a grass carp growth performance test reagent, which comprises a substance for detecting the molecular marker combination of the grass carp insulin receptor alpha subtype according to the first aspect of the present invention.

In some embodiments of the invention, the growth performance includes higher body weight, body length, body height and body width compared with normal grass carps, and the individual is uniform and not too obese (moderate fullness).

According to a third aspect of the invention, in some embodiments of the invention, the detection comprises detection at the gene level and at the protein level.

In some preferred embodiments of the present invention, the substance is a primer and/or a probe for detecting the molecular marker combination of grass carp insulin receptor alpha subtype according to the first aspect of the present invention.

Wherein, the corresponding relation between each grass carp insulin receptor alpha subtype molecular marker and the primer is shown in the instruction table 1. Wherein, in the embodiment of the invention, the insertion and deletion are represented by SNP 9.

In some preferred embodiments of the present invention, the nucleotide sequence of the primer is shown in SEQ ID NO. 1-10.

The fourth aspect of the invention provides a method for screening high-quality grass carp growth seeds, which comprises the following steps:

detecting the genotype of the locus where the grass carp insulin receptor alpha subtype molecular marker combination is located according to the first aspect of the invention, and judging whether the grass carp sample is a high-quality growing species or not according to the combination condition of the genotypes.

In some preferred embodiments of the present invention, the grass carp insulin receptor alpha subtype molecular marker combination is (1) - (4) (i.e. SNP 1-4 in the examples of the present invention) of the grass carp insulin receptor alpha subtype molecular marker combination according to the first aspect of the present invention.

According to a fourth aspect of the present invention, in some embodiments of the present invention, the criteria for determining whether a grass carp sample is a good-growing species are:

referring to table 7 in the examples of the present invention, if the genotype combination of the grass carp sample is shown in H4, it is a high-quality grass carp growth species, otherwise, it is a low-quality grass carp growth species.

The key of molecular marker assisted breeding is to find a marker closely linked with a character, and the markers in the invention are all based on a candidate gene method, namely, genes which are considered to have direct physiological functions on a character are used for searching sex character linked markers, and a plurality of important character linked markers are obtained by adopting the method, so that the method can be effectively and practically applied to genetic breeding.

Quantitative traits are commonly regulated and controlled by multiple micro-effect genes and multiple sites, and in order to improve the real correlation between genes or alleles and traits, haplotype or double-type analysis needs to be carried out by combining the comprehensive effects of the multiple sites.

Multiple sites within a single gene can often constitute a genotype that has a greater effect on the phenotype.

In a fifth aspect of the present invention, an application of the reagent for measuring growth performance of grass carp in the third aspect of the present invention in preparing a test product for measuring growth performance of grass carp is provided.

According to a fifth aspect of the invention, in some embodiments of the invention, the product comprises a test kit, a gene chip and a test strip.

The invention has the beneficial effects that:

the grass carp insulin receptor alpha subtype molecular marker combination is selected from grass carp proinsulin genes (grass carp insulin receptor alpha subtype), the relation between the grass carp insulin receptor alpha subtype and grass carp growth traits is obtained through general linear model analysis, H4 double type formed by the grass carp insulin receptor alpha subtype molecular marker combination is found to be remarkably higher than other genotype individuals (P <0.05) in weight, body length, body height and body width, the average value of the fullness is 1.59 and is not higher, the H4 double type individuals are shown to not only have high growth speed but also have long body type, and therefore the H4 double type of the grass carp proinsulin genes can be considered as a candidate marker for high-quality grass carps with high growth speed to be applied to grass carp molecular marker assisted breeding.

Drawings

FIG. 1 is a diagram of linkage disequilibrium analysis of 10 SNPs sites of the alpha subtype of grass carp insulin receptor, wherein A is a D' value diagram and B is r²A graph of values; the upper part of the graph is the relative position of 10 SNPs sites in the alpha subtype gene sequence of the insulin receptorThe number of linkage disequilibrium between 10 sites is D' and r²The value multiplied by 100 indicates the linkage between markers, the darker the color, the more unbalanced the linkage.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Experimental Material

(1) SNPs screening samples:

in the following examples, the grass carp samples for screening the SNPs of the insulin receptor alpha gene of grass carp were obtained from the cultured grass carp groups of Ningxiang, Yuansuan, Hubei, Jianli, Shisho, Hubei, Honghu and Guangdong province in Hunan province, respectively, and 4 grass carp samples were taken from each cultured grass carp group, 24 grass carp samples were collected, and tail fins were cut out for genomic DNA extraction.

(2) Growth trait association analysis sample:

in the following examples, the grass carp samples for the growth trait association analysis were all obtained from platinum germchit, inc. The grass carp samples for the growth trait association analysis are all grass carp individuals randomly selected from the group of 17-month-old grass carps bred in the same batch and bred in the same pond. And (4) co-selecting 296 tail grass carp samples for trait association analysis. The growth data of the grass carp individuals such as weight, body length, body height, body width and the like are uniformly measured according to a conventional method in the field, and tail fins are cut and stored in absolute ethyl alcohol for later use.

Extraction of grass carp sample genome

In this example, grass carp sample genomes were extracted using the guangzhou meiji marine animal tissue genome DNA extraction kit. The specific operation is carried out according to the instruction, and total DNA of the fin ray tissues (tail fins) of the SNPs screening samples is extracted. The integrity of the extracted DNA was checked by electrophoresis on a 1.0% agarose gel, and the DNA quality and concentration were determined by a Nanodrop 2000(Thermal) microplate reader, stored at-20 ℃ for future use.

Screening of grass carp insulin receptor alpha subtype SNP molecular marker

(1) Obtaining the complete genome sequence of the grass carp insulin receptor alpha subtype:

obtaining mRNA sequences of grass carp insulin receptor alpha subtypes from a grass carp transcriptome database (https:// www.ncbi.nlm.nih.gov/SRA/.

(2) Screening of grass carp insulin receptor alpha subtype SNP molecular marker

In order to verify the accuracy of the obtained complete sequence of the grass carp insulin receptor alpha subtype genome and further use the complete sequence for SNPs site screening, the primer group described in the table 1 is adopted to amplify the total DNA (representing different geographical source groups) of the obtained 24-tailed SNPs screening sample. The P1 and P2 primers were located in fragment 1, P3 in fragment 2, P4 and P5 in fragment 3.

TABLE 1 primer set for screening of grass carp insulin receptor alpha subtype SNPs

The PCR reaction system is as follows:

the total reaction volume for PCR was 50. mu.L, including: PrimeSTAR GXL DNA Polymerase (Takara) (1.25U/. mu.L), 1. mu.L; 5 × PrimeSTAR GXL Buffer (Mg)²⁺plus), 10 μ L; dNTP mix (2.5 mM each), 4. mu.L; any one group of middle-upper and lower primers (20 muM/L) from P1 to P5, 1 muL each; SNPs screened sample genomic DNA (40ng), 2. mu.L.

Amplification was performed using a TC-96/G/H (B) B-Amplifier (Bori life), using the following protocol: pre-denaturation at 94 ℃ for 4min, 35 cycles of amplification (denaturation at 94 ℃ for 30s, renaturation at 55 ℃ for 90s, extension at 72 ℃ for 120s) and final extension at 72 ℃ for 7 min.

Detecting the length of the amplified fragment by using 1.5 percent agarose gel electrophoresis, sending the amplified fragment to Guangzhou Egyi Biotechnology Limited for sequencing identification, and further analyzing the sequenced fragment to obtain SNPs sites.

The resulting SNPs sites are shown in Table 2.

TABLE 2 genetic diversity of SNPs sites

Wherein SNP6 is insertion (I)/deletion (D); SNP9 is a completely linked single nucleotide polymorphism, with A designating TG and B designating CA.

Wherein the positions of the corresponding targets of the primer groups P1-P5 and the positions of SNPs are shown as follows:

grass carp insulin receptor alpha subtype nucleotide sequence fragment 1:

5’-CCCAATCTGTGGTTTGTGCACATTGTTGGCACGGCTTCAGATCTGGTGTTATATTGTGACACTAGTATGATTGTCAAGACTGTGAAAACCACACCTCCATATCCTTTGTTTTGCTTGTGGGTTCACATTTTATTAAAGTTGTCGTACAAACATGCGTTGCATAGTTTCACAGGAAACAGTGGCGCAGAAAGCATCCTGCACAGAGGACAAACAGTTCCTCATTTAGGAACGACAAATCTACAAAAACAATCAGTGATTAAGTTGTGTCTCTGGCACGTCTGAGAGAGTGTCTCATTCATCCTTGATTGCACCCTCGGCCTGCCCTGCAAGGTCACCCGCTCCCACCGAAACAGCGCTGGCACTTTGCGCAACAGGATCCATGTCCTTGACACACACAATCCTTTATTGTCTTAATGCTGCATATTTGCCTTAACGTCAACGAAAATAGAAACTCTCCATCAAATCTGTATTTCTTTGTGTTCACAGCAAGTGTAGATGAATGAATGACTTGTTTTTTGTCATGTGGCTTTGTGTACATGTGACTTTGTGTTCTGTGCTGCAGGTTGAACTGCACCCCGTGCGCTGGCCTCTGTCCCAAAGTGTGCATGGGGCTGAAGACAGTTGACTCAGTCACTGCGGCGCAGGCTCTGAGAGGATGCACTGTGATCAATGGCAGTTTGGTCATCAACATCCGCGGAGGGAGTAAGAACAAACAAGCTCTCTAATACTATTTCTCCTTTTTGTGCAGCATAGAAGGGCGACTTTAATACACATCTCTCTGGAATTCATAGTTCTGATTGGTTAATCAGCATTCTGCAAATGTTTTTATATAATGATGACTAAACTATATAATTGGCTTTTGTGGCAACCATTCTCCTATACTG(P1 upstream primer) TTAGATTCAGATCACCATCAGTGTTCTGATCACCCTGTTGCTAACTGGAGGTGTATTTACCTTCCAGTTACTTAATAATAATATTCTGATTTTTATTATTAAGAAATATTTAGCTCTGGAAAAAATTTGGAGACCACTGCAAAATTTGCAGTTTCTCTGAATTTATTATTTATATGTTTCTGTTTGAGGAAAACAGACATTTTTTATTTAACTACTGACAATATTTCTTCCAAATTTCAAATAAAAATGTTGTCATTTATTTGCAGAAAATGACAATTGGTCAAAATAATGAAAAAAGAGGTTTTCTGACCTCGAATAATGCCAAGAAAACAAGTTTATATTCATTTTTAAACAACACAATACTAATGTTTTAACTTAGGAATAGTTCAGAAATCAGTATTTGGTG(SNP1，A/G)GAATAACCCTGATTTCCAGTCACAGCTTTCCTGCGTCTTGGCATGCTCCCTATCAGTCAGCCACATTGCTGTTGGGAGACTTTATGCCATTCCTGGCGAAAAAATTCAAGCAGTTTGGCTTTACTTAATGGCTGGTGACCCTCTTGCTTCAGGTCTGGAGATTGTGATGGACATGACAGGGTCTTGATCTGGTGGTCCTCCATCCACACCTTGATTGGTCTGGCTGTGTGGATTTGAAAACATGGCATCTTTTTCCGTTATTTTGACCGGTTGTAATTTTCAGCAAATAAATTCTCTAAATGACAATATTTTTATTTGAAATATGGAAGAAATGTTGTCAGTAGTTTGTAGAATAAATCAAAAATGTTCATTTTCCTCAAACAAATACATTCACTTCCAATAATTCATTGTAATCAAATTACAGAGTAGCCTTATTGTACTTCATTGTACATTTTAACTTTCATAAATTTTACTTTCATAAATTCATAGATTTTAAACTTGCTCTTCTTTTCACCTCTCTCTGTTAGATAACATAGCCGCAGAGCTGGAA(P2 upstream primer) GCTAACCTGGGCCAGCTGGAGGAGATCACCGGCTACCTGACAGTGCGACACTCATATGCCCTAGTCTCCCTATCTTTCCTTCGCAAGCTCCGAATCATCCATGGAGAGACACAGGAAGTTGGGTATGAGTCTTCTTCATTGGATGTCT(P1 downstream primer) AGGTTTTCTTTGCCACCATTTGATGTAAAGAAACTGT(SNP2，C/T)TGGCTTTATCCATTTGGGATGTCAGGTCTTGTTTGTCAACCATAACGATT(SNP3，A/T)CCATGTCATTTTTCTATTCCTTCCTTTTCCCTTCTCACATGTGTGCTGTTTTCTTTCCCATCAGGAATTATTCATTCTACGCGCTCGATAACCAGAACCTGCGTCAGCTGTGGGACTGGAACAAACACAACCTGACCATCCAGCAAGGTCGCATGTTTTTCCATCATAACTCCAAACTTTGCATGTCGGAGATCCGGAAGATGGAGGAGGTGACTGGCACTAAGAACCGGCAAACAAAGAATGACATTGTCTCAAAGACCAATGGAGATCAGGCTTCATGTGAGGAGGACATATCTTTGTGTTTCCTTTATGCTCTTGTGCTCTTTTTCTGAGTAAATCTGAACGCATTGTGGTTGGTGTTTCACCAGGTGAGAGCCAGGTTTTGAAGTTCACACAGATTCGCACACTTTCAGACAAGATCATAATTAAATGGGAACCTTTCTGGCCGCCAGATTTTCGAGACCTCCTGGGATTCATGGTTTTATACAAAGAGGCGTAAGTGACTCAACTTTTTTTTTTTTTTTTTTTCAAATGGACCTTAAAGGTGCACTGCAGGATTTTCCTGACTCGTACAAAAATAATGCAAATGTTGCTGTTGACTTTTTGACTGCTATCTGTTACTTGGCATTGTAATTAGGTTTTTATGATGACTC(SNP4，A/ C)GTAATTAGTTGG(SNP5，A/G)CTAAACACAAATTACAGCTAGTCATCTAACTATTAGCGTAACTGTTCTTTGATATGTACAAAATATGCTATTTATGCATGATAAGCATAATATTTAAACAAATACTTGCAAAATAGTTCATCCCACTGGTATTCTCTTGAGTCGGTTAGGCTGCGTGTGCAACTGAAAAATCATGTCAGGGCCTGAGCTTCTGGCGGTGCTTCGCCTGCGAGGATGGGCCAGGTTTGAGTGTTGTGATTACAAACAGTAACTGAAAATCCTGCATAATGCACCCATAATCAAGATAGACAGCATATAATGTGTTGTGAATGAAATTAAGACTGTGAGGGATAGGGGATGAAGTACCA(P2 downstream primer) -3' (SEQ ID NO. 11).

Grass carp insulin receptor alpha subtype nucleotide sequence fragment 2:

5’-ATGGATGGATGTACTTTTTTGGGCTTCAAAATCTCGAGCCCCATTCACTCCCATTATAAAGCTTTGAAGACCCTGAATATTTTTTAATATAACTCCGATTGTGTTTGACTGAAAGAAAAAAGTCATATACACGTAGGATGGCTTATAATTTTCATTTTTTTTATTAACTAATCCTTTAATGATACACACCAAGTTATACGGACTGTATTATCCCTCACATCCTCGTTTTCATTTTCACCATTTTAACAATGGTGTCTACCCTGACTTTAAGGTCCATTTTATGGCTGTGCTTGGTTTGTAGATTGAGTTCAGTGCAATGCTCTGTTAGTGTTTGATAACATTTCTCTCTGCTTCTCTAGGCCATATAAGAATGTGACTGAGTTCGATGGTCAGGATGCATGTGGCTCTAACAGCTGGGTCATAGCAGATGTGGACCCTCCGCCTCGCGCCACAGAGGGAAAAGAACAGCAGGAGCCTGGATACCTCATCCTTCCTCTGAAGCCATGGACGCAGTATGCCATCATGGTCAAAACCCAGCTCTCCGCTTCTGATGAGCACCAGGTCCATGGGGCAAAGAGCGAAATCATCTATGTCCGCACCAATGCTACCAGTAAGAGCTTCATAAGCAGTGTTGTAGGAAGGTTATGATTTTTTCAAGGAAAAGGAAGGAAAATGAAACTTTAGCTTAAAACTAAATTTAGTCATTTCTGAAGTCTTTTCTGTAAGTTGATTTCAGTAGCAGTGTTTATCGTGCAGGCTTGGCATGAAAGAGGTCAGAGGCTGGTAAAATAGGTCACTGGTGACTTTTAGTGGTGTGAAATCACTCTGCAGTATGCAGGATGTCAGGCCGTGATTTAGATGAAAAAATAAATGTGGTTTGCTTTCAGTTTTGCCAGGTGTCTGTCTGACTTCCTTTTGAGCTTGTTTTGTCTTTCCTTCCTGATAAACCACATAACCATTCACTAAGCTGAGAACAT TGTTTCG(P3 upstream primer) GCAGCTGGTTGGCAAGAGCTGAAAGTAGCGCTTCAGGGTTTTTCTCTTTCTTTTTTTTTCTGGTTTGGCAAACGGTTGGGTTCACACACTGTGACATCGACGGTGCCCCTCTACTTCTTCCTTTTTAGGAGGAAGCTGTCGTTATTCTTCTTGTCTCGTTGGTGACCCAGTCTGACAGTGCGGTCTAACCAGAATTGAGTTTTGTTAAGAGTCTACTAGTCGTCTACTTTTTTTTTTTTTGGCTTGGACTTCCTCAAAATCGTTAATTTTTTTCTACTTACTAATGGGTGAGTTGTTTGTTGTTGTTTTTCAGAACCCTCTGTGCCCCTGGACCCCATATCCTCGTCAAACTCCTCATCTCAGATCATTCTGAAGTGGAAGCCTCCTAACGATCCAAACGGAAACATCACACACTACTTAGTCTTCTGCCAGCAGCAACCAGAGGCCAGCGAGCTCTACAAGTTTGATTACTGTCAGAAAGGTTAGAAAACATAAAGGAACATAATATAAAACATACACACTACCGTTCAGAAGTTTGGGGTTGGTAAGATTTGAAATGTTATCTATGGAAGCTTGTTTCCACCATGAAATAAAAAATAAAAAAGGTAATTGCGACTTTTTATCTCACAATTCTGACTTTTTTTTCTCACAATTGTGAGTTATAAAGTCAGAATTGTGTGATATAAAG (SNP6, deletion/insertion TCAGAATTGTGTGATTAAAAAC) TCAGAATTGGGAGATATAAAGTCAGAATTGGGAGATATAAAGTCAGAATTGCGAGTTATAAAGTCAGAATTGCGAGATATAAAGTCAGAATTGGGAGATATAAAGTCAGAATTGCGAGATAAAAAGTCAGAATTGCGAGATAAAAACCTTTTTTTACAGGCTTTTTTGTATTTTTAACTATTGATACATGGGTCATAGCACCTAATTAGCTGGAAGTTAGGGGTGTGCGAGACAACGATTTTTGATCGTGGACGATTAAAATGTGAGAACTTTAATACATTAGCTTTATTAAGAATCAATGTATAATTTATACAGAGAAGTCTAGTGTTATATTTTTATATTTGTTCATTTAATTTTTTACTTGAATGCTACTGTTAAATACATCTACTG(P3 downstream primer) TAGCTGAAAAAATAAATCATTTTATTAATTTCATTTGTATAATGTGTAGCCTATGTTTTTCTTCTTTTTTAATAACAAAGATAAAATTAATAACAAATAATAACAAATAACATAAAAAATAATGACAAAGATTTTTATCTTCGCCACTTTGGCCTAAATGTCTGCCATTTTTAATTTTATTTTATATTTTGTTATTTTGTAAGGCTTTGTTTTTAAAATAGGGAAATTAGTTTGGCTGTAATGCTCAGTCACTTGCAAAATAGTAAAAACAACATGAAGAATCGTGATAAAAATCGTGAATCGTGATTTTCCTGAAAAAAATCGTGATATGATATTTTTGCCATATCGCCCACCCCTACTGGAAGTGCTTTGATTGGAGAGCAAAATCGAAGCCTTATGTTTCTGTTGTTCAGGTATGAAGCTGCCCACCAGAGCTCCCACACAAGTGGACAACGAAGAGGAACAGAAGTGGAACCAAACCGAGGAGCAAGGCCAAGATAGCCGCTGCTGCGCCTGCCCCAAAACAGAGAAACAGCTGAAGAAAGAGGCAGAAGAGACAGAGTACCGCAAAACCTTTGAGAACTACCTCCATAATGAAGTCTTTGAGCTCAGGTAAATTTCACAGTTCCTCATTTAACTAGTCTAGATTAGAACTGCAAAAATGAATCATATAGACTTTTTTATTTGAAAGAATTTTTCATATATAGTCAGTATATATTGTACATTATATAAACTTATGCAGACCGTTTTCTCATAGTTTGAAGGAGAGCATTGTGCAAGATGTGAAATATCTAATTAATATATGAAATAATCAACAGATTGATCAATTATCAAAATAGGTGGAGATTCTCAAGTGGTTCATGTTGTTACGGTGTTCATCTGTTTTGTTTAGAGTTCTAAACTCAGGGTTGAACGTCTTAGTAGTGGTCCGAACACTTTCCCTCGCTGGGTTGTTTTTGACTCCAAGACCTTGAGTACATGTTTTTGTTAGTTGCATTTCCACCTTCTTGGTGTATCTTT-3' (SEQ ID NO. 12).

Grass carp insulin receptor alpha subtype nucleotide sequence fragment 3:

5’-GTGGAGTTGATTCAACTCATCGACTAGCATGTGCCGTCATGTTAATCTTTTGTGCAAATCCAGTGTTGAATTGACCCTCGTTTGTGAAGCAGTCCGGCGTAAAATGACAGCATGGCAACAACACTCTACTACAACAACTCTTCCTCTTCTCTAAAGCAGCCCAACATGGCCTCACCCCCTTTGTTGCGTGTTCTCTGGCGCAGGGTTTATGTAAATTTTGGGGTTAGTGATGTCACTAACCCGGGAAGAAGCTCGTTGTAGTCCCTACCAGCCGTTTGTTGTAGTCCTTAAACAGAGAATTCTTTAAAAAAAATATCTCCCTTTGCATTGAACTTTGAGCGTCGTAACTTTGCAGATGTTGTTTATGCTCAAACAGCAACATTACACACTAACTAAAGTTAAAAAAGTGAAATCAAAATCAAGGACCCCTTTAAGACTGAACCTAAATCTAGACTGTCTTCCTTCTCTTTACATGTTTCTGTGTACCCAGGACATCAAGACAGCGCAGATCTCTGGTGGGCATTGCCAATAGGACCTCCTCTCGTCTTTTCACTACACCCTCCTCGTTGCCCAATGGCTCTGCCACTCGTAGCCCAGAGGAAGAAGCAGAGGCCAATAAGATTTCACTGATTGTTCGTGCCAAGGAGTCGACAGTCATCTCCAATCTGCGCCATTTCACCAGCTACCAGATAGAAATCCATGCCTGCAACCATGAGAGTGACCCCACTCGCTGCAGTATGGCGGCCTACGTCAGTGCCCGCACCATGCCCGAAGGTGTGGGATTGTAATTTTATAATGCATTATTTCATATAGTATAGTTGTTTTTATTGACATATAGTCTTCTGTTTTTGTACAGAAAAAGCTGATGACATTGTGGGTCCGATAACCTATGAGGTGTCGGAGTACTCTGTACACATCAGGTGGATGGAGCCCAAGGCCCCTAATGGCATGATTATTCTCTATGAAGTCCATTACAAGAGACTGGGAGATACAGAGGTGAGATGTTACACAGGATAGAGATGGAAGCCTGCACTCTGCTCCCATGAATGCTGGCTGCACTTTTAAACTGATTTTTATCAGCCCTGATGAAATCTCCAAATAGCTCCAAATAAACAAGCTTTTTTAACATAATTGCTTCAGCTGTGTATTTCTGTAACCATGGAGAAATCCAGCCAGAAACAGAGGAAATGTTCCTAATGCTGTTTTTGAAAAGCCTCGGATAAACAAGGGATGATTATATAAAGGTTAATAATTGTTATAAGCAAAACAAAATGTGATGGTTTTGCACTTAGCGGCACATGCACCTGTTTGGGTTGATGGATTTGAAACGCTCTGTTTACACTGACCTCTAGCAGTTTCATTAAAATCTGATGAGTAATATTTATATGTATTGTTGTGAGATGGACTTTAAAAACATGATGATAACTAAGCCTTTGCCACCGAATAATGGCCGTCTTGAGTTCGATGTGCACAGCAATATGGGTTATCTAACTGCATGCCTTTATGTCTAAGGAGCTGCACCACTGCGTATCCAGGAAGAATTATATTGCAGACGGAGGCTGTAAGCTGAGAGTGGTGCACCCTGGGAACTATACAGTGCGGATCAGAGCAACCTCCTTGGCAGGCAATGGATCGTGGACAGAGCCAACACACTTCTATGTTCAGGACCTACGTAAGACGGCTTGTCTATAATGCCTATGAATATCATATCTTCAGTTATTAAATTATGCATGAGACACACATAACTGAATTTTGAAGAGGTTTGTGTGTGTGTGTGTATCTGGAAAAAAACTAAATATTCACACACTGATACTATTGAGTTATATTGAACAATTTGAAAGTTTCAACCTCTACGTCTATAGTATGGAAAAATGGTCAAAATGTTGCAGTTGTTCAATATGGAAGCCCTAAGATGC(P4 upstream primer) CATGTTGCAATTCAGTTTTTTTTAAACCATAAATTGGTTTTGTTTTGTGGTCATTTTTATTGTTTATTATTGGTTAATTTTGTTGTGGGTTTTATTTTAATTATTTTTTATATTAAAGGCACAATATGTAAGATTTTTGGATGAAAATATCCCAAAACCACTAGAACAGTGTTATTTATTTTGTTGACTTGTGTACTTACACTATCCCAAATGTTTCCAAGAATGTTTAAATACAGAGAAATAAGCAATTTTAACCAGCGTCGCCTATCAATGACATC(SNP7，C/T)ATTACCCTCGATTTCCGGTTTTATTTTGTAGAAACCATGGAAACACCAAAGACGCTTTAATATAGTATGTGTTTTATTAGACAGGTGAGCAACTGTTTGGATACATTCATTGACAGAGAACTAATCATGTTATATAGGTCAACACAGTAAGTCTTATTGTTTAAATCTCGTTTTCTTGATTTACCGCCATATTTTACCATGACTAATTTCGATCTAGCTTACTGCAGTGTGT(SNP8，C/T)AACAAGTGTCTCGTAGTAGCTG(SNP9，TG/CA)CCGAGCGAATGCACAGAGTAGCATTATAACAACTGTCAACACACAAATGTATCTAATATGATAAACAGCGCTGTGTTACCCCACATACTCATGAGCGGAAGAAGAAGAAGCGTTCGTCTGTGGCATAATAAATGTTCCACTGCTCTTGAGCCGTGTGTCGCACTCGTCTCTCATTAGCAATTGCTTCAGCGGCCTCGTTCCGTTCCAATGTCTTTCAGCTCCACCCTGCTTCATACTACAGTAATGTTAATAGTTCTTTAAAACATTAGCTCATCCATGAATATGATTTCTGCATGAGTCCCATTGGATTCGATTCCATTGGCTGTAGATGTGAAGACTACACCTCCCATGCGTCATTAAATTACGCCTTTGTTATGAATTAGTGACCTCTAGTGGTGAAAAATTACATATTGTGCCTTTAATTGATTTTTTTTATTCCTTGGCATTTTAATGCTTCCTGT TAATATCAGTATA(P5 upstream primer) TAGAGAATATTTTTTATTTAATCTATTAAGTATTATTTGCTCCATTGTGTCCTGCACCTGTATACAACTTAGTTTGGAAGTGCACAACTTTTATATATTTTTTGGTTGTATCTATGTGAACAGGTGTGGACCCATCCAACATGCTGAAGATTGTGATTGGTCCG(P4 downstream primer) GTGATCTGTGTCTTTTTACTCCTTGTGATGGGAGGTGTGGGCTTTTTCATGTTCAAGAAAAAGTAAGTGTTGTGATTGTCCTGAAAACTCAAGCATAAAACTATT(SNP10，C/T)CTTTATGAGGTAAGTAACGAATTTCATTAATTTTTGACCAGGCAAACAGAAGGTCCAACTGGGCGACTTTATACCTCCCCAAATCCAGAGTATTTCAGCCCAGATGAGGGTGAGCAACTTTCCTTTCCCTGCGTGTGTGACCTTTAGCAGAAAATGCTGGGGTCGATGCACATTTAATTTAGTTCTAATAGAAGAGTTGATCACATTAACGATCGATTTAAGCAGATTATCACATTTAAAATGACTTTCTCTTTTGCCTGAGATCTGCTCCATCTCAAATATGACACTGTGTGTCCTGCATGTGTGTTGTGTTTGTGCAGTGTATGAGCCAGACGAGTGGGAGGTGCCCCGTGAAAAGATTAACCTATTGCGAGAGTTAGGCCAGGGATCTTTCGGCATGGTGTATGAGGGCATCGGAAAGGACATAGTCAAAGGCGAGCCACAGACCCGTGTTGCAGTGAAGACGGTTAACGAGTCGGCCAGCCTCAGAGAGAGGATCGAGTTCCTCAACGAAGCTTCTGTGATGAAGGCGTTCAGCTGCCACCATGTGGTAAGACATGTCTCTTACATATCAGCCTATGTGTTTTGTAGTCTTATTTTAGTATTTGTTTATATATTGTTTATTATTATTTTGAATAAGCTTTTATTTTATATTTTCATTTAAATGTTTAATTTTAGTAGTTTGTGCTTTTATCTTTTTTTTTCTTTTTTTTTTTTAATATTTCCAATTTAATTTTATATCTGTTTTAATTTTAGAATTTATTAATTTCAGTTAGTTACCAAGGCAACATTTCTTTTAATCTAATATTTATATATAGACACTACTGCTCAAAAGTTTGGAATAATTAAGATTTTTAATCTTTTTTGAAAGATGTCTCACCATCGCTGCATTTATTTGATCTAAAGTACAGTAAAAACAGTAATACTGTGAAATGTTATTACAATTTAAAATAACAGTTTTCGGTTTGAATATATTTTAAAATGTGATTTATTCCTGTAATATAAAAGCTAAATTTTCAGCATCATTAATCCATTCTTCAGTGTCACATGA(P5 downstream primer) TCCTTCAAAAATCATTCTGATATTCTGATTTGGTTGTCAGTCATTATCAATTATTATTATTAATTATTATTGGTGCTCAAATATTGATAATGGTCCTTATTATGAGTGTTGAAAAGTTTTTGCTGCTTAATATTTTTGTGGAAACCAAGGAATACATTTTTCAGGATTCTTATATGAATTGAAAGTTTAAAAGAATAGAATTAATTTAAAATAGAAATCTTTTGTAACATTATAAATGTCTTTACTGTCAGTTTTGAATGACAGTGTATCACGGTTTCCACAAAACTATTCGAACTATTCAACATATACTTTTTAGGTTTTATTTTAATTACTGAGAATGATTTTGATATTTTTAGGTTTAGTTGTAGTTAATAATAACACTGGTTGGACTGTGGCATATTCTGATGGTAACTGACATGATCTTGTGGGTTAGGTGAGGCTGCTGGGGGTGGTGTCTAAAGGCCAGCCCACCCTAGTGGTGATGGAGCTCATGACACATGGAGATCTGAAGAGTTTCCTGCGTTCTCTCAGACCTGACTCAGAGGTAACAAATCAGAAGTGTCACTATCAGACCTGCTAGAGTTTTTAGATACTGTATGTATATATATATATATATATATATATATATATATATATATATATAAAAAATAATGTATTTTTGTTTTACTGTGTTGTTTGATTGCTTTGTTACAGAATAACCCAGGTCGGCCTCCTCCAACGCTGAAAGAGATGATTCAAATGGCCGCAGAGATAGCAGATGGCATGGCTTACTTGAACGCCAAGAAGTTTGTGCACCGAGACCTTGCTGCCAGGAACTGCATGGTGGCCGAAGACCTGACCGTAAAAATTGGAGGTACATTACTAGTGACGGGTGTACATTTGTTTATTTAAGCTTAAAGGATTAGTCCACTTTCAAATAAAAATTTCCTGATAATTTACTCACCCCCATATCATCCAAGATGTCCATGTCCTTCTTTCTTCAGTCCAAAAGAAATTAAGGTTTTTGATGAAAACATTCCAGGATTATTCTCCTTATAGTGGACTTCAATGGTCTCCAAACGGTTGAAGGTCAAAATTACAGTTTCAGTGCA-3' (SEQ ID NO. 13).

(3) Genotyping of grass carp insulin receptor alpha subtype SNP molecular marker:

the SNPs sites obtained by the above screening were subjected to SNaPshot typing. Typing primers were designed with the primer sequences shown in Table 3.

Table 310 SNPs-labeled typing primer set

The specific method comprises the following steps:

the SNP6 typing primer is used for carrying out PCR amplification on 296 grass carp samples directly according to the amplification conditions of the P1-P5 primer, the amplification products are directly sent to Guangzhou Egypti Biotechnology limited company for sequencing, and the genotype of each sample is determined according to the sequencing result.

And performing multiple PCR amplification on 296 tail grass carp samples by adopting typing upstream and downstream primers for other SNPs markers to obtain PCR amplification products.

An amplification step: purifying 3 μ L PCR amplification product with Exo I and FastAP (Fermentas), incubating at 37 deg.C for 15min, and inactivating at 80 deg.C for 15 min; after purification of the amplification product, an extension reaction was performed by using the SNaPshot kit from ABI (USA) according to the procedure described in the specification. Taking 1 mu L of extension product, adding 10 mu L of loading buffer, denaturing at 95 ℃ for 3min, immediately performing ice bath, and detecting by using an ABI 3730XL sequencer. The genotype of each sample was determined according to the typing results.

And calculating genetic parameters such as effective allele factors (Ne), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information quantity (PIC), Hardy-Weinberg equilibrium state (Hardy-Weinberge guiliberium), allele frequency and the like of the SNPs by using Popgene 32(version 3.2) software.

The results are shown in tables 4 and 5.

TABLE 4 genetic diversity analysis of SNPs sites

TABLE 5 genotype and allele frequencies at SNPs loci

296 tail grass carp samples were typed by the SnapShot method according to 0.25<PIC<0.50 is a medium polymorphism principle, and the analysis finds that SNP5, SNP7 and SNP9 do not belong to medium polymorphism sites, and the rest sites all accord with the medium polymorphism principle. Jingchi type²The test shows that the 3 sites of SNP8, SNP9 and SNP10 are not in Hardy-Weinberg equilibrium (P)>0.05), the rest of the sites are in Hardy-Weinberg equilibrium (P)>0.05)。

Relevance of grass carp insulin receptor alpha subtype SNP molecular marker and grass carp growth traits

And (3) carrying out relevance research on the obtained 10 SNPs sites and the growth traits of the grass carps, and specifically comprising the following steps:

the correlation between the genotypes of the different SNPs sites of the insulin receptor alpha gene fragment and the growth traits (body weight, body length, body height, body width) and fullness was calculated using a General Linear Model (GLM) of the SPSS 17.0 software and subjected to least squares analysis (LSD), wherein the fullness formula is:

wherein K represents the fullness;

w represents the weight (g) of the grass carp;

l represents the body length (cm) of the grass carp.

The results are shown in Table 6.

TABLE 6 correlation of different genotypes of SNPs with growth traits

Wherein different lower case letters indicate significant difference (P < 0.05).

The SNP 1-4 and SNP 6-10 have insignificant differences in genotype, body weight, body length, body height and body width traits (P >0.05), and the SNP5 locus has significant differences in genotype and body height (P < 0.05). The GG genotype at SNP1 site is very obvious in body length and fat percentage compared with AA type and AG type (P <0.01), and the allele G frequency (63.01%) is higher than the allele A (36.99%), which indicates that the GG type gene shows body shape growth in the population. The CC genotype at SNP2 only differed significantly from CC and CT types in fullness (P <0.05), but the CC type growth advantage was not significant. Similarly, the SNP3 site AA genotype is significantly higher than AT and TT genotypes in fertility (P <0.01), but the AA genotype frequency is not high, only 28.72%. The SNP4 locus CC genotype is obviously higher than AA genotype and AC genotype in the difference of body height and fertility (P < 0.05). The SNP6 site II genotype is obviously superior to the ID and DD genotypes in individual growth traits and shows obvious difference in fullness (P < 0.05). The SNP9 site AB genotype is more obvious than AA and BB genotype in individual growth traits and shows obvious difference in fullness (P <0.05), and the heterozygote at the site is proved to be more advantageous in fullness. The SNP10 site II genotype is more obvious than the ID and DD genotypes in individual growth traits and shows very obvious difference in fertility (P < 0.01).

Relevance of double SNP (Single nucleotide polymorphism) type of grass carp insulin receptor alpha subtype and growth traits

And (3) researching the association between the SNP double type of the grass carp insulin receptor alpha subtype and the growth traits by utilizing a population genetics research method (linkage disequilibrium (LD) analysis).

LD analysis was performed using SHESIS, where r is given²Is a metric index.

r²The larger the value, the greater the degree of LD. When r is²>0.33, indicating the presence of a strong LD.

The results are shown in FIG. 1.

After linkage disequilibrium analysis of 10 SNPs using SHESIS, SNP5 was found to be unlinked with the other 9 SNPs (r)²<0.33), linkage equilibrium between SNP10 and other 9 SNPs (r2 ≈ 0), linkage imbalance between SNP4 and 6 SNPs including SNPs 1-3, SNP and SNP 8-9 (r)²>0.33). Strong linkage (r) among 4 SNPs of SNP 1-4²>0.5), therefore, the association between the double type and the growth trait was analyzed using the combination of 4 SNPs, SNPs 1-4.

The results of the association analysis of the double-type and growth traits for combinations of 4 SNPs, SNPs 1 to 4, are shown in Table 7.

TABLE 7 different doubling and growth relationships of SNP 1-4 compositions

Wherein different lower case letters indicate significant difference (P <0.05) and different upper case letters indicate significant difference (P < 0.01).

The 4 SNPs sites of SNP 1-4 constitute 10 double types, wherein the effective double type is H1-H8 and 8 (the other double types have low frequency and do not participate in statistical analysis and multiple comparison).

The double H4 gene is the dominant growth genotype, with the highest data on various traits including body weight, body length, body height and body width, and the medium fertility being 1.60. The H1 type gene is a growth disadvantage gene, the individual body is light and short, and is fat (the fatness is obviously higher than that of the double type gene except H3 (P <0.05)), and the actual breeding benefit is low. Compared with the body weight alone, the optimal allele TT, TT and CC type average values of SNP2, SNP3 and SNP4 are 1628.14g, 1628.14g and 1629.36g respectively, and the dominant double type H4 body weight average value of SNP 1-4 allele combination is 1699.79g, which can also show that the dominant genotype character is more obvious along with the increase of the number of polymerization markers. By analyzing the fullness, the average value of the AA type of the SNP4 optimal allele is 1.56, and the average value of the dominant duplex H4 of the four allele combinations is 1.59, which also can show that the dominant genotype traits are more obvious and the fullness combination is lower with the increase of the number of the polymerization markers.

Therefore, according to the results of the different association analysis of the double type and the growth, the double type H4 has the maximum value on the data of various characters of body weight, body length, body height and body width, and is the growth dominant genotype. From specific values, body weight: h4> H6> H3> H7> H8> H5> H2> H1; body length: h4> H8> H6> H5> H7> H3> H2> H1; body height: h4> H3> H1> H6> H2> H5> H7> H8; body width: h4> H3> H6> H8> H7> H1> H2> H5; and (3) fullness: h3> H1> H2> H4> H7> H6> H5> H8.

H4 was very significantly higher than H5, H2, H1(P <0.01) and significantly higher than H8(P <0.05) on body weight. In body length, H4 is very significantly higher than H3, H2, H1(P < 0.01). Likewise, H6, H8 were also very significantly higher than H1(P < 0.01). In body height, H4 is very much higher than H8(P <0.01) and significantly higher than H2, H5, H7(P < 0.05). H4 is also significantly higher in body height than H2, H5(P < 0.05). In terms of fullness, H8, H5 were significantly lower than H2(P <0.01), and H8, except H5, were significantly lower in fullness than other double-type genes. From the above, it is presumed that the H1 type gene is a growth-disadvantaged gene, and the individual is slightly short and slightly obese (the fullness is significantly higher than that of the double type gene except H3 (P <0.05)), and the actual breeding efficiency is low. The H4 gene has certain advantages in weight, body length, body height and body width, and is a growth dominant genotype, the individual body is uniform and does not lean, the weight is small, and the actual culture benefit is high.

Verification of growth dominant genotype of grass carp insulin receptor alpha subtype

Reselecting 2-year-old grass carps bred in the same pond and in the same batch, randomly selecting 230 carps to perform typing on the SNP1, SNP2, SNP3 and SNP4 markers by using a SNapShot typing method, wherein the typing results are shown in Table 8.

TABLE 8 correlation of different doublet types of SNP 1-4 composition with grass carp growth

Wherein, different lower case letters represent significant difference (P <0.05)

Body weight was seen from the typing results: h4> H6> H3> H8> H7> H5> H1> H2; body length: h4> H8> H6> H3> H5> H7> H1> H2; body width: h6> H4> H3> H1> H5> H8> H7> H2; body height: h7> H4> H5> H3> H6> H8> H1> H2; and (3) fullness: h2> H1> H3> H6> H7> H4> H5> H8. It can be seen that the double type H4 has significant advantages in the data of body weight, body length, body height and body width, and the fullness of 1.77 is only higher than that of the double type H5 and the double type H8.

Comprehensive analysis shows that the four alleles are combined into the double type H4 which belongs to the dominant double type on the growth traits, the combined grass carp is low in fullness, the body growth is proved, the market demand is met, and H4 can be used as a candidate marker for molecular marker assisted breeding of fast-growing high-quality grass carps.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Zhujiang aquatic research institute of Chinese aquatic science research institute

<120> grass carp insulin receptor alpha subtype SNP molecular marker combination and application thereof

<130>

<160> 37

<170> PatentIn version 3.5

<210> 1

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<212> DNA

<213> Artificial sequence

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gtggcaacca ttctcctata ctg 23

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agacatccaa tgaagaagac tcat 24

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acatagccgc agagctggaa 20

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tggtacttca tcccctatcc ctc 23

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cactaagctg agaacattgt ttcg 24

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cagtagatgt atttaacagt agc 23

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caatatggaa gccctaagat gc 22

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cggaccaatc acaatcttca g 21

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<213> Artificial sequence

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ggcgcttcct gttaatatca gtata 25

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tcatgtgaca ctgaagaatg gatta 25

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<213> insulin receptor

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cccaatctgt ggtttgtgca cattgttggc acggcttcag atctggtgtt atattgtgac 60

actagtatga ttgtcaagac tgtgaaaacc acacctccat atcctttgtt ttgcttgtgg 120

gttcacattt tattaaagtt gtcgtacaaa catgcgttgc atagtttcac aggaaacagt 180

ggcgcagaaa gcatcctgca cagaggacaa acagttcctc atttaggaac gacaaatcta 240

caaaaacaat cagtgattaa gttgtgtctc tggcacgtct gagagagtgt ctcattcatc 300

cttgattgca ccctcggcct gccctgcaag gtcacccgct cccaccgaaa cagcgctggc 360

actttgcgca acaggatcca tgtccttgac acacacaatc ctttattgtc ttaatgctgc 420

atatttgcct taacgtcaac gaaaatagaa actctccatc aaatctgtat ttctttgtgt 480

tcacagcaag tgtagatgaa tgaatgactt gttttttgtc atgtggcttt gtgtacatgt 540

gactttgtgt tctgtgctgc aggttgaact gcaccccgtg cgctggcctc tgtcccaaag 600

tgtgcatggg gctgaagaca gttgactcag tcactgcggc gcaggctctg agaggatgca 660

ctgtgatcaa tggcagtttg gtcatcaaca tccgcggagg gagtaagaac aaacaagctc 720

tctaatacta tttctccttt ttgtgcagca tagaagggcg actttaatac acatctctct 780

ggaattcata gttctgattg gttaatcagc attctgcaaa tgtttttata taatgatgac 840

taaactatat aattggcttt tgtggcaacc attctcctat actgttagat tcagatcacc 900

atcagtgttc tgatcaccct gttgctaact ggaggtgtat ttaccttcca gttacttaat 960

aataatattc tgatttttat tattaagaaa tatttagctc tggaaaaaat ttggagacca 1020

ctgcaaaatt tgcagtttct ctgaatttat tatttatatg tttctgtttg aggaaaacag 1080

acatttttta tttaactact gacaatattt cttccaaatt tcaaataaaa atgttgtcat 1140

ttatttgcag aaaatgacaa ttggtcaaaa taatgaaaaa agaggttttc tgacctcgaa 1200

taatgccaag aaaacaagtt tatattcatt tttaaacaac acaatactaa tgttttaact 1260

taggaatagt tcagaaatca gtatttggtg gaataaccct gatttccagt cacagctttc 1320

ctgcgtcttg gcatgctccc tatcagtcag ccacattgct gttgggagac tttatgccat 1380

tcctggcgaa aaaattcaag cagtttggct ttacttaatg gctggtgacc ctcttgcttc 1440

aggtctggag attgtgatgg acatgacagg gtcttgatct ggtggtcctc catccacacc 1500

ttgattggtc tggctgtgtg gatttgaaaa catggcatct ttttccgtta ttttgaccgg 1560

ttgtaatttt cagcaaataa attctctaaa tgacaatatt tttatttgaa atatggaaga 1620

aatgttgtca gtagtttgta gaataaatca aaaatgttca ttttcctcaa acaaatacat 1680

tcacttccaa taattcattg taatcaaatt acagagtagc cttattgtac ttcattgtac 1740

attttaactt tcataaattt tactttcata aattcataga ttttaaactt gctcttcttt 1800

tcacctctct ctgttagata acatagccgc agagctggaa gctaacctgg gccagctgga 1860

ggagatcacc ggctacctga cagtgcgaca ctcatatgcc ctagtctccc tatctttcct 1920

tcgcaagctc cgaatcatcc atggagagac acaggaagtt gggtatgagt cttcttcatt 1980

ggatgtctag gttttctttg ccaccatttg atgtaaagaa actgttggct ttatccattt 2040

gggatgtcag gtcttgtttg tcaaccataa cgattccatg tcatttttct attccttcct 2100

tttcccttct cacatgtgtg ctgttttctt tcccatcagg aattattcat tctacgcgct 2160

cgataaccag aacctgcgtc agctgtggga ctggaacaaa cacaacctga ccatccagca 2220

aggtcgcatg tttttccatc ataactccaa actttgcatg tcggagatcc ggaagatgga 2280

ggaggtgact ggcactaaga accggcaaac aaagaatgac attgtctcaa agaccaatgg 2340

agatcaggct tcatgtgagg aggacatatc tttgtgtttc ctttatgctc ttgtgctctt 2400

tttctgagta aatctgaacg cattgtggtt ggtgtttcac caggtgagag ccaggttttg 2460

aagttcacac agattcgcac actttcagac aagatcataa ttaaatggga acctttctgg 2520

ccgccagatt ttcgagacct cctgggattc atggttttat acaaagaggc gtaagtgact 2580

caactttttt tttttttttt tttcaaatgg accttaaagg tgcactgcag gattttcctg 2640

actcgtacaa aaataatgca aatgttgctg ttgacttttt gactgctatc tgttacttgg 2700

cattgtaatt aggtttttat gatgactcgt aattagttgg ctaaacacaa attacagcta 2760

gtcatctaac tattagcgta actgttcttt gatatgtaca aaatatgcta tttatgcatg 2820

ataagcataa tatttaaaca aatacttgca aaatagttca tcccactggt attctcttga 2880

gtcggttagg ctgcgtgtgc aactgaaaaa tcatgtcagg gcctgagctt ctggcggtgc 2940

ttcgcctgcg aggatgggcc aggtttgagt gttgtgatta caaacagtaa ctgaaaatcc 3000

tgcataatgc acccataatc aagatagaca gcatataatg tgttgtgaat gaaattaaga 3060

ctgtgaggga taggggatga agtacca 3087

<210> 12

<211> 3082

<212> DNA

<213> insulin receptor

<400> 12

atggatggat gtactttttt gggcttcaaa atctcgagcc ccattcactc ccattataaa 60

gctttgaaga ccctgaatat tttttaatat aactccgatt gtgtttgact gaaagaaaaa 120

agtcatatac acgtaggatg gcttataatt ttcatttttt ttattaacta atcctttaat 180

gatacacacc aagttatacg gactgtatta tccctcacat cctcgttttc attttcacca 240

ttttaacaat ggtgtctacc ctgactttaa ggtccatttt atggctgtgc ttggtttgta 300

gattgagttc agtgcaatgc tctgttagtg tttgataaca tttctctctg cttctctagg 360

ccatataaga atgtgactga gttcgatggt caggatgcat gtggctctaa cagctgggtc 420

atagcagatg tggaccctcc gcctcgcgcc acagagggaa aagaacagca ggagcctgga 480

tacctcatcc ttcctctgaa gccatggacg cagtatgcca tcatggtcaa aacccagctc 540

tccgcttctg atgagcacca ggtccatggg gcaaagagcg aaatcatcta tgtccgcacc 600

aatgctacca gtaagagctt cataagcagt gttgtaggaa ggttatgatt ttttcaagga 660

aaaggaagga aaatgaaact ttagcttaaa actaaattta gtcatttctg aagtcttttc 720

tgtaagttga tttcagtagc agtgtttatc gtgcaggctt ggcatgaaag aggtcagagg 780

ctggtaaaat aggtcactgg tgacttttag tggtgtgaaa tcactctgca gtatgcagga 840

tgtcaggccg tgatttagat gaaaaaataa atgtggtttg ctttcagttt tgccaggtgt 900

ctgtctgact tccttttgag cttgttttgt ctttccttcc tgataaacca cataaccatt 960

cactaagctg agaacattgt ttcggcagct ggttggcaag agctgaaagt agcgcttcag 1020

ggtttttctc tttctttttt tttctggttt ggcaaacggt tgggttcaca cactgtgaca 1080

tcgacggtgc ccctctactt cttccttttt aggaggaagc tgtcgttatt cttcttgtct 1140

cgttggtgac ccagtctgac agtgcggtct aaccagaatt gagttttgtt aagagtctac 1200

tagtcgtcta cttttttttt ttttggcttg gacttcctca aaatcgttaa tttttttcta 1260

cttactaatg ggtgagttgt ttgttgttgt ttttcagaac cctctgtgcc cctggacccc 1320

atatcctcgt caaactcctc atctcagatc attctgaagt ggaagcctcc taacgatcca 1380

aacggaaaca tcacacacta cttagtcttc tgccagcagc aaccagaggc cagcgagctc 1440

tacaagtttg attactgtca gaaaggttag aaaacataaa ggaacataat ataaaacata 1500

cacactaccg ttcagaagtt tggggttggt aagatttgaa atgttatcta tggaagcttg 1560

tttccaccat gaaataaaaa ataaaaaagg taattgcgac tttttatctc acaattctga 1620

cttttttttc tcacaattgt gagttataaa gtcagaattg tgtgatataa agtcagaatt 1680

gggagatata aagtcagaat tgggagatat aaagtcagaa ttgcgagtta taaagtcaga 1740

attgcgagat ataaagtcag aattgggaga tataaagtca gaattgcgag ataaaaagtc 1800

agaattgcga gataaaaacc tttttttaca ggcttttttg tatttttaac tattgataca 1860

tgggtcatag cacctaatta gctggaagtt aggggtgtgc gagacaacga tttttgatcg 1920

tggacgatta aaatgtgaga actttaatac attagcttta ttaagaatca atgtataatt 1980

tatacagaga agtctagtgt tatattttta tatttgttca tttaattttt tacttgaatg 2040

ctactgttaa atacatctac tgtagctgaa aaaataaatc attttattaa tttcatttgt 2100

ataatgtgta gcctatgttt ttcttctttt ttaataacaa agataaaatt aataacaaat 2160

aataacaaat aacataaaaa ataatgacaa agatttttat cttcgccact ttggcctaaa 2220

tgtctgccat ttttaatttt attttatatt ttgttatttt gtaaggcttt gtttttaaaa 2280

tagggaaatt agtttggctg taatgctcag tcacttgcaa aatagtaaaa acaacatgaa 2340

gaatcgtgat aaaaatcgtg aatcgtgatt ttcctgaaaa aaatcgtgat atgatatttt 2400

tgccatatcg cccaccccta ctggaagtgc tttgattgga gagcaaaatc gaagccttat 2460

gtttctgttg ttcaggtatg aagctgccca ccagagctcc cacacaagtg gacaacgaag 2520

aggaacagaa gtggaaccaa accgaggagc aaggccaaga tagccgctgc tgcgcctgcc 2580

ccaaaacaga gaaacagctg aagaaagagg cagaagagac agagtaccgc aaaacctttg 2640

agaactacct ccataatgaa gtctttgagc tcaggtaaat ttcacagttc ctcatttaac 2700

tagtctagat tagaactgca aaaatgaatc atatagactt ttttatttga aagaattttt 2760

catatatagt cagtatatat tgtacattat ataaacttat gcagaccgtt ttctcatagt 2820

ttgaaggaga gcattgtgca agatgtgaaa tatctaatta atatatgaaa taatcaacag 2880

attgatcaat tatcaaaata ggtggagatt ctcaagtggt tcatgttgtt acggtgttca 2940

tctgttttgt ttagagttct aaactcaggg ttgaacgtct tagtagtggt ccgaacactt 3000

tccctcgctg ggttgttttt gactccaaga ccttgagtac atgtttttgt tagttgcatt 3060

tccaccttct tggtgtatct tt 3082

<210> 13

<211> 5333

<212> DNA

<213> insulin receptor

<400> 13

gtggagttga ttcaactcat cgactagcat gtgccgtcat gttaatcttt tgtgcaaatc 60

cagtgttgaa ttgaccctcg tttgtgaagc agtccggcgt aaaatgacag catggcaaca 120

acactctact acaacaactc ttcctcttct ctaaagcagc ccaacatggc ctcaccccct 180

ttgttgcgtg ttctctggcg cagggtttat gtaaattttg gggttagtga tgtcactaac 240

ccgggaagaa gctcgttgta gtccctacca gccgtttgtt gtagtcctta aacagagaat 300

tctttaaaaa aaatatctcc ctttgcattg aactttgagc gtcgtaactt tgcagatgtt 360

gtttatgctc aaacagcaac attacacact aactaaagtt aaaaaagtga aatcaaaatc 420

aaggacccct ttaagactga acctaaatct agactgtctt ccttctcttt acatgtttct 480

gtgtacccag gacatcaaga cagcgcagat ctctggtggg cattgccaat aggacctcct 540

ctcgtctttt cactacaccc tcctcgttgc ccaatggctc tgccactcgt agcccagagg 600

aagaagcaga ggccaataag atttcactga ttgttcgtgc caaggagtcg acagtcatct 660

ccaatctgcg ccatttcacc agctaccaga tagaaatcca tgcctgcaac catgagagtg 720

accccactcg ctgcagtatg gcggcctacg tcagtgcccg caccatgccc gaaggtgtgg 780

gattgtaatt ttataatgca ttatttcata tagtatagtt gtttttattg acatatagtc 840

ttctgttttt gtacagaaaa agctgatgac attgtgggtc cgataaccta tgaggtgtcg 900

gagtactctg tacacatcag gtggatggag cccaaggccc ctaatggcat gattattctc 960

tatgaagtcc attacaagag actgggagat acagaggtga gatgttacac aggatagaga 1020

tggaagcctg cactctgctc ccatgaatgc tggctgcact tttaaactga tttttatcag 1080

ccctgatgaa atctccaaat agctccaaat aaacaagctt ttttaacata attgcttcag 1140

ctgtgtattt ctgtaaccat ggagaaatcc agccagaaac agaggaaatg ttcctaatgc 1200

tgtttttgaa aagcctcgga taaacaaggg atgattatat aaaggttaat aattgttata 1260

agcaaaacaa aatgtgatgg ttttgcactt agcggcacat gcacctgttt gggttgatgg 1320

atttgaaacg ctctgtttac actgacctct agcagtttca ttaaaatctg atgagtaata 1380

tttatatgta ttgttgtgag atggacttta aaaacatgat gataactaag cctttgccac 1440

cgaataatgg ccgtcttgag ttcgatgtgc acagcaatat gggttatcta actgcatgcc 1500

tttatgtcta aggagctgca ccactgcgta tccaggaaga attatattgc agacggaggc 1560

tgtaagctga gagtggtgca ccctgggaac tatacagtgc ggatcagagc aacctccttg 1620

gcaggcaatg gatcgtggac agagccaaca cacttctatg ttcaggacct acgtaagacg 1680

gcttgtctat aatgcctatg aatatcatat cttcagttat taaattatgc atgagacaca 1740

cataactgaa ttttgaagag gtttgtgtgt gtgtgtgtat ctggaaaaaa actaaatatt 1800

cacacactga tactattgag ttatattgaa caatttgaaa gtttcaacct ctacgtctat 1860

agtatggaaa aatggtcaaa atgttgcagt tgttcaatat ggaagcccta agatgccatg 1920

ttgcaattca gtttttttta aaccataaat tggttttgtt ttgtggtcat ttttattgtt 1980

tattattggt taattttgtt gtgggtttta ttttaattat tttttatatt aaaggcacaa 2040

tatgtaagat ttttggatga aaatatccca aaaccactag aacagtgtta tttattttgt 2100

tgacttgtgt acttacacta tcccaaatgt ttccaagaat gtttaaatac agagaaataa 2160

gcaattttaa ccagcgtcgc ctatcaatga catcattacc ctcgatttcc ggttttattt 2220

tgtagaaacc atggaaacac caaagacgct ttaatatagt atgtgtttta ttagacaggt 2280

gagcaactgt ttggatacat tcattgacag agaactaatc atgttatata ggtcaacaca 2340

gtaagtctta ttgtttaaat ctcgttttct tgatttaccg ccatatttta ccatgactaa 2400

tttcgatcta gcttactgca gtgtgtaaca agtgtctcgt agtagctgcc gagcgaatgc 2460

acagagtagc attataacaa ctgtcaacac acaaatgtat ctaatatgat aaacagcgct 2520

gtgttacccc acatactcat gagcggaaga agaagaagcg ttcgtctgtg gcataataaa 2580

tgttccactg ctcttgagcc gtgtgtcgca ctcgtctctc attagcaatt gcttcagcgg 2640

cctcgttccg ttccaatgtc tttcagctcc accctgcttc atactacagt aatgttaata 2700

gttctttaaa acattagctc atccatgaat atgatttctg catgagtccc attggattcg 2760

attccattgg ctgtagatgt gaagactaca cctcccatgc gtcattaaat tacgcctttg 2820

ttatgaatta gtgacctcta gtggtgaaaa attacatatt gtgcctttaa ttgatttttt 2880

ttattccttg gcattttaat gcttcctgtt aatatcagta tatagagaat attttttatt 2940

taatctatta agtattattt gctccattgt gtcctgcacc tgtatacaac ttagtttgga 3000

agtgcacaac ttttatatat tttttggttg tatctatgtg aacaggtgtg gacccatcca 3060

acatgctgaa gattgtgatt ggtccggtga tctgtgtctt tttactcctt gtgatgggag 3120

gtgtgggctt tttcatgttc aagaaaaagt aagtgttgtg attgtcctga aaactcaagc 3180

ataaaactat tctttatgag gtaagtaacg aatttcatta atttttgacc aggcaaacag 3240

aaggtccaac tgggcgactt tatacctccc caaatccaga gtatttcagc ccagatgagg 3300

gtgagcaact ttcctttccc tgcgtgtgtg acctttagca gaaaatgctg gggtcgatgc 3360

acatttaatt tagttctaat agaagagttg atcacattaa cgatcgattt aagcagatta 3420

tcacatttaa aatgactttc tcttttgcct gagatctgct ccatctcaaa tatgacactg 3480

tgtgtcctgc atgtgtgttg tgtttgtgca gtgtatgagc cagacgagtg ggaggtgccc 3540

cgtgaaaaga ttaacctatt gcgagagtta ggccagggat ctttcggcat ggtgtatgag 3600

ggcatcggaa aggacatagt caaaggcgag ccacagaccc gtgttgcagt gaagacggtt 3660

aacgagtcgg ccagcctcag agagaggatc gagttcctca acgaagcttc tgtgatgaag 3720

gcgttcagct gccaccatgt ggtaagacat gtctcttaca tatcagccta tgtgttttgt 3780

agtcttattt tagtatttgt ttatatattg tttattatta ttttgaataa gcttttattt 3840

tatattttca tttaaatgtt taattttagt agtttgtgct tttatctttt tttttctttt 3900

ttttttttaa tatttccaat ttaattttat atctgtttta attttagaat ttattaattt 3960

cagttagtta ccaaggcaac atttctttta atctaatatt tatatataga cactactgct 4020

caaaagtttg gaataattaa gatttttaat cttttttgaa agatgtctca ccatcgctgc 4080

atttatttga tctaaagtac agtaaaaaca gtaatactgt gaaatgttat tacaatttaa 4140

aataacagtt ttcggtttga atatatttta aaatgtgatt tattcctgta atataaaagc 4200

taaattttca gcatcattaa tccattcttc agtgtcacat gatccttcaa aaatcattct 4260

gatattctga tttggttgtc agtcattatc aattattatt attaattatt attggtgctc 4320

aaatattgat aatggtcctt attatgagtg ttgaaaagtt tttgctgctt aatatttttg 4380

tggaaaccaa ggaatacatt tttcaggatt cttatatgaa ttgaaagttt aaaagaatag 4440

aattaattta aaatagaaat cttttgtaac attataaatg tctttactgt cagttttgaa 4500

tgacagtgta tcacggtttc cacaaaacta ttcgaactat tcaacatata ctttttaggt 4560

tttattttaa ttactgagaa tgattttgat atttttaggt ttagttgtag ttaataataa 4620

cactggttgg actgtggcat attctgatgg taactgacat gatcttgtgg gttaggtgag 4680

gctgctgggg gtggtgtcta aaggccagcc caccctagtg gtgatggagc tcatgacaca 4740

tggagatctg aagagtttcc tgcgttctct cagacctgac tcagaggtaa caaatcagaa 4800

gtgtcactat cagacctgct agagttttta gatactgtat gtatatatat atatatatat 4860

atatatatat atatatatat atataaaaaa taatgtattt ttgttttact gtgttgtttg 4920

attgctttgt tacagaataa cccaggtcgg cctcctccaa cgctgaaaga gatgattcaa 4980

atggccgcag agatagcaga tggcatggct tacttgaacg ccaagaagtt tgtgcaccga 5040

gaccttgctg ccaggaactg catggtggcc gaagacctga ccgtaaaaat tggaggtaca 5100

ttactagtga cgggtgtaca tttgtttatt taagcttaaa ggattagtcc actttcaaat 5160

aaaaatttcc tgataattta ctcaccccca tatcatccaa gatgtccatg tccttctttc 5220

ttcagtccaa aagaaattaa ggtttttgat gaaaacattc caggattatt ctccttatag 5280

tggacttcaa tggtctccaa acggttgaag gtcaaaatta cagtttcagt gca 5333

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence

<400> 14

cttaggaata gttcagaaat cagt 24

<210> 15

<211> 18

<212> DNA

<213> Artificial sequence

<400> 15

atgccaagac gcaggaaa 18

<210> 16

<211> 24

<212> DNA

<213> Artificial sequence

<400> 16

tgtgactgga aatcagggtt attc 24

<210> 17

<211> 24

<212> DNA

<213> Artificial sequence

<400> 17

ttctttgcca ccatttgatg taaa 24

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence

<400> 18

tgagaaggga aaaggaagga atag 24

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence

<400> 19

tgacatccca aatggataaa gcca 24

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence

<400> 20

gtcttgtttg tcaaccataa cgat 24

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence

<400> 21

gcaaatgttg ctgttgactt t 21

<210> 22

<211> 24

<212> DNA

<213> Artificial sequence

<400> 22

caaagaacag ttacgctaat agtt 24

<210> 23

<211> 24

<212> DNA

<213> Artificial sequence

<400> 23

tgtaattasg tttttatgat gact 24

<210> 24

<211> 24

<212> DNA

<213> Artificial sequence

<400> 24

tgactagctg taatttgtgt ttag 24

<210> 25

<211> 23

<212> DNA

<213> Artificial sequence

<400> 25

gggttggtaa gatttgaaat gtt 23

<210> 26

<211> 21

<212> DNA

<213> Artificial sequence

<400> 26

tgctatgacc catgtatcaa t 21

<210> 27

<211> 21

<212> DNA

<213> Artificial sequence

<400> 27

attttaacca gcgtcgccta t 21

<210> 28

<211> 23

<212> DNA

<213> Artificial sequence

<400> 28

gcggtaaatc aagaaaacga gat 23

<210> 29

<211> 24

<212> DNA

<213> Artificial sequence

<400> 29

ataaaaccgg aaatcgaggg taat 24

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence

<400> 30

gatttaccgc catattttac 20

<210> 31

<211> 18

<212> DNA

<213> Artificial sequence

<400> 31

tgtgttgaca gttgttat 18

<210> 32

<211> 24

<212> DNA

<213> Artificial sequence

<400> 32

ttcgatctag cttactgcag tgtg 24

<210> 33

<211> 24

<212> DNA

<213> Artificial sequence

<400> 33

atgctactct gtgcattcgc tcgg 24

<210> 34

<211> 24

<212> DNA

<213> Artificial sequence

<400> 34

gtgttgtgat tgtcctgaaa actc 24

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence

<400> 35

ccttctgttt gcctggtcaa 20

<210> 36

<211> 24

<212> DNA

<213> Artificial sequence

<400> 36

aaattcgtta cttacctcat aaag 24

<210> 37

<211> 22

<212> DNA

<213> Artificial sequence

<400> 37

tcagaattgt gtgattaaaa ac 22

Claims

1. The grass carp insulin receptor alpha subtype molecular marker combination is characterized in that the grass carp insulin receptor alpha subtype molecular marker combination is covered in a grass carp insulin receptor alpha subtype genome;

the genome of the grass carp insulin receptor alpha subtype preferably comprises a sequence shown in SEQ ID NO.1 and/or SEQ ID NO.2 and/or SEQ ID NO. 3.

2. The grass carp insulin receptor alpha-subtype molecular marker combination according to claim 1, wherein the grass carp insulin receptor alpha-subtype molecular marker combination comprises: a SNP and at least one of an insertion and a deletion.

3. The grass carp insulin receptor alpha subtype molecular marker combination according to claim 2, wherein the SNP comprises at least one of (1) to (10):

(1) the alpha subtype 5 intron s.588G > A of the grass carp insulin receptor;

(2) the s.64C > T intron 6 of grass carp insulin receptor alpha subtype;

(3) the s.114A > T intron 6 of grass carp insulin receptor alpha subtype;

(5) the s.170G > A intron 8 of grass carp insulin receptor alpha subtype;

4. The grass carp insulin receptor alpha subtype molecular marker combination according to claim 2, wherein the insertions and deletions are: the s.209 located in the 10 th intron of the grass carp insulin receptor alpha subtype is mutated into TCAGAATTGTGTGATTAAAAAC or the base is deleted.

5. The use of the molecular marker combination of grass carp insulin receptor alpha subtype according to any one of claims 1 to 4 in the following (1) to (5):

(1) the application in grass carp variety screening;

(2) the application in grass carp variety identification;

(3) the application in grass carp breeding;

(4) the application in germplasm resource protection;

(5) application in germplasm resource improvement.

6. A grass carp growth performance detection reagent, which is characterized by comprising a substance for detecting the grass carp insulin receptor alpha subtype molecular marker combination according to any one of claims 1 to 4;

the assays include both gene-level and protein-level assays.

7. The reagent for detecting grass carp growth performance according to claim 6, wherein the substance is a primer and/or a probe for detecting the molecular marker combination of grass carp insulin receptor alpha subtype according to any one of claims 1 to 4.

8. The grass carp growth performance detection reagent according to claim 7, wherein the nucleotide sequence of the primer is shown in SEQ ID No. 1-10.

9. A method for screening high-quality grass carp growth seeds comprises the following steps:

detecting the genotype of the locus where the grass carp insulin receptor alpha subtype molecular marker combination is located according to any one of claims 1 to 4, and judging whether a grass carp sample is a high-quality growing species or not according to the combination condition of the genotypes;

the molecular marker combination of grass carp insulin receptor alpha subtype is preferably (1) to (4) in claim 3.

10. The use of the reagent for measuring the growth performance of grass carp as defined in any one of claims 6 to 8 in the preparation of a test product for measuring the growth performance of grass carp;

the product preferably comprises a detection kit, a gene chip and a test strip.