CN112795661B - Microsatellite marker, primer, method and application related to fugu obscurus growth traits - Google Patents

Abstract

The invention discloses a microsatellite marker, primers, a method and application related to the growth traits of fugu obscurus, wherein the microsatellite marker comprises one or more of XH11, XH47, XH63, XH94 and XH96, and the primers for amplifying the microsatellite marker related to the growth traits of fugu obscurus comprise 5 pairs of specific microsatellite primers shown as SEQ ID NO. 1-10. According to the method, the microsatellite primers and a plurality of growth traits of the takifugu obscurus are subjected to correlation analysis, so that 5 microsatellite markers (XH 11, XH47, XH63, XH94 and XH 96) which are obviously related to the growth traits are screened out. The advantages and the inferior genotypes of different microsatellite loci screened by the method can effectively assist the breeding of good parents of the fugu obscurus and accelerate the breeding efficiency, and the method has accurate results, is simple and practical.

Description

Microsatellite marker, primer, method and application related to growth traits of fugu obscurus

Technical Field

The invention belongs to the technical field of aquaculture, and particularly relates to a microsatellite marker, a primer, a method and application related to the growth traits of takifugu obscurus.

Background

Takifugu obscurus (Takifugu fasciatus) is commonly called puffer, belongs to Osteichthyes, actinopterygii, fugu morphes (Letrodontiforms), takifudae (Tetraodontidae) and Takifugu eastern (Takifugu), and is mainly distributed in yellow sea, bohai sea, offshore sea area and rivers and lakes in China. The fugu obscurus meat-quality fresh and tender food is good, has high nutritional value, is deeply popular with consumers, and is an aquaculture fish with higher economic, nutritional and culture values.

The body weight and body length of the female and male fugu obscurus of 3-4 years old are obviously less than those of the cultured population. In the culture process, the situation that the difference of growth traits of the male and female genders of the same Fugu obscurus population is obvious also commonly exists. Many small individuals are usually eliminated in one breeding period, and the takifugu obscurus has the characteristic of mutual cannibalism, so that the small individuals are easy to be injured and have high death rate, and the small individuals can be injured to infect other fish bodies. Therefore, the economic traits of fish can be genetically improved by screening genes or markers that control growth traits.

Microsatellites (also known as Simple Sequence Repeats) or Short Sequence Tandem Repeats (STRs) are generally formed by 1-6 base Tandem Repeats, widely exist in genomes of eukaryotes and prokaryotes, and are an excellent second-generation molecular marking technology. The method is widely applied to researches such as population genetic diversity analysis, genetic relationship identification, sex marker screening, genetic map construction and the like. Therefore, the research on the difference of the growth traits of the fugu obscurus by using the microsatellite marker not only provides conditions for the breeding of improved varieties of the fugu obscurus, but also has profound significance for improving the culture yield and the economic benefit of the fugu obscurus.

Due to the lack of genome and microsatellite sequences of the takifugu obscurus and other fugu fish of the family of the globefish, many researchers can only use the takifugu rubripes microsatellite design primer to carry out related researches on the function marker screening of related fugu rubripes, the genetic information evaluation of different groups and the like, which hinders the progress of various researches on the fugu rubripes to a certain extent.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a microsatellite marker related to the growth performance of the Fugu obscurus, and the molecular marker is a molecular marker assisted breeding basis of the Fugu obscurus.

The invention also provides 5 pairs of specific primers, a kit and application for amplifying the microsatellite marker related to the growth characteristics of the fugu obscurus.

The technical scheme is as follows: in order to achieve the aim, the microsatellite markers related to the growth traits of the takifugu obscurus comprise one or more of microsatellite markers XH11, XH47, XH63, XH94 and XH 96.

Wherein said XH11 locus is significantly associated with the body height, body width, body weight and body length of Takifugu obscurus (P < 0.05); the XH47 site was significantly associated with the body length of Takifugu obscurus (P < 0.05); the XH63 site is significantly related to the full length, caudal stalk length and body weight of Fugu obscurus (P < 0.05), and extremely significant related to head length and trunk length (P < 0.01); the XH94 site was strongly associated with the weight of Fugu obscurus (P < 0.01); the XH96 site is significantly related to the head length, tail stalk length and body length of Fugu obscurus (P < 0.05), and is significantly related to the total length, body height, eye distance and body weight (P < 0.01).

The invention is used for amplifying the primers of the microsatellite markers related to the growth traits of the takifugu obscurus, and the primers comprise 5 pairs of specific microsatellite primers which are respectively as follows:

the forward primer for amplifying XH11 is shown in SEQ ID NO 1 (5'-TGTAAAACGACGGCCAGTAGTCTGGGCTTCTCTTCTTAGGCT-3') and the reverse primer is shown in SEQ ID NO 2 (5'-GAGCAAACCAGAAAGGGAGGTTGA-3');

the forward primer for amplifying XH47 is shown as SEQ ID NO 3 (TGTAAAACGACGGCCAGTCGGCGGACTATTAACGATGGAGTC-3 '), and the reverse primer is shown as SEQ ID NO 4 (5'-CGACAAACATGCGCCGTTCATAAA-3');

the forward primer for amplifying XH63 is shown in SEQ ID NO. 5 (5'-TGTAAAACGACGGCCAGTTAGCCACATGTCCCTCGTCTGTAA-3') and the reverse primer is shown in SEQ ID NO. 6 (5'-CCACAACACAGGTCCTGACAGTAA-3');

the forward primer used for amplifying XH94 is shown as SEQ ID NO. 7 (5'-TGTAAAACGACGGCCAGTGTGTCACAACTGGAATGGCAGGTA-3') and the reverse primer is shown as SEQ ID NO. 8 (5'-GTGCTCAAGTGTGCCTTCAACAC-3');

the forward primer used for amplification of XH96 is shown in SEQ ID NO 9 (5'-TGTAAAACGACGGCCAGTCGCAGCGGGGCCAATATTAAATTA-3') and the reverse primer is shown in SEQ ID NO 10 (5'-TATGCTGGTTCCCATACGGATCAC-3').

The microsatellite markers related to the growth characteristics of the fugu obscurus comprise microsatellite markers XH11, XH47, XH63, XH94 and XH96, and the 5 pairs of microsatellite primers are shown as SEQ ID: 1-10, wherein the primer SEQ ID NO:1 and SEQ ID NO:2, the repetitive motif of the amplified XH11 microsatellite fragment is (TGGA) n, the value range of n is 38-42, and the XH11 microsatellite fragment is associated with the body height, the body width, the body weight and the body length of the takifugu obscurus; primers SEQ ID NO:3 and SEQ ID NO:4, the repetitive motif of the amplified XH47 microsatellite fragment is (AAAC) n, the value range of n is 37 to 39, and the XH47 microsatellite fragment is associated with the body length of the takifugu obscurus; primers SEQ ID NO:5 and SEQ ID NO:6, the repetitive motif of the amplified XH63 microsatellite fragment is (CA) n, the value range of n is 68-93, and the XH63 microsatellite fragment is related to the full length, the caudal peduncle length, the weight, the head length and the trunk length of the fugu obscurus; primers SEQ ID NO:7 and SEQ ID NO:8, the repetitive motif of the amplified XH94 microsatellite fragments is (GT) n, the value range of n is 79-85, and the XH94 microsatellite fragments are related to the weight of the fugu obscurus; primers SEQ ID NO:9 and SEQ ID NO: the repetitive motif of the amplified XH96 microsatellite fragment 10 is (TG) n, the value range of n is 83-98, and the XH96 microsatellite fragment Wei Xingpian segment is associated with the head length, tail stalk length, body length, full length, body height, eye space and body weight of the takifugu obscurus.

When a sample to be detected is amplified, if an AC (152bp, 156bp) genotype (wherein (152bp, 156bp) represents the size of an allele) is amplified by adopting a microsatellite marker XH11 primer, the sample to be detected is a strain which is associated with the genotype AC and has poor body height, body width, body weight and body length; if the genotype AB (136bp, 146bp) is amplified by adopting the microsatellite marker XH63 primer, the sample to be detected is a strain with better overall length, tail stalk length, body weight, head length and trunk length which are associated with the genotype AB; if the CC (160bp ) genotype is amplified by adopting the microsatellite marker XH94 primer, the sample to be detected is a better quality line which is related to the genotype CC; if the BC (174bp, 178bp) and CD (166bp, 178bp) genotypes are amplified by adopting the microsatellite marker XH96 primer, the sample to be detected is a strain with superior head length, tail stalk length, body length, full length, body height, eye spacing and body weight related to the BC and CD genotypes.

The kit for amplifying the microsatellite marker related to the growth traits of the takifugu obscurus comprises the specific primer.

The microsatellite marker, the primer or the kit disclosed by the invention are applied to analysis of the growth traits of takifugu obscurus.

The microsatellite marker, the primer or the kit disclosed by the invention are applied to the Takifugu obscurus molecular breeding.

The invention relates to a whole genome microsatellite marking method related to the growth characteristics of Fugu obscurus, which is used for the association analysis of the growth characteristics of the Fugu obscurus and the molecular assisted breeding of the Fugu obscurus and comprises the following steps:

(a) Developing the takifugu obscurus whole genome microsatellite primers in batches;

(b) Extracting individual genome DNA of the takifugu obscurus;

(c) Using the DNA as a template, and adopting screened polymorphic microsatellite primers (such as SEQ ID NO. 1-10) to carry out PCR amplification respectively;

(d) Loading the PCR amplification product into an ABI analyzer for genotype analysis;

(e) And (3) analyzing the correlation between the microsatellite locus genotype which has obvious difference in the extremely large and small groups and a plurality of growth traits of the takifugu obscurus.

Preferably, the whole genome microsatellite marking method related to the growth characteristics of the takifugu obscurus comprises the following steps: (a) Screening all microsatellite locus sequences in the fugu obscurus whole genome by using microsatellite screening software MISA, and designing fugu obscurus whole genome microsatellite primers in batches by using Primer design software Primer 5; (b) extracting the individual genome DNA of the takifugu obscurus; (c) Amplifying 70 randomly selected microsatellite primers by taking the DNA as a template, and checking the quality and polymorphism of the primers; (d) Respectively carrying out PCR amplification on 12 individuals of a 'maximum population' and a 'minimum population' of the fugu obscurus by using screened 42 pairs of microsatellite primers, loading an amplification product into an ABI analyzer for genotype analysis, and carrying out difference analysis on 12 individuals of the maximum population and the minimum population of each microsatellite locus by using chi-square test; (e) Random population validation analysis was performed on 7 microsatellite loci that were significantly different in the two populations. The association of the fugu obscurus growth traits and 7 microsatellite loci is subjected to least square analysis by utilizing GLM (general linear model) in SPSS, and the significance of the growth trait index difference between the same marker genotypes is tested and subjected to multiple comparison. And (3) screening the microsatellite loci related to the growth traits of the fugu obscurus, namely the microsatellite markers related to the growth traits of the fugu obscurus.

The invention utilizes the characteristics of microsatellite marker polymorphism and codominance, firstly develops microsatellite primers in batches from the whole genome of the fugu obscurus, amplifies the microsatellite primers with excellent quality and polymorphism obtained by screening in the fugu obscurus individuals, analyzes the distribution difference of the genotypes of the microsatellite loci in the maximum population and the minimum population by using chi-square test, and selects the microsatellite loci with obvious difference to perform correlation analysis between the genotypes and a plurality of growth traits of the random population of the fugu obscurus. The invention utilizes the microsatellite marker which is obtained by screening and is related to the growth characteristics of the fugu obscurus to reserve fugu obscurus individuals with dominant genotypes in production and eliminate the individuals with inferior genotypes. Individuals with a plurality of dominant genotypes are selected as parents, and excellent germplasm is selected for breeding, so that the excellent germplasm of offspring is ensured, the breeding efficiency can be effectively accelerated, various growth indexes of the offspring of the takifugu obscurus are improved, and the economic benefit of the breeding industry is improved. The method has accurate result, and is simple and practical.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. all the microsatellite primers used in the method are microsatellite primers developed in batch from the whole genome of the fugu obscurus, all microsatellite loci in the whole genome are covered, and compared with microsatellite markers obtained by other microsatellite development methods, the microsatellite markers obtained by the method are more random, more comprehensive and richer.

2. The 5 pairs of microsatellite marked primers provided by the invention have the characteristics of stable PCR amplification result and high polymorphism, and the microsatellite marks can be effectively used for analysis of the growth traits of the fugu obscurus and auxiliary breeding of fugu obscurus molecular markers.

3. The dominant and the disadvantaged genotypes of different microsatellite markers screened by the invention can be utilized to retain takifugu obscurus individuals with the dominant genotypes in production and eliminate the individuals with the disadvantaged genotypes. Individuals with a plurality of dominant genotypes are selected as parents, and excellent germplasm is selected for breeding purposefully, so that the excellent quality of offspring is ensured, the breeding efficiency can be effectively accelerated, various growth indexes of the offspring of the takifugu obscurus are improved, and the economic benefit of the breeding industry is improved. The method has accurate result, and is simple and practical.

Drawings

FIG. 1 is the result of typing of a disadvantaged genotype AC for an XH11 microsatellite locus;

FIG. 2 is the result of typing of the dominant genotype AB at the XH63 microsatellite locus;

FIG. 3 is the result of typing of the disadvantaged genotype BB of XH63 microsatellite loci;

FIG. 4 is the result of typing the dominant genotype CC for the XH94 microsatellite locus;

FIG. 5 is the result of typing the dominant genotype BC for the XH96 microsatellite locus;

FIG. 6 is the result of typing of dominant genotype CD of XH96 microsatellite loci;

FIG. 7 is the results of typing of the disadvantaged genotype AB of XH96 microsatellite locus;

FIG. 8 is the result of typing of a disadvantaged genotype CC for the XH96 microsatellite locus;

FIG. 9 shows the results of typing of the disadvantaged genotype BG for the XH96 microsatellite locus.

Detailed Description

The invention is further illustrated by the following figures and examples.

Example 1

Screening of microsatellite marker related to growth traits of fugu obscurus

(1) Sources of materials

300 pieces of Takifugu obscurus (cultivated in the same cultivation pond) of about 5 months old from Shengang Sanxian cultivation Co., ltd in Jiangyin city are selected and respectively subjected to measurement and recording of 9 pieces of growth character data of total length, body height, body width, head length, eye spacing, trunk length, tail stalk length, body weight and body length. The SPSS software is utilized to establish 9 normal distribution graphs of growth traits for the 300 takifugu obscurus, and the results are all in accordance with the normal distribution. Taking 16% of high-value individuals (48 individuals), namely, individuals with the weight of more than 42g as an extremely large population, taking 16% of low-value individuals (48 individuals), namely, individuals with the weight of less than 26g as an extremely small population, and randomly selecting 12 individuals from the extremely large and extremely small populations for preliminary screening of microsatellite loci related to the growth of the takifugu obscurus. Then, randomly selecting 96 individuals from the culture ponds of the remaining 204 individuals for growth trait association analysis, and recording the takifugu obscurus as a random population.

(2) Extraction of fugu obscurus genome DNA

10-30 mg of tail fin is taken from each takifugu obscurus, and a DNA extraction kit (purchased from Beijing Baitaike biology company) is utilized to extract genome DNA.

(3) Screening for microsatellite loci

And (3) screening microsatellite loci in the fugu obscurus whole genome by using microsatellite screening software MISA, and designing microsatellite primers in the fugu obscurus whole genome in batches by using microsatellite position information (MISA files) obtained by using Primer design software Primer 5 and MISA software and combining scripts. Randomly selecting 70 pairs of microsatellite primers of 2-4 base types for screening and testing microsatellite markers related to the growth traits of the fugu obscurus.

(4) Microsatellite PCR amplification

And (3) carrying out PCR amplification by using the DNA of the takifugu obscurus extracted in the step (2) and adopting a microsatellite system, and carrying out genotype detection on a PCR product on an ABI analyzer. The specific process is as follows:

1) Design and Synthesis of microsatellite primers

In order to realize the fluorescent semi-automatic detection of the microsatellite PCR product, an 18bp fluorescent modification group mark is added to the 5 'end or the 3' end of a common primer during primer design. Prior to fluorescent primer synthesis, PCR amplification was performed with common primers to verify the quality of the primer amplification. And subsequently adding 18bp fluorescent fragments to the microsatellite primer forward primer capable of being stably amplified, and adding a fluorescent reagent in an amplification system. The 4 commonly used fluorescent modifying groups are FAM (green), HEX (blue), TAMRA (yellow) and ROX (red), respectively. The agarose gel electrophoresis result shows that 42 of the 70 pairs of primers can be successfully amplified and have polymorphism, and can be used for subsequent microsatellite marker screening experiments related to the growth traits of the takifugu obscurus.

2) Microsatellite PCR amplification system

The microsatellite PCR amplification reaction system (10. Mu.L) was as follows: sterilized water (3.64. Mu.L), 2 XTaq Plus Mix (5. Mu.L) (purchased from Shanghai Probiotics technology Co., ltd.), 10. Mu.M forward primer (0.04. Mu.L) with an 18bp addition fluorescent fragment, reverse primer (0.16. Mu.L), FAM/HEX/TAMRA/ROX (0.16. Mu.L), takifugu obscurus tail fin DNA (1. Mu.L).

3) Microsatellite PCR amplification reaction

Pre-denaturing at 95 ℃ for 5min on a PCR amplification instrument; 40 cycles: denaturation at 94 ℃ 30sec, annealing at 59 ℃ 30sec, extension at 72 ℃ 30sec; finally, the product is extended at 72 ℃ to 7min and stored at 4 ℃.

(5) Data statistics and analysis

And (4) counting the genotype size and frequency of each microsatellite locus by using GeneMarker software. Differential analysis was performed on 12 individual genotypes for each microsatellite locus in the maximal and minimal populations using the chi-square test in the SPSS software. Analysis showed that 7 of the 42 microsatellite loci had a genotype distribution that was nearly significant in the extremely large and small cohorts (P < 0.1). Wherein the loci XH11, XH47 and XH96 were significantly different in genotype distribution in both populations (P < 0.05), and the XH55, XH63, XH93 and XH94 differences were approximately significant (0.05P-bags 0.1). Analytical results table 1 is as follows:

TABLE 1 42 pairs of microsatellite primer information

* Indicating that the microsatellite locus P <0.1.

Example 2

Application of microsatellite molecular marker obviously related to growth traits of fugu obscurus in molecular breeding

The 7 microsatellite loci used in example 1 were used for the next validation analysis of the random population. The least square analysis is carried out on the relevance of the fugu obscurus growth character and 7 microsatellite loci by utilizing GLM (general linear model) in SPSS, and the significance of the growth character index difference between the same marker genotype is tested and multiple comparisons are carried out. Genotypes with less than 3 observations were not statistically analyzed here due to lack of analytical value.

(1) Correlation analysis between 7 microsatellite loci and 9 growth traits of Fugu obscurus

Performing relevance analysis on the microsatellite loci and 9 growth traits of the takifugu obscurus by using a least square method, wherein the XH11 loci are obviously related to the height, the width, the weight and the length of the body (P is less than 0.05); the XH47 site is significantly associated with body length (P < 0.05); the XH63 site was significantly associated with full length, caudal stalk length and body weight (P < 0.05), with head length, trunk length (P < 0.01); XH94 sites were very significantly associated with body weight (P < 0.01); the XH96 site was significantly associated with head length, tail stalk length and body length (P < 0.05), with full length, body height, inter-ocular distance and body weight (P < 0.01), with the results shown in table 2.

TABLE 2 Association analysis between 7 microsatellite loci and 9 growth traits of Fugu obscurus

Note: the values in the table are the probability values for the association analysis of 9 growth traits with microsatellite loci. The no-superscript data indicates that the trait was not associated with the microsatellite marker (P > 0.05), "indicates that the trait was significantly associated with the microsatellite marker (P < 0.05) and" "indicates that the trait was significantly associated with the microsatellite marker (P < 0.01).

(2) Multiple comparisons of growth traits between different genotypes were performed for the 5 microsatellite loci with significant differences (XH 11, XH47, XH63, XH94 and XH 96) and the results are shown in tables 3 and 4.

The growth trait indicator difference significance between the same marker genotypes was examined and multiple comparisons were made using multivariate analysis in the SPSS software GLM (general linear model). Analysis shows that the mean value of the AC genotype (152, 156bp) (shown in figure 1) in the XH11 locus is lower than that of other three genotypes except the body length, the mean value of the body length, the body weight, the head length and the eye distance traits of the AC genotype is obviously lower than that of the genotype AA individual (P < 0.05), and the mean value of the body height and the body width traits is extremely obviously lower than that of the genotype AA individual (P < 0.01); the mean values of body height and length traits are significantly lower than those of genotype AB individuals (P < 0.05), and the mean values of body width and weight traits are significantly lower than those of genotype AB individuals (P < 0.01). Indicating that the AC genotype is a disadvantaged genotype in the XH11 locus.

The mean body length of the BC genotype in the locus XH47 is higher than the other three genotypes. Wherein the mean length of the BC genotype is significantly higher than AB and BB. Other genotypes had no significant difference in the comparison of the mean values of different traits.

The AB genotype (136,146bp) in site XH63 (as in FIG. 2) averaged higher in all growth traits than the other four genotypes. In the full-length character, the AB genotype mean value is obviously higher than AA, BB and CC genotypes, and is extremely obviously higher than AC genotype; in the head length trait, the AB genotype mean is significantly higher than the CC genotype, and very significantly higher than the BB and AC genotypes. The AA genotype mean value is obviously higher than that of a BB genotype and is extremely obviously higher than that of an AC genotype; in the trunk length trait, the mean value of AB genotypes is obviously higher than CC and AC genotypes, and the genotypes AB and AA are obviously higher than BB genotypes; in the caudal peduncle length trait, the mean value of the BB genotype (146,146bp) (as in fig. 3) is significantly lower than the AB genotype, and extremely significantly lower than the AA genotype; in the body weight traits, the AB genotype mean value is obviously higher than AA, CC and AC genotypes, and is extremely obviously higher than BB genotype; in the body length trait, the AB genotype mean is significantly higher than the BB, CC and AC genotypes. The AB genotype is shown as the XH63 locus dominant genotype, and BB is the disadvantaged genotype.

In addition to trunk length, CC genotypes (160,160bp) in site XH94 (as shown in FIG. 4) averaged over the other eight genotypes for all other growth traits. In the high trait, the CC genotype is significantly higher than the AB, BD and AC genotypes, and is significantly higher than the CD, AD and DD genotypes; in the body width trait, the CC genotype is significantly higher than the AD, BD and AC genotypes, and very significantly higher than the DD genotype. The BB genotype is obviously higher than the DD and AC genotypes; in the body length trait, the CC genotype is significantly higher than the CD, BD and AC genotypes; in the body weight traits, the CC genotype is obviously higher than AA and AB genotypes, and is extremely obviously higher than CD, AD, BD, DD and AC genotypes; in the head length trait, the CC genotype is significantly higher than the AA, CD and AB genotypes, and very significantly higher than the AC genotype. The AC genotype is significantly lower than the BB and AD genotypes; in the caudal peduncle length trait, the BD genotype is significantly lower than the CC and DD genotypes; in the interocular distance trait, the CC genotype is significantly higher than BB, AD and BD genotypes, and is significantly higher than DD genotype; in the full-length trait, the CD genotype is significantly lower than the BB, CC and DD genotypes. The AC genotype is significantly lower than the CC and DD genotypes. The CC genotype is shown as the XH94 locus dominant genotype.

Of the 11 genotypes at the XH96 locus, most of the growth traits of the BC genotype (174, 178bp) (shown in FIG. 5) and the CD genotype (166, 178bp) (shown in FIG. 6) were higher in mean value than those of the other 9 genotypes. In the full-length character, the BC genotype is obviously higher than AA, BB, BG and AD genotypes, and is extremely obviously higher than AB and DF genotypes. The CD genotype is obviously higher than AA, DD and AE genotypes, and is extremely obviously higher than BB, AB, DF, BG and AD genotypes. The AB genotype is significantly lower than the CC and DD genotypes; in the high trait, the BC genotype is significantly higher than the DD and AE genotypes, and is significantly higher than the BB, CC, AB and BG genotypes. The CD genotype is obviously higher than AE and DF genotypes, and is extremely obviously higher than DD, BB, CC, AB, BG and genotypes. The AB genotype is significantly lower than AA, BC and AD genotypes; in the body width character, the CD genotype is obviously higher than the DD, CC and AB genotypes, and is extremely obviously higher than the BB and BG genotypes; in the long shape, the BC genotype is significantly higher than the AA, DD, BB, CC and BG genotypes, and very significantly higher than the AB and DF genotypes. The genotype of CD is obviously higher than that of DD, BB and CC, and is extremely obviously higher than that of AA, AB, DF and BG. The DF genotype is significantly lower than BB and DD genotypes; in the body weight traits, the BC genotype is significantly higher than the DD, AB, DF and BG genotypes, and is significantly higher than the BB, CC and AD genotypes. The genotype of CD is obviously higher than that of DD, BB and CC, and is extremely obviously higher than that of AA, AB, DF and BG. The CD genotype is obviously higher than other 9 genotypes except for BC;

in the head length trait, the AB genotype (174,190bp) (as in fig. 7) was significantly lower than the DD, BB, AE, CC, DF and BG genotypes, and very significantly lower than the BC and CD genotypes. The BC genotype was significantly higher than the AA, BB and AE genotypes. The CD genotype is obviously higher than AA, DD, AE and BG genotypes, and is extremely obviously higher than BB genotypes; in the tail-stalk length trait, the CC genotype (176,176bp) (as in fig. 8) is significantly lower than the BC, DD and AD genotypes, and very significantly lower than the BB, CD and BG genotypes. The CD genotype is obviously higher than AA, DD and AB genotypes; in the interocular trait, the AA genotype is significantly higher than BB and AD genotypes, and very significantly higher than DD, CC and BG genotypes. The BC genotype is obviously higher than DD and CC, and is extremely obviously higher than BG genotype. The genotype of CD is obviously higher than that of AD, and is extremely obviously higher than that of DD, BB, CC and BG. The BG genotype (164,174bp) (as in fig. 9) is significantly lower than the AE and DF genotypes, and very significantly lower than the AA, BC and CD genotypes. The genotype of BC and CD is shown to be the dominant genotype of XH96 locus, and the genotypes of AB, CC and BG are shown to be the disadvantaged genotypes. The multiple comparative analysis was as follows:

TABLE 3 multiple comparisons of various genotypes at microsatellite loci with Fugu obscurus full length, body height, body width and head length

TABLE 4 multiple comparisons of various genotypes of microsatellite loci with eye spacing, trunk length, tail stalk length, body weight and body length of Takifugu obscurus

Note that the numerical values in the table are the mean value (LSM.) + -standard deviation (SD.) of each trait. In the same column of values, the superscript with the same letter indicates no significant difference between the two genotypes (P > 0.05), different lower case letters indicate significant difference (P < 0.05), and different upper case letters indicate very significant difference (P < 0.01).

The above results indicate that the XH11 site AC is a disadvantaged genotype. The XH63 site AB is the dominant genotype and BB is the disadvantaged genotype. The XH94 site CC is the dominant genotype. Two XH96 loci BC and CD are dominant genotypes, and three of AB, CC and BG are inferior genotypes. In the production process, the takifugu obscurus individuals with dominant genotypes can be retained, and the individuals with inferior genotypes are eliminated. Individuals with a plurality of dominant genotypes are selected as parents, and excellent germplasm is purposefully selected for breeding, so that the excellent germplasm of offspring is ensured, the breeding efficiency can be effectively accelerated, various growth indexes of the offspring of the takifugu obscurus are improved, and the economic benefit of the breeding industry is improved. The method has accurate result, and is simple and practical.

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Claims (7)

1. The application of microsatellite markers related to the growth traits of the takifugu obscurus in the analysis of the growth traits of the takifugu obscurus and the molecular breeding of the takifugu obscurus is disclosed, wherein the microsatellite markers comprise one or more of microsatellite markers XH11, XH47, XH63, XH94 and XH96, XH11 is a microsatellite fragment with a repetitive motif (TGGA) n, and the value range of n is 38 to 42; XH47 is a microsatellite fragment with a repeating motif (AAAC) n, the value range of n is 37 to 39, XH63 is a microsatellite fragment with a repeating motif (CA) n, and the value range of n is 68 to 93; XH94 is a microsatellite fragment with a repeating motif (GT) n, the value range of n is 79 to 85, XH96 is a microsatellite fragment with a repeating motif (TG) n, and the value range of n is 83 to 98.

2. The use of claim 1, wherein the XH11 site is significantly associated with the body height, body width, body weight and body length of Takifugu obscurus; said XH47 locus is significantly associated with the body length of Fugu obscurus; the XH63 site is significantly associated with the full length, caudal stalk length and body weight of Takifugu obscurus, and with a very significant association of head length and trunk length; the XH94 site was associated with a very significant body weight in Fugu obscurus; the XH96 site is significantly associated with the head length, tail stalk length and body length of Takifugu obscurus, and is highly significant in relation to full length, body height, inter-ocular distance and body weight.

3. Primers for amplifying the microsatellite markers related to the growth traits of takifugu obscurus of claim 1, comprising 5 pairs of specific microsatellite primers:

the forward primer for amplifying XH11 is shown in SEQ ID NO 1 and the reverse primer is shown in SEQ ID NO 2;

the forward primer for amplifying XH47 is shown as SEQ ID No. 3, and the reverse primer is shown as SEQ ID No. 4;

the forward primer for amplifying XH63 is shown in SEQ ID NO. 5 and the reverse primer is shown in SEQ ID NO. 6;

the forward primer for amplifying XH94 is shown as SEQ ID NO. 7 and the reverse primer is shown as SEQ ID NO. 8;

the forward primer for amplification of XH96 is shown in SEQ ID NO 9 and the reverse primer is shown in SEQ ID NO 10.

4. A kit for amplifying the microsatellite marker associated with the growth trait of takifugu obscurus of claim 1 comprising the specific microsatellite primers of claim 3.

5. Use of the primer of claim 3 or the kit of claim 4 in analysis of growth traits of takifugu obscurus.

6. Use of the primer of claim 3 or the kit of claim 4 in the molecular breeding of takifugu obscurus.

7. A microsatellite marking method for association analysis of growth traits of Fugu obscurus and Fugu obscurus molecular assisted breeding by using the primer of claim 3, which comprises the following steps:

(a) Developing the takifugu obscurus whole genome microsatellite primers in batches;

(b) Extracting individual genome DNA of the takifugu obscurus;

(c) Using the DNA as a template and adopting the screened polymorphic microsatellite primers to carry out PCR amplification respectively;

(d) Loading the PCR amplification product into an ABI analyzer for genotype analysis;

(e) And (3) analyzing the correlation between the microsatellite locus genotype which has obvious difference in the extremely large and extremely small groups and a plurality of growth traits of the takifugu obscurus.

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