A gene combination related to Pacific oyster growth and its application in breeding

Abstract

The present invention provides a gene combination related to the growth of Pacific oysters and its application in breeding. The provided gene combination can be used for screening research of Pacific oysters with different growth rates, and provides molecular markers for oyster breeding screening. The gene combination related to the growth of Pacific oysters, the gene combination includes BMP3 gene, BMP4 gene and I-Smad gene. The present invention analyzes Pacific oyster populations with different growth rates and finds that the group with low expression of the three genes BMP3, BMP4 and I-Smad has a faster growth rate, which can be used as a molecular marker for screening Pacific oysters with rapid growth potential.

Description

Technical Field

[0001] The invention belongs to the technical field of fish fry and fish species propagation, and in particular relates to a gene combination related to Pacific oyster growth and an application thereof in breeding. Background Art

[0002] The Pacific oyster (Crassostrea gigas) is a valuable marine invertebrate with significant economic and ecological value due to its special biological characteristics. It is a key species and dominant species in the offshore estuarine reef habitats of countries in the Americas, Europe, Asia and Africa, and is the world's leading farmed mollusk in terms of production. Genetic resources are the source of the development of marine aquaculture and a strategic resource for the country's bio-industry. Improving the production and performance traits of Pacific oysters through genetic means is crucial to ensuring the health and sustainable development of the oyster industry and meeting the growing global demand for Pacific oysters. Despite this, research on the genetic basis of key traits such as growth and quality of elite varieties, trait evaluation, molecular breeding and large-scale seed production is still lacking. Traditional breeding has made slow progress in improving quality traits and cannot support a large and strong oyster market.

[0003] Molecular breeding has made great progress in the aquatic seed industry, and has also promoted the continuous innovation of aquaculture models and the high-quality development of the industry. Whole-genome modular breeding technology will effectively improve breeding efficiency and achieve genetic improvement of quality traits of aquatic animals. Based on de novo prediction, homology prediction and transcriptome assembly prediction, 28,027 oyster candidate genes were annotated, and all genes were annotated with functional domains, functional enrichment and KEGG annotations. These genes involved 296 signaling pathways. 8,953 genes related to resistance traits, 4,320 genes for growth and development traits, and 903 candidate genes for quality traits such as glycogen, amino acids and fatty acids were screened. Research on the resource genomics basis for the creation of high-quality oysters mainly focuses on the species composition and geographical distribution of Chinese economic oysters. Although existing studies have shown that Pacific oyster populations under different temperature gradients have stress adaptation differences in fitness traits such as growth, heat tolerance, and physiological metabolism, the fine molecular regulatory mechanisms of these differences are still unclear. Rapid growth is one of the most important breeding goals for all economic species, but research on the molecular regulatory mechanisms and genetic network analysis of growth in Pacific oysters is still scarce. Summary of the invention

[0004] The purpose of the present invention is to provide a gene combination related to Pacific oyster growth and its application in breeding. The provided gene combination can be used for screening research of Pacific oysters with different growth rates, and provide molecular markers for oyster breeding screening.

[0005] The invention firstly provides a gene combination related to the growth of Pacific oysters, wherein the gene combination comprises a BMP3 gene, a BMP4 gene and an I-Smad gene.

[0006] Furthermore, the amino acid sequence of the protein encoded by the BMP3 gene is SEQ ID NO: 1; the amino acid sequence of the protein encoded by the BMP4 gene is SEQ ID NO: 5; the amino acid sequence of the protein encoded by the I-Smad gene is SEQ ID NO: 9;

[0007] Furthermore, the nucleic acid sequence encoding CDS of the BMP3 gene is SEQ ID NO:2; the nucleic acid sequence encoding CDS of the BMP4 gene is SEQ ID NO:6; and the nucleic acid sequence encoding CDS of the I-Smad gene is SEQ ID NO:10.

[0008] Each gene in the gene combination provided by the present invention can be used as a molecular marker to screen Pacific oysters with different growth rates.

[0009] The present invention also provides a use of a preparation for detecting the expression level of each gene in the above gene combination, wherein the use is used in preparing a preparation for screening Pacific oysters with different growth rates;

[0010] The product is a fluorescent quantitative PCR amplification detection kit.

[0011] The present invention also provides a method for screening Pacific oysters with different growth rates, which is to screen Pacific oyster individuals by detecting the expression levels of the three genes in the above gene combination;

[0012] The screening is to screen individuals with low expression levels of BMP3 gene, BMP4 gene and I-Smad gene.

[0013] The screening is carried out by a fluorescent quantitative PCR amplification method; wherein a primer pair for detecting the BMP3 gene has upstream and downstream primer sequences of SEQ ID NO:3 and SEQ ID NO:4; a primer pair for detecting the BMP4 gene has upstream and downstream primer sequences of SEQ ID NO:7 and SEQ ID NO:8; a primer pair for detecting the I-Smad gene has upstream and downstream primer sequences of SEQ ID NO:11 and SEQ ID NO:12.

[0014] The present invention analyzes Pacific oyster populations with different growth rates and finds that the population with low expression of three genes, BMP3, BMP4 and I-Smad, grows faster, which can be used as molecular markers for screening Pacific oysters with rapid growth potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are photos of two different sizes of Pacific oysters.

[0016] Figure 2 The expression levels of different genes in six different tissues of two different sizes of Pacific oysters, group L and group B.

[0017] Figure 3 Phylogenetic tree constructed for the Pacific oyster INHBA, Nodal 1, BMP2, BMP3, BMP4, BMP7, GDF2 (also known as BMP9), BMP10, and GDF8 genes and other closely related species.

[0018] Figure 4 Phylogenetic tree constructed for the Pacific oyster BMPR2, BMPR1B, ACVR1, TGFBR1, and TGFBR3 genes and other closely related species.

[0019] Figure 5 Phylogenetic tree constructed for the Pacific oyster Smad1, Smad5, Smad3, Smad4, and Smad6 genes and other closely related species.

[0020] Figure 6 This is the principal component analysis of the expression levels of 15 genes in two different sizes of oysters. DETAILED DESCRIPTION

[0021] The applicant screened Pacific oysters and identified two populations of different sizes of Pacific oysters, which were named Group L and Group B. Figure 1 ), the average wet weight of the soft parts of Pacific oysters in group L was 3.76±0.58g, and the average wet weight of the soft parts of Pacific oysters in group B was 11.32±1.88g. By combining the results of principal component analysis of Pacific oysters in groups L and B with the relative expression of mRNA, it was found that the relative expression of BMP3 gene in the visceral mass of group L was 15.82 times that of group B; the relative expression of I-Smad gene in the heart of group L was 10.52 times that of group B; the relative expression of BMP4 gene in gonads and adductor muscles of group L was more than 10 times that of group B. Therefore, these three genes can be used as markers to screen Pacific oyster parents with different growth potentials.

[0022] The present invention is described in detail below in conjunction with embodiments and drawings.

[0023] Example 1: Screening and cluster analysis of Pacific oyster growth-related genes

[0024] The present invention analyzes the transcriptome and finds the differentially expressed gene fragments in the L group and the B group of Pacific oysters, and performs fluorescence quantitative analysis to determine the genome related to the growth of Pacific oysters ( Figure 2 ).

[0025] The genes with differential expression were isolated. Specifically, the differentially expressed gene fragments obtained by screening were used as queries to perform nucleotide homology alignment or amino acid homology alignment in the sea cucumber genome using BLAST, and fragments with high similarity to known conserved domain sequences (Identity>50%) were isolated from the genome. After extending these fragments 20 kb before and after the genome, GENSCAN was used to predict the open reading frame of the target gene online, and then BLAS (http: / / blast.ncbi.nlm.nih.gov / ) was used for survey, and finally the complete amino acid sequence of the gene was isolated.

[0026] The amino acid sequences of the screened genes were subjected to phylogenetic analysis with the genomes of other species, and the gene information of the selected species was as follows: nodal1 Plakobranchus ocellatus, GFO10962.1; nodal1 Myaarenaria, XP_052805284.1; nodal1 Pecten maximus, XP_033752862.1; nodal1 Ostreaedulis, XP_048780111.1; nodal1X1 Crassostrea virginica, XP_022336385.1; nodalCrassostrea gigas, XP_011422588.2; GDF8 Pecten maximus, XP_033736807.1; GDF8Mizuhopecten yessoensis, XP_021351561.1; GDF8X1 Crassostrea virginica, XP_022336385.1; virginica,XP_022313076.1; GDF8X2 Crassostrea virginica,XP_022313077.1; 2,XP_019920628.2; INHBA Haliotis rufescens,XP_046380636.1; yessoensis,XP_021347108.1;INHBA Pectenmaximus,XP_033762331.1; INHBA Ostrea edulis, 1,XP_011453034.2; GDF2 Crassostrea angulata,XP_052683361.1;2;BMP3 Haliotis rubra,XP_046567559.1;BMP3Mytilus californianus,XP_052069049.1;BMP3 / 3B Mytilus galloprovincialis,VDI68563.1;BMP3 Mytilus edulis,CAG2218645.1;BMP3 Pecten maximus,XP_033742698.1;BMP3 Mizuhopecten yessoensis,XP_021343121.1;BMP3 Pinctada fucata,ANJ60739.1;BMP3 Ostrea edulis,XP_048753450.1;BMP3 Crassostrea gigas,XP_011415620.2;BMP4 Mytilus coruscus,CAC5414513.1;BMP4likeX1 Crassostrea gigas,XP_034311484.1;BMP4likeX2 Crassostrea gigas,XP_034311485.1;BMP4X1Crassostreagigas,XP_011416937.2;BMP4X2 Crassostrea gigas,XP_011416938.2;TGFB1 Ictidomystridecemlineatus,KAG3256109.1;TGFB 1 Marmota monax,ADP44690.1;TGFB1 Vicugnapacos,AHI87689.1;TGFB1 Ctenopharyngodon idella,ABU84814.1;TGFB1 Sparusaurata,AAN76665.1;TGFB1 Sus scrofa,AAL57902.1;TGFB1 Horse caballus,AAD49431.1;TGFB1 Loxodonta africana,QQK85124.1;TGFB1 Cyprinus carpio,AAF22573.1;TGFB1 Mustela putorius furo,ABB16434.1;BMP7Tegillarca granosa,AFP57673.1;BMP7 Mytilus californianus,XP_052086249.1;BMP7Mytilus galloprovincialis,VDI37599.1;BMP7 Mytilus edulis,CAG2230021.1;BMP7 Mizuhopecten yessoensis,XP_021377502.1;BMP7 Pecten maximus,XP_033726914.1;BMP7 Pinctada imbricata,AGS32053.1;BMP7 Ostrea edulis,XP_048779745.1;BMP7 Crassostrea gigas,XP_011428157.1;BMP10 Mizuhopectenyessoensis,XP_021353852.1;.

[0027] BMP10 Pecten maximus, XP_033731896.1;BMP10 Pinctada fucata, AOT85458.1;BMP10 Mytilus edulis, CAG2235065.1;BMP10 Mytilus coruscus, CAC5423908.1;BMP10 Mytilus californianus, XP_052069425.1;BMP10 Ostrea edulis, XP_048764000.1;BMP10 Crassostrea virginica, XP_022313556.1;BMP10 Crassostrea angulata, XP_052687905.1;BMP10 Crassostrea gigas, XP_034313024.1;BMP2 Sinonovacula constricta, QFF91640.1;BMP2 Solen grandis,UWM22940.1;BMP2 Mytilus coruscus,CAC5412150.1;BMP2 Azumapecten farreri,AGF68558.1;BMP2 Mizuhopectenyessoensis,OWF43831.1;BMP2X2 Pecten maximus,XP_033734741.1;BMP2X1 Pectenmaximus,XP_033734740.1;BMP2 Pinctada fucata,BAD16731.1;BMP2 Crassostreavirginica,XP_022333695.1;BMP2 Ostrea edulis,XP_048736596.1;BMP2X2Crassostreagigas,XP_011434596.1 and BMP2X1 Crassostrea gigas,XP_019924915.2.GenBankaccession numbers for the receptor protein sequences are as follows: BMPR2Mercenaria mercenaria,XP_045209130.2;BMPR2X1 Dreissena polymorpha,XP_052265234.1;BMPR2X2 Dreissena polymorpha,XP_052265235.1;BMPR2X2Octopussinensis,XP_029646711.1;BMPR2 Haliotis rubra,XP_046573372.1;BMPR2X1 Haliotisrufescens,XP_046329579.1;BMPR2 Crassostrea gigas,CAD20574.1;BMPR2X2Crassostrea gigas,XP_034327883.1;BMPR2X1 Crassostrea gigas,XP_034327876.1;BMPR2 Mytilus galloprovincialis,VDI15534.1;BMPR2 Mytilus edulis,CAG2222821.1;BMPR2 Mytilus californianus,XP_052092005.1;BMPR2 Mytilus coruscus,CAC5402056.1;TGFBR3X2 Haliotis rufescens,XP_046371438.1;TGFBR3X1Haliotisrufescens,XP_046371437.1;TGFBR3 Pecten maximus,XP_033731749.1;TGFBR3Mizuhopecten yessoensis,XP_021347454.1;TGFBR3 Mytilus coruscus,CAC5417350.1;TGFBR3 Mytilus californianus,XP_052095384.1;TGFBR3 Ostrea edulis,XP_048775648.2;TGFBR3X1 Crassostrea virginica,XP_022321264.1;TGFBR3X2 Crassostrea virginica,XP_022321265.1;TGFBR3X2 Crassostrea angulata,XP_052720273.1;TGFBR3X1 Crassostrea angulata,XP_052720272.1;TGFBR3X1Crassostrea agigas,XP_034302484.1;TGFBR3X2 Crassostrea gigas,XP_034302485.1;ACVR1 Pectenmaximus,XP_033728767.1;ACVR1 Mytilus edulis,CAG2187787.1;ACVR1 Mytilusgalloprovincialis,VDI65000.1;ACVR1X2 Mytilus californianus,XP_052080275.1;ACVR1X1 Mytilus californianus,XP_052080274.1;ACVR1 Pinctada fucata,ADD80738.1;ACVR1X3 Ostrea edulis,XP_056011015.1;ACVR1X2 Ostrea edulis,XP_056011013.1;ACVR1X1 Ostrea edulis,XP_056011012.1;ACVR1Crassostrea virginica,XP_022324124.1;ACVR1 Crassostrea gigas,CAC85263.1;ACVR1X2 Crassostreaangulata,XP_052672815.1;ACVR1X1 Crassostrea angulata,XP_052672814.1;ACVR1 LowCrassostrea gigas,XP_011451303.2;BMPR1B Pecten maximus,XP_033745345.1;BMPR1BMizuhopecten yessoensis,XP_021354771.1;BMPR1B Mytilus galloprovincialis,VDI34739.1;BMPR1B Mytilus californianus,XP_052068713.1;BMPR1B Mytiluscoruscus,CAC5409385.1;BMPR1B Ostrea edulis,XP_048760143.1;BMPR1B Crassostreavirginica,XP_022317289.1;BMPR1b Crassostrea gigas,CAE11917.1;BMPR1BCrassostrea gigas,XP_034312252.1;BMPR1B Crassostrea angulata,XP_052687167.1;TGFBR1X2 Mercenaria mercenaria,XP_045179301.1;TGFBR1X1 Mercenaria mercenaria,XP_045179300.1;TGFBR1X1 Dreissena polymorpha,XP_052235549.1;TGFBR1X2 Dreissenapolymorpha,XP_052235550.1;TGFBR1X2 Ostrea edulis,XP_056011203.1;TGFBR1X1 Ostrea edulis,XP_048778749.2;TGFBR1 Crassostrea virginica,XP_022324495.1;TGFBR1X1 Crassostrea gigas,XP_011414113.1;TGFBR1 Crassostrea gigas,CAD20573.1;TGFBR1 Crassostrea angulata,XP_052672666.1;TGFBR1 Pecten maximus, XP_033727883.1;TGFBR1 Mizuhopecten yessoensis, XP_021365929.1;TGFBR1 Azumapectenfarreri, AFQ23184.1;TGFBR1 Mytilus edulis, CAG2217787.1;TGFBR1 Mytiluscoruscus, CAC5414192.1;TGFBR1 Mytilus californianus, XP_052079115.1.GenBankaccession numbers for the Smad protein sequences are as follows:Smad6Pinctada fucata, AGI96395.1;Smad6 Ostrea edulis, XP_048749813.1;Smad6Crassostrea gigas, NP_001295807.1;Smad6X1 Crassostrea gigas, XP_011453272.2;Smad6 Patella vulgata, XP_050397169.1;Smad6 Haliotis rufescens, XP_046331908.1;Smad6 Haliotis rubra, XP_046578483.1;Smad6 Pecten maximus, XP_033725000.1;Smad6Mizuhopecten yessoensis,XP_021377964.1;Smad6Mytilus galloprovincialis,VDI21705.1;Smad6Mytilus edulis,CAG2211855.1;Smad6Mytilus coruscus,CAC5365765.1;Smad6Mytilus californianus,XP_052096574.1;Smad4X1Ostremae edulis,XP_048749179.1;Smad4X2.

[0028] Ostrea edulis, XP_048749181.1;Smad4X3 Ostrea edulis, XP_056003268.1;Smad4X1Crassostrea virginica, XP_022311791.1;Smad4 Crassostrea gigas, AHB37077.1;Smad4X3 Crassostrea gigas, XP_034321159.1;Smad4X1 Crassostreagigas, XP_034321156.1;Smad4X2 Crassostrea gigas, XP_034321158.1;Smad4X4Crassostrea gigas, XP_034321160.1;Smad4 Mytilus edulis, CAG2217664.1;Smad4X1Mytilus californianus, XP_052084009.1;Smad4X1 Mizuhopecten yessoensis, XP_021379467.1;Smad4X2 Mizuhopecten yessoensis, XP_021379468.1;Smad4X1 Pectenmaximus, XP_033724907.1;Smad4X2 Pecten maximus, XP_033724908.1;Smad4X1 Haliotisrufescens, XP_048251185.1;Smad4X3 Haliotis rufescens, XP_048251190.1;Smad4X4Haliotis rufescens, XP_048251191.1;Smad4X2 Haliotis rufescens, XP_048251189.1;Smad4X4 Patella vulgata, XP_050395776.1;Smad4X1 Patella vulgata, XP_050395772.1;Smad4X3 Patella vulgata,XP_050395775.1;Smad4X2 Patellavulgata,XP_050395774.1;Smad1 / 5 Lottia gigantea,BAQ19235.1;Smad5 Hyriopsiscumingii,ARV76495.1;Smad5Mya arenaria,XP_052793321.1;Smad1X3 Dreissenapolymorpha,XP_052278712.1;Smad5X1 Dreissena polymorpha,XP_052278710.1;Smad1Tegillarca granosa,ALG64476.1;Smad5X1 Mizuhopecten yessoensis,XP_021360276.1;Smad1 Mytilus coruscus,CAC5390568.1;Smad1 Mytilus californianus,XP_052078142.1;Smad5Pinctada fucata,AGI96394.1;Smad5 Crassostrea gigas,XP_011448163.2;Smad5Crassostrea virginica,XP_022324128.1;Smad5 Ostrea edulis,XP_048779087.1;Smad1 / 5 / 8 Crassostrea gigas,AHB37076.1;Smad3X1 Ostrea edulis,XP_048750135.1;Smad3X2 Ostrea edulis,XP_048750136.1;Smad3X1 Crassostreavirginica,XP_022306393.1;Smad3X2 Crassostrea virginica,XP_022306394.1;Smad3X3Crassostrea virginica,XP_022306396.1;Smad3X1 Crassostrea gigas,XP_011441244.1;Smad3X2 Crassostrea gigas,XP_011441245.1;Smad3X3 Crassostreagigas,XP_034321760.1;Smad3 Mya arenaria,XP_052786611.1;Smad3 Meretrixmeretrix, APW85816.1;Smad3X1 Mercenaria mercenaria, XP_045183780.1;Smad3X2 Mytilus californianus, XP_052096427.1;Smad3 Mytilus coruscus, CAC5425638.1;Smad3X1 Mytilus californianus, XP_052096426.1; Smad3X1 Octopus sinensis, XP_029653407.1; Smad3X2 Octopus sinensis, XP_036371093.1; Smad3 Azumapectenfarreri, AMQ47456.1; Smad3X2 Pecten maximus, XP_033724945.1 and Smad3X1 Pectenmaximus, XP_033724944.1. The EditSeq program contained in the Lasergene software package (DNAStar, USA) was used to edit and save all amino acid sequences. The protein sequences were aligned using ClustalX 1.83 software, and the phylogenetic tree was constructed using the Neighbor-Joining (NJ) method using MEGA 7.0 software. Bootstrap value 1000 represents the credibility of the phylogenetic tree, which is the probability of a clade appearing in 1000 calculations. .

[0029] Figure 3 Phylogenetic tree constructed for Pacific oyster INHBA, Nodal 1, BMP2, BMP3, BMP4, BMP7, GDF2 (also known as BMP9), BMP10, and GDF8 genes and other closely related species; 14 known Pacific oyster TGFβ ligand sequences were divided into 9 categories.

[0030] Figure 4 Phylogenetic tree constructed for the Pacific oyster BMPR2, BMPR1B, ACVR1, TGFBR1, and TGFBR3 genes and other closely related species. According to the phylogenetic tree, ACVR1X2 and ACVR1X1 of the Portuguese oyster are more closely homologous to ACVR1Low of the Pacific oyster, while ACVR1 of the Pacific oyster is more distantly homologous.

[0031] Figure 5 Phylogenetic tree constructed for the Pacific oyster Smad1, Smad5, Smad3, Smad4, and Smad6 genes and other closely related species. Based on the phylogenetic tree, the 12 Smads of the Pacific oyster are divided into five categories. The Pacific oyster has a very rich Smad4 subtype, with five types identified so far.

[0032] Example 2: Differentially expressed genes in group L and group B

[0033] The mRNA levels of TGFβ signaling pathway genes in the mantle, gills, digestive glands, gonads, adductor muscles and heart of groups L and B were shown in Figure 2 Figure 2 shown. Figure 2The figure shows the expression heat map of different genes in six different tissues of two different sizes of Pacific oysters. From the tissue distribution heat map, we can find the expression difference of the same gene in the same tissue of group L and group B. Among them, Smad1 / 5 / 8 in mantle, BMP10 in gill, INHBA in digestive gland, TGFBR1 in gonad and adductor muscle, and BMPR2 in heart are the most significantly different genes in these tissues (p<0.05).

[0034] In order to find growth biomarkers for Pacific oysters, PCA analysis was performed on the expression levels of 15 TGFβ signaling pathway genes in two sizes of Pacific oysters. Figure 6 The principal component analysis diagram of the expression levels of 15 genes in two different sizes of oysters. The results of the principal component analysis combined with the relative expression of mRNA showed that the relative expression of the BMP3 gene in the visceral mass was 15.82 times that of the B group in the L group; the relative expression of the I-Smad gene in the heart was 10.52 times that of the B group; the relative expression of the BMP4 gene in the gonad and adductor muscle was more than 10 times that of the B group in the L group. Therefore, the three genes, BMP3, I-Smad and BMP4, can be used as biomarkers to screen Pacific oyster parents with different growth potentials. That is, in Pacific oyster individuals, the lower the expression of these three genes, the greater the growth potential of the individual.

[0035] The protein sequence of the BMP3 gene is as follows (SEQ ID NO: 1):

[0036] MRFCNLVYFMCTFLGVTFGKADQKNVLLHEAVRTLLGFQESPDRYQKLQTVPNVLSEQAPQYMMDLYEKFKNNRISKGHLSGNTVRSIHAEIAVVNGETMFVFNLSSVSRTEQLLSAEIHLYKRKPKRFNHKKDIELKMYEVAPHYMSDEGKITMRAESFGWQWYDVTEAIDSCLAARRRVPHLFALNFKTEKQNGKLKDIALKKFIRHHSMPFLIVYTNDTQENRFDQLDSLADKIKSDSPVKKETVKKIIPDEVTEVKLRDKRSIYDNEIPENPVDYQRIERKFNIPQTHPGILQTRRQNRNKNSGSRLIPYPGKGKRKKNRKRKNRRRKNRRRNKVFPKEWKDVQQNMATEGKHGQLCGRQKLIVDFADIGWGDWIISPKSFEAHYCAGSCPFPLTQRLKPSNHATIQSMVHAIGIYTDVPAPCCVPDSLSSITLLYFDENRNVVLKNYPSMTVNSCAVQVDSKK;

[0037] The sequence of its coding CDS is as follows (SEQ ID NO: 2):

[0038] ATGAGATTTTGTAACTTAGTGTATTTTATGTGCACCTTTCTTGGTGTGACTTTTGGAAAAGCTGATCAGAAAAACGTGCTTCTTCACGAGGCCGTTCGAACGTTGCTGGGATTTCAAGAATCTCCGGATCGCTATCAGAAACTACAGACAGTTCCCAATGTCTTATCGGAACAGGCGCCCCAGTATATGATGGATTTGTACGAGAAGTTTAAAAACAATCGAATCTCCAAAGGACACTTATCTGGAAACACTGTCAGAAGCATCCACGCTGAAATAGCTGTGGTCAACGGAGAGACTATGTTTGTATTCAACCTATCCTCAGTTAGTCGAACAGAACAGCAGCTTTTGAGCGCAGAAATTCATTTGTACAAACGAAAACCAAAGCGGTTCAATCATAAAAAAAGACATAGAATTGAAAATG TATGAGGTCGCCCCTCATTACATGTCGGATGAAGGAAAGATAACGATGCGGGCCGAGTCTTTTGGTTGGCAGTGGTATGACGTAACAGAAGCCATTGACAGTTGTCTTGCAGCTAGAAGAAGGGTTCCCCATTTATTTGCTTTGAATTTTTTAAAACAGAGAAACAAAATGGAAAATTGAAAGACATTGCTTTAAAAATATTCATCCGCCA TCATTCCATGCCATTTTTAATTGTTTACACTAACGATACTCAAGAAAATCGTTTCGACCAGTTAGATTCTCTAGGCAGACAAAATCAAAAGTGACTCTCCAGTTAAAAAAGAAACGGTTAAAAATTATACCAGACGAGGTGACTGAGGTCAAACTAAGAGACAAGAGATCAATATACGACAATGAAATTCCGGAGAATCCAGTAGATT;

[0039] The sequence information of a primer pair used to detect the expression of BMP3 gene is as follows:

[0040] Upstream primer: 5′-CGGATGAAGGAAAGATAACG-3′ (SEQ ID NO: 3),

[0041] Downstream primer: 5′-CATACCACTGCCAACCAAA-3′ (SEQ ID NO: 4).

[0042] The BMP4 gene protein sequence is as follows (SEQ ID NO: 5):

[0043] MTKGNNWLCFLVITAVVGWKGGDGSLRLAEEALHRLGIDPNRPVQRETQINSFVQHVFNQIQEKQNDLRISPDKDPRVQFYDSLSENSNGRVDFDLEQGPIVGNVTKVEVAFTGHLHRNFIIDITSPSQLSRVVTTARTPHGKVADITTLVADKLSGSTLSLSISVKGAGNWIRHLRPVLAIFLDLDASVLNRRRKRFIVQTGNGSVSADTAESGNYCRLVPWTVSLRDLGWYPQFIYSPESYQANLCSGGCPRAILESYFNVTNHGIFKAMLEKKLCCTPIEYNDQSLLFGNTIQTVKGMSVARCGCR;

[0044] The sequence encoding the CDS is as follows (SEQ ID NO:6):

[0045] ATGACCAAAGGTAACAATTGGTTGTGCTTCTTGGTAATAACTGCAGTTGTTGGATGGAAAGGCGGTGATGGTTCCCTCCGCCTGGCCGAAGAGGCCCTTCACAGGCTGGGCATTGACCCTAACCGTCCAGTTCAAAGGGAAACGCAGATAAATTCGTTCGTGCAGCACGTGTTTAACCAGATTCAGGAGAAACAAAACGATTTACGGATATCTCCCGACAAAGACCCCAGGGTGCAGTTTTATGACTCCCTGTCAGAAAATTCAAACGGAAGAGTGGATTTCGACCTTGAGCAGGGTCCTATCGTCGGCAATGTCACCAAGGTGGAGGTTGCATTCACCGGTCATCTCCATAGAAACTTTATC ATTGATATCACATCTCCAAGTCAGCTGAGCAGAGTAGTCACCACTGCACGGACTCCACACGGCAAGGTGGCAGACATTACGACTCTCGTTGCAGACAAGCTTTCTGGTTCTACTCTTTCTCTCTCCATCTCCGTCAAGGGTGCAGGGAACTGGATCAGACATCTACGTCCCGTTCTAGCAATATTTCTGGATCTTGATGCTTCTGTTTTGAATCGACGTCGAAAGCGTTTTATTGTTCAGACAGGAAATGGTTCGGTTTCCGCGGATACGGCTGAAAGTGGAAATTACTGCCGTTTAGTACCATGGACGGTTAGTCTTCGGGATCTGGGGTGGTATCCACAATTTATTTACTCCCCAGAATCTTATCAGGCAAATCTTTGTTCCGGTGGGTGTCCCCGAGCAATATTAGAGAGTTATTTCAATGTCACAAACCACGGAATCTTCAAGGCGATGTTGGAGAAGAAACTTTGTTGTACCCCTATTGAGTACAACGATCAGTCTCTGCTGTTTGGAAATACAATCCAGACGGTCAAGGGCATGTCTGTGGCAAGGTGTGGGTGTCGCTGA。

[0046] The sequence information of a primer pair for detecting the expression level of the BMP4 gene is as follows:

[0047] Upstream primer: 5′-TCTCTCTCCATCTCCGTCA-3′ (SEQ ID NO: 7),

[0048] Downstream primer: 5′-ACCGAACCATTTCCTGTCT-3′ (SEQ ID NO: 8).

[0049] The protein sequence of the I-Smad gene is as follows (SEQ ID NO: 9):

[0050] MFRSKRSSLVRELWKLRIPQNDTESQNEKESHECKAVIHSMLKRLKEKNLEILLRSVESKGREETPCVLLPNVELKMGKMSTDPHVLCCQIWRWPELTLDTTLRRLPCCETDKDPLYVCCNPYHWSIQQNAESSYCAEKANCLTSGINWPSDISMEPDSTETGLTPTCRRNHGGDISDLS EGEEPGSEVTTHWCSVAYWELRQRVGRLYTLHEPYLCIFQDLPHGNGLSLSLVQEPTSVDCVKRTREKIGLGLVLSREADGVWLYNRSGYPVFVNSPTLDNPSSRASVVIKVNPGYSMNIFNYDMASVLRAVKERHCLDGPYDPTSIRISFAKGWGPSYHRQFITSCPAWLEVLLNVNR,

[0051] The sequence of the CDS encoding the I-Smad gene is as follows (SEQ ID NO: 10):

[0052] ATGTTCCGTTCCAAGAGATCGTCTCTAGTGCGAGAGCTCTGGAAACTCCGAATTCCACAAAATGACACCGAAAGTCAAAATGAAAAAGAATCCCATGAGTGTAAAGCTGTCATTCATTCTATGTTGAAGAGATTAAAAGAGAAGAACTTGGAAATTCTGCTTCGGAGCGTCGAATCCAAGGGGCGGGAAGAGACCCCTTGTGTGCTGCTTCCGAACGTGGAATTAAAGATGGGGAAGATGTCCACGGACCCCCATGTTTTGTGTTGTCAGATCTGGCGATGGCCAGAATTGACGCTGGACACAACATTACGGCGATTGCCGTGTTGTGAAACGGACAAAGATCCGTTATACGTTTGTTGCAATCCGTATCATTGGAGTATACAACAAAACGCAGAATCTTCATATTGCGCTGAAAAAGCAAATTGTCTTACCTCAGGAATTAATTGGCCCAGTGATATTTCAATGGAACCTGACTCCACAGAAACAGGACTGACTCCTACTTGTCGTAGAAACCACGGCGGGGATATATCAGATCTGAGTGAGGGCGAGGAGCCGGGGTCAGAGGTCACGACCCACTGGTGCTCCGTGGCCTATTGGGAGTTACGTCAGAGGGTGGGGCGCCTGTACACGCTGCACGAGCCCTACCTGTGTATATTTCAGGATCTACCTCACGGGAACGGTTTGTCCCTCAGTCTGGTCCAAGAACCCACCTCCGTGGATTGTGTCAAACGGACGCGCGAAAAAATCGGCCTTGGCCTCGTTTTGAGCCGAGAAGCAGACGGCGTGTGGCTGTACAACCGCTCCGGGTACCCGGTGTTCGTCAACTCGCCCACCCTGGACAACCCTAGCTCGCGGGCGTCGGTTGTGATCAAAGTGAACCCTGGATACTCCATGAACATATTTAACTACGATATGGCCAGTGTGCTTCGGGCAGTGAAAGAGCGGCATTGTCTGGACGGACCGTACGACCCGACCTCCATTAGAATCAGCTTCGCCAAGGGCTGGGGACCTTCTTATCACAGACAGTTCATTACGTCATGTCCGGCCTGGCTCGAGGTCTTACTCAACGTCAATAGGTGA,

[0053] The sequence information of a primer pair for detecting the expression level of the I-Smad gene is as follows:

[0054] Forward primer: 5′-CGGATGAAGGAAAGATAACG-3′ (SEQ ID NO:11),

[0055] Reverse primer: 5′-CATACCACTGCCAACCAAA-3′ (SEQ ID NO:12).

[0056] Based on the correlation rules of the above three gene expressions, the growth of Pacific oysters can be regulated in a targeted manner and the effects of corresponding breeding methods can be evaluated.

[0057] Example 3: Screening parents based on BMP3, I-Smad and BMP4 expression levels

[0058] The Pacific oysters with the largest shell height and regular shell shape were selected as the selection criteria, and 100 individuals were selected as parents to construct a fast-growing breeding line; the Pacific oysters with the smallest shell height and regular shell shape were selected as the selection criteria, and 100 individuals were selected as parents to construct a slow-growing breeding line. Because some parents died during the gonadal ripening process or their gonads were not mature enough when the breeding line was constructed, they were not used. The actual number of parents in each breeding line was 81-90. Sampling and analysis of spats after six consecutive generations of breeding were carried out. The mantle, gills, digestive glands, gonads, adductor muscles and hearts of Pacific oysters of different sizes were carefully peeled off for mRNA detection. The sample size of each group was 20 oysters. The remaining spats continued to be raised into adult shellfish, and the wet weight of their soft parts after breeding was weighed to verify whether the screened gene combination indicated that the oysters could grow rapidly.

[0059] In order to obtain accurate and reliable gene expression results, we first performed an internal reference gene validation experiment. The following seven housekeeping genes were selected as candidate genes: RPS18, RPL7, EF-1a, ACTB, 18S, 28S, and GAPDH. Finally, in the geNormv3.5 software analysis, ACTB was the most stable gene in both group L and group B, so it was used as an internal reference gene. Primers were synthesized by Invitrogen Biotechnology Co., Ltd. Total RNA was extracted using TRIzol reagent according to the instructions of the Invitrogen manufacturer, and contaminating genomic DNA was removed using RNase-free DNase. RNA samples were reverse transcribed using Prime Script RT-PCR Kit. Equal amounts of cDNA were taken for real-time fluorescence quantitative RT-PCR in the PikoReal 96-well RT-PCR system. The total volume of the amplification reaction was 10 μL, including 5 μL 2×SYBR Green Master Mix, 1 μL diluted cDNA, 0.4 μL primers, and 3.2 μL PCR-grade water. The PCR cycle conditions were 94°C pre-denaturation for 5 min, 95°C denaturation for 15 s, 60°C annealing for 30 s, 72°C extension for 1 min, 30 cycles, and a final extension at 72°C for 7 min. Each sample was repeated 3 times with the internal reference gene (ACTB). The PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide and developed on a UV transilluminator.

[0060] The results of relative mRNA expression analysis showed that the relative expression of BMP3 gene in visceral mass was 16.76 times that of fast growth group in the slow growth group. The relative expression of I-Smad gene in heart was 14.68 times that of fast growth group in the slow growth group. The relative expression of BMP4 gene in gonad was 11.37 times that of fast growth group in the slow growth group, and the relative expression in adductor muscle was 16.27 times that of fast growth group in the slow growth group.

[0061] After the remaining juveniles were grown into oysters, the oyster shells were opened with a scalpel, the seawater in the inner and outer shell walls and shell cavities was dried with absorbent paper, and the weight of the soft parts was weighed. It was found that the fast-growing population with low expression of the three genes BMP3, BMP4 and I-Smad had an average wet weight of the soft parts of more than 7.13g. The slow-growing population with high expression of the above three genes had an average wet weight of the soft parts of less than 4.82g. The results show that the three genes BMP3, BMP4 and I-Smad can be used as molecular markers for screening Pacific oyster growth.

Claims

1. A gene combination related to the growth rate of Pacific oysters, characterized in that: The gene combination is BMP3 gene, BMP4 gene and I-Smad gene; The amino acid sequence of the protein encoded by the BMP3 gene is SEQ ID NO: 1; the amino acid sequence of the protein encoded by the BMP4 gene is SEQ ID NO: 5; the amino acid sequence of the protein encoded by the I-Smad gene is SEQ ID NO: 9; The nucleic acid sequence encoding CDS of the BMP3 gene is SEQ ID NO:2; the nucleic acid sequence encoding CDS of the BMP4 gene is SEQ ID NO:6; and the nucleic acid sequence encoding CDS of the I-Smad gene is SEQ ID NO:10.

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