CN110129455B

CN110129455B - Application of growth-related molecular marker in genetic breeding of litopenaeus vannamei

Info

Publication number: CN110129455B
Application number: CN201910402931.7A
Authority: CN
Inventors: 李富花; 王全超; 于洋; 相建海; 张晓军
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2019-05-15
Filing date: 2019-05-15
Publication date: 2022-05-06
Anticipated expiration: 2039-05-15
Also published as: CN110129455A

Abstract

The invention belongs to the technical field of aquatic product breeding, and particularly relates to a weight-related molecular marker of a Litopenaeus vannamei LvMMD2 gene and application thereof in weight trait breeding. One marker (LvMMD2-132-C/T) selected from the coding region of LvMMD2 gene was significantly correlated with shrimp body weight (P2.42X 10) by methods such as whole genome correlation analysis and candidate gene correlation analysis^‑8). The marker is located at the 132bp position of SEQIDLVMMD2 and is C/T type mutation, and the TT genotype of the marker is a growth dominant genotype. The marker can be used as a molecular marker related to the growth traits to assist the genetic breeding of the growth traits of the prawns.

Description

Application of growth-related molecular marker in genetic breeding of litopenaeus vannamei

Technical Field

The invention belongs to the field of aquatic animal breeding, and particularly relates to a weight-related molecular marker on a Litopenaeus vannamei LvMMD2 gene and application thereof in weight character breeding.

Background

Litopenaeus vannamei (Litopenaeus vannamei), also known as Penaeus vannamei (white prawn), is one of the most important aquaculture species in the world, occupies 76% of the total yield of the world, and the aquaculture area is distributed all over the world. The Litopenaeus vannamei is also the leading variety for the culture of the Litopenaeus vannamei in China, the variety is introduced into China from the United states in 1988, large-scale culture is carried out in China from 1999, the yield can reach about 140 ten thousand tons in 2013 years, and the yield accounts for more than 80% of the total yield of the Litopenaeus vannamei culture in China.

With the continuous expansion of the breeding scale, the demand of the breeding industry for the excellent germplasm of the litopenaeus vannamei is increasingly urgent. When the excellent germplasm of the prawn is cultivated, the growth traits are not only important indexes, but also the most direct breeding targets. However, for a long time, the genetic improvement of the prawns mainly depends on manual selection and a traditional breeding method based on quantitative genetics, and the traditional genetic breeding also has the problems of long breeding period, low selection accuracy and the like, so that the practical requirement of accelerating the genetic improvement progress is difficult to meet. Compared with the traditional genetic breeding, the molecular marker assisted breeding has the characteristics of short breeding interval, high accuracy and reliability and the like, and can obviously accelerate the genetic breeding progress of important economic characters. In (1).

The molecular marker assisted breeding is a technical means for carrying out assisted selection in the process of applying molecular markers to genetic improvement of relevant traits of animals and plants, and the essence is that the molecular markers closely linked with target genes are utilized to carry out target region and whole genome screening on individuals to obtain expected individuals, so that the breeding efficiency and accuracy are improved. Earlier, in prawns, researchers located some growth-related QTL sites by QTL mapping methods, such as found in QTL mapping of Litopenaeus vannamei (Andriantahina F, Liu X, huangg h. genetic map construction and Quality Trail Locations (QTL) detection of growth-related traits in litoae van narrow for selective breeding applications, plos One,2013,8: e75206), but due to lower marker density, no genes or markers directly related to growth were located. The invention aims to provide a marker which is obviously related to growth traits and can be used for auxiliary breeding of the growth traits of litopenaeus vannamei.

Disclosure of Invention

The invention aims to provide a molecular marker related to the growth of litopenaeus vannamei, and the molecular marker is applied to the molecular marker-assisted breeding of fast-growing varieties of the litopenaeus vannamei.

In order to achieve the purpose, the invention adopts the technical scheme that:

a molecular marker related to growth and located on the LvMMD2 gene of Litopenaeus vannamei and its application in genetic breeding of growth traits are characterized in that (1) DNA of parent of prawn is extracted; (2) the following primer LvMMDF was used: ACAATCGCTTCAACCAACCTG and LvMMDR: TCCTGGGCGTGATCTTCTTT amplifying the DNA of the parent material; (3)

sequencing the amplified fragment by using LvMMDF primer to obtain a sequence shown as SEQIDLLvMMD 2, and analyzing the genotype of SNP site (LvMMD2-132-C/T) at 132bp position, wherein if the LvMMD2-132-C/T site in a sequencing peak map has only C peak, the individual genotype is CC, if C and T double peaks, the individual genotype is CT, and if only T peak, the individual genotype is TT. (4) And (3) designing a mating strategy according to a typing result, preparing TT genotype individuals with growth advantages, and assisting in genetic breeding of growth traits. Furthermore, due to the application of molecular markers related to the growth of prawns, TT genotype individuals grow at the fastest speed, and CT individuals grow at the slowest speed.

In the process of breeding prawns, after the marker is typed by an SNP typing method, TT type individuals are selected for breeding to assist growth, so that the yield of the prawns is improved.

The invention has the following advantages: the molecular marker related to growth provided by the method can select individuals with fast growth on a molecular level, is not limited by external environment and the size of the individuals, can select in an early stage, improves the selection efficiency and accuracy, reduces the breeding cost and shortens the breeding period.

Drawings

FIG. 1 mean body weight distribution plots of the three genotypes at LvMMD2-132-C/T locus in the polyculture population.

Detailed Description

Example 1: acquisition of LvMMD2-132-C/T marker

(1) Genome-wide association analysis (GWAS) of litopenaeus vannamei growth traits:

the material used for GWAS analysis was a mixed population consisting of 13 holosible families. In 7 months in 2015, 13 full-sibling families were constructed in total by oriented mating at Guangtai ocean breeding company of Hainan (Wenchang, Hainan). Transferring to 5m after each family hatching larva²The pond is independently cultivated. In the period, in order to reduce the influence of the culture environment on different families, each family is cultured in a standardized culture mode as much as possible, so that the conditions of salinity, temperature, larva density, bait, aeration and the like are kept consistent as much as possible. In 2015, 9 months, 50 litopenaeus vannamei were randomly picked from each of 13 families, mixed and transferred to 10m²The water pools are cultured in the same environment to form a mixed population. In 11 th month, 200 shrimps were randomly sampled in 2015, and their weights were measured on an electronic balance to determine their sexes.

DNA of all materials was extracted using a plant genome extraction kit (Tiangen, Beijing), and then genome-wide SNP typing was performed on all individuals separately using the method of 2b-RAD (Wang S, Meyer E, McKay JK, Matz MV,2b-RAD: a single and flexible methods for genome-wide typing, Nature methods,2012,9,808, 810), GWAS analysis of shrimp body weight was performed using GenABEL software using the obtained SNP typing data, and growth-related SNP markers were located (Aulchenko Y.S., Ripke S., Isaacs A., van Duijn C.M.ABEL: an R package for genome-wide analysis. Biolnomatics.23: 1294-6), (2007).

(2) Association analysis of LvMMD2 as a growth-related candidate gene

A panel of significant body weight correlations (P) was obtained by GWAS analysis<0.01). By gene annotation of these markers, it was found that one of the SNPs is located in an intron region of the monocyte to major differentiation factor 2(LvMMD2) gene. The gene (LvMMD2) is used as a growth related candidate gene, SNP detail examination is carried out on the coding region of the gene, and association analysis of candidate sites is carried out on non-synonymous mutation SNPs of the coding region. The analysis result shows that the SNP locus at the position of 132bp of the SEQIDLVMMD2 sequence is extremely obviously related to the weight of the prawns (P is 2.42 multiplied by 10)^-8) The interpretation rate of the locus on the phenotypic variation of the body weight can reach 10.5 percent, and the TT genotype of the marker is the dominant genotype.

SEQ IDLvMMD2 in sequence Listing

ACAATCGCTTCAACCAACCTGCACTGGGAGCATACCCATCCTACAAGCTAGAGCACTCAGGCAAAGATGCATCACATGTCTCAGGTGCCCTGCCTTCAACTCCCCCCACTATCCCCACAGCTGAGGGGGTG[C/T]TCTCAGATGCTGAAGGAAGAATCAGGTAGGTGAGGACAGCATAGTAATGGATAAAGGCTGCCAGGACCACAAAGAGATGCCATATGGCATGTGCCAGAGGAATGCGGCCATCCAGCTTAAAGAAGATCACGCCCAGGA

Example 2: the use of the LvMMD2-132-C/T marker in breeding.

(1) Typing of candidate parent LvMMD2-132-C/T locus

In 2016, after taking 60-tailed parent shrimps of Hainan Guangtai ocean breeding company (Wenchang, Hainan) as candidate male and female parents, extracting DNA from a pair of swimming feet of each shrimp, and amplifying the target region sequence of each individual by using the following primer pairs:

LvMMDF：ACAATCGCTTCAACCAACCTG

LvMMDR：TCCTGGGCGTGATCTTCTTT

and performing first-generation sequencing on the PCR amplification product by using an ABI3730xl sequencer, wherein the sequencing primer is an LvMMDF primer, parting is performed on LvMMD2-132-C/T according to a sequencing peak diagram, if the position has only 1C base peak, the parting result is CC type, if the position has only one T base peak, the parting result is TT type, and if the position has both C base peak and T base peak, the parting result is CT type.

(2) Preparation of family material

According to the typing results, CC type individuals and TT type individuals are respectively selected as parents to prepare CC and TT type homozygous families. In addition, CT type families were prepared by directional mating with CC type and TT type individuals as parents. Transferring to 5m after each family is hatched to larva²The pond is independently cultivated, when the family material grows to 1cm, 50 litopenaeus vannamei boone are randomly picked out from each family and put in 10m²And (5) polyculturing in a water pool. After 4 months of cultivation, 200 individuals are randomly selected from the mixed culture population for weight measurement.

(3) LvMMD2-132-C/T marker assisted selection

The individuals were genotyped at the LvMMD2-132-C/T locus. Of the above materials, individuals with three genotypes, CC, CT and TT, were detected, which corresponded to an average body weight of 8.77. + -. 3.22g, 9.52. + -. 3.21g and 12.48. + -. 2.37g, respectively (FIG. 1). Therefore, by selecting the TT genotype in the LvMMD2-132-C/T marker, the growth rate of the breeding population can be improved.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Therefore, it is intended that the present invention cover such modifications and variations as fall within the true spirit of the invention.

Sequence listing

<110> oceanographic institute of Chinese academy of sciences

<120> application of growth-related molecular marker in genetic breeding of litopenaeus vannamei

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<212> DNA/RNA

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acaatcgctt caaccaacct gcactgggag catacccatc ctacaagcta gagcactcag 60

gcaaagatgc atcacatgtc tcaggtgccc tgccttcaac tccccccact atccccacag 120

ctgagggggt gytctcagat gctgaaggaa gaatcaggta ggtgaggaca gcatagtaat 180

ggataaaggc tgccaggacc acaaagagat gccatatggc atgtgccaga ggaatgcggc 240

catccagctt aaagaagatc acgcccagga 270

Claims

1. The application of a growth-related molecular marker in the genetic breeding of the weight traits of the litopenaeus vannamei is characterized in that:

the molecular marker related to growth is positioned in the Litopenaeus vannameiLvMMD2The gene has a nucleotide sequence shown by SEQID NO.1 in a sequence table, the marker is positioned at the 132bp position of the SEQID NO.1 sequence and is a C/T type SNP marker, and the TT genotype of the marker is an advantageous genotype.

2. Use according to claim 1, characterized in that:

in the process of prawn breeding, after the marker is typed by an SNP typing method, TT type individuals are selected for seed reservation and can assist in selection of weight traits, so that the yield of prawns is improved.

3. Use according to claim 2, characterized in that: the specific operation process of SNP typing is as follows,

(1) extracting DNA of parent of prawn;

(2) the following primer LvMMDF was used: ACAATCGCTTCAACCAACCTG and LvMMDR: TCCTGGGCGTGATCTTCTTT amplifying the DNA of the parent material;

(3) sequencing the amplified fragment by using LvMMDF primer to obtain a sequence shown by SEQ ID No.1, analyzing the genotype of SNP sites LvMMD2-132-C/T at 132bp position, if the LvMMD2-132-C/T sites in a sequencing peak map only have C peak, the individual genotype is CC, if the sites are C and T double peaks, the individual genotype is CT, and if the sites only have T peak, the individual genotype is TT.