CN116497129B - Gene molecular marker related to stichopus japonicus growth traits and application thereof - Google Patents
Gene molecular marker related to stichopus japonicus growth traits and application thereof Download PDFInfo
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- 241000965254 Apostichopus japonicus Species 0.000 title claims abstract description 62
- 239000003147 molecular marker Substances 0.000 title claims abstract description 32
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 14
- 238000009395 breeding Methods 0.000 claims abstract description 28
- 230000001488 breeding effect Effects 0.000 claims abstract description 26
- 101150099086 sif gene Proteins 0.000 claims abstract description 15
- 230000035772 mutation Effects 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 7
- 108700009124 Transcription Initiation Site Proteins 0.000 claims abstract description 5
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- 235000008434 ginseng Nutrition 0.000 claims description 2
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- 108020004414 DNA Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
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- 241000258955 Echinodermata Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- XLYOFNOQVPJJNP-PWCQTSIFSA-N Tritiated water Chemical compound [3H]O[3H] XLYOFNOQVPJJNP-PWCQTSIFSA-N 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
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- 238000009364 mariculture Methods 0.000 description 2
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- 238000012257 pre-denaturation Methods 0.000 description 2
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Abstract
The invention belongs to the field of molecular marker assisted breeding, and particularly relates to a molecular marker combination related to stichopus japonicus growth traits and application thereof. The invention establishes the stichopus japonicus molecular marker assisted breeding method by screening stichopus japonicus sif gene molecular markers, and provides gene markers and technical methods for the genetic improvement of stichopus japonicus growth traits. The molecular marker is located in an exon region of the stichopus japonicus sif gene. Screening by Sanger sequencing method to obtain molecular markers which are obviously related to the growth traits of stichopus japonicus, wherein the molecular markers comprise the following sites: the transcription initiation site starts a T > C mutation at 1538, a T > C mutation at 1574, a T > C mutation at 29283. When in use, one or more molecular markers are used for breeding stichopus japonicus offspring seed with excellent phenotype and fast growth speed. The invention provides a molecular marker for improving the growth characters of the stichopus japonicus from the genetic level, and has important significance for improving the breeding benefit of the stichopus japonicus.
Description
Technical Field
The invention belongs to the field of molecular marker assisted breeding, and particularly relates to a gene molecular marker related to stichopus japonicus growth traits and application thereof.
Background
Apostichopus japonicus (Apostichopus japonicus) belongs to Apostichopus japonicus (Holothuroideaia) belonging to Echinodermata (Echinodermata), is mainly distributed on Pacific coastal, has extremely high nutritive and medicinal value, and increases market demand, so that wild resource of Apostichopus japonicus is seriously degenerated. In recent years, the stichopus japonicus culture industry is rapidly developed, becomes a key supporting industry for mariculture, and brings remarkable economic benefit for China. However, as the industrial scale is gradually increased, problems such as germplasm degeneration, slow growth, disease occurrence and the like are increasingly prominent, wherein slow growth is one of the most remarkable problems. The stichopus japonicus population growth variation coefficient exceeds 50%, and has very high genetic improvement potential. Therefore, the genetic improvement of stichopus japonicus, and the cultivation of new varieties with excellent characters such as high growth speed are of great importance for the healthy and sustainable development of the industry.
Since the 90 th century of the 20 th century, the problem of lack of improved variety gradually becomes an important factor for restricting the development of mariculture industry, and breeders in all countries of the world actively explore new ideas and methods for breeding, pay attention to genetic analysis of important economic characters, and aim to promote the transition from a traditional breeding platform to a modern molecular breeding platform. Compared with the livestock industry, the genetic improvement of the aquatic industry is late, and the molecular breeding work of stichopus japonicus is more lagging behind other aquatic organisms. Phenotype-based selective breeding and cross breeding are used as traditional type breeding technologies, the inbreeding recession phenomenon exists after multi-generation breeding, the purification effect is poor, and the development of industry breeding is hindered. Molecular marker assisted breeding and genetic engineering breeding are just started in stichopus japonicus.
Sun Wenjing et al (2010) developed for the first time 13 SNPs for describing the structure of the stichopus japonicus population using stichopus japonicus expression sequence tags (Expressed Sequence Tag, EST); team Zhou Zunchun developed 15 SNPs using EST (Yang et al 2012), followed by 142,511 potential SNPs by high-throughput transcriptome sequencing technology (Zhou et al 2014); bao Zhenmin team (2012) screens out 54,000 potential SNPs sites according to transcriptome sequencing technology and develops 101 SNPs sites using high resolution melting (High Resolution Melting, HRM) genotyping technology; jung-Ha Kang et al (2011) developed 6 SNP molecular markers that can be used for body color traits; team Yang Gongsheng analyzed the regulatory mechanism of HSP family genes on heat resistance of stichopus japonicus and developed 3 SNP markers associated with high temperature resistance (Xu et al, 2014b; xu et al, 2015; xu et al, 2014 a).
At present, the stichopus japonicus genetic research is mainly based on resistance characters, molecular marker assisted breeding is mainly based on molecular marker development, and the developed markers are not subjected to subsequent expansion verification.
Disclosure of Invention
The invention aims to provide a gene molecular marker related to stichopus japonicus growth traits and application thereof.
In order to achieve the above purpose, the invention adopts the technical scheme that:
a gene molecular marker related to the growth trait of stichopus japonicus, wherein the molecular marker is an exon region of a sif gene in a stichopus japonicus genome No. 18 linkage group.
The molecular marker is one or more of T > C mutation at 1538 (e 2-2.1538), T > C mutation at 1574 (e 2-4.1574) and T > C mutation at 29283 (e 9-2.29283) of apostichopus japonicus genome 18 linkage group sif gene, which is started at the transcription initiation site of the gene.
The molecular marker detects (CC), (TT) and (CT) 3 genotypes at 1538 of sif gene sequence;
The molecular marker detects (CC), (TT) and (CT) 3 genotypes at 1574 of sif gene sequences;
The molecular markers detected the (CC), (CT) and (TT) 3 genotypes at 29283 of the sif gene sequence.
The molecular marker is located in the exon region of the stichopus japonicus growth gene sif, and the nucleotide sequence of the molecular marker is shown in the Seq ID No. 1.
The application of the gene molecular marker related to the stichopus japonicus growth trait in stichopus japonicus breeding.
A primer for screening and SNP molecular marker genotyping is provided, wherein the primer pair is
AJ_e2-2_F:CTCTCCAAGAATGGGGACCG
AJ_e2-2_R:CGCAGATCAGGACCAACTGT
AJ_e2-4_F:ACATAACCGATGGAGCGACA
AJ_e2-4_R:TTCCGGGTCAAAGGTCGTG
AJ_e9-2_F:AGCACAGCGTTTGGAGATTC
AJ_e9-2_R:CCGTGTTGTCTCGTAAATGCC
The PCR amplification procedure and system are: 50ng/ul 1ul of DNA template, 7.5 ul of Premix Taq enzyme, 0.5 ul of forward and reverse primers, 5.5 ul of RNase-FREE WATER, respectively; the PCR reaction procedure was: pre-denaturation at 95℃for 5min, denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, total of 35 cycles, extension at 72℃for 7min and preservation at 4 ℃.
The application of the primer in identifying or assisting in identifying stichopus japonicus growth genes.
A kit for screening a rapidly growing stichopus japonicus, comprising the primer for screening a rapidly growing stichopus japonicus of claim 6 and a screening molecular marker locus.
A method for breeding stichopus japonicus strain with high growth speed by utilizing sif gene molecular markers related to stichopus japonicus growth characteristics is characterized in that:
(1) Amplifying the growth-related sif gene of claim 1 of an individual stichopus japonicus to be detected using the primers set forth in claim 6 or claim 8;
(2) The molecular marker is CT type and CC type at 1538, CT type and CC type at 1574 and CT type at 29283 of the gene, and one or more positions are selected for breeding stichopus japonicus growth characters;
(3) The individuals selected according to the steps are used as seed ginseng for breeding, and stichopus japonicus strain with excellent phenotype and fast growth speed is cultivated
Compared with the prior art, the invention has the advantages that:
1. The invention provides 3 SNP molecular markers and dominant genotypes related to the growth traits of the stichopus japonicus for the auxiliary breeding of the stichopus japonicus molecular markers. The molecular marker is verified in other stichopus japonicus groups, and stichopus japonicus improved varieties with high growth speed can be more efficiently bred.
2. The method is used for breeding stichopus japonicus, the target is accurate, the selection efficiency is higher, the purification effect is better, and the breeding characters are stable.
Drawings
FIG. 1 shows PCR amplification of SNPs detection primers of Apostichopus japonicus (M: DL2000, marker, lane 1, site e2-2.1538T > C, site e2-4.1574T > C, lane 4, amplified fragment of site e 9-2.283T > C).
Detailed Description
The technical contents of the present invention will be described in detail by examples, but the scope of the present invention is not limited in any way by the examples.
The test materials used in the examples of the present invention are all conventional in the art and are commercially available. The experimental procedure, without specifying the detailed conditions, was carried out according to the conventional experimental procedure or according to the operating instructions recommended by the suppliers.
According to the invention, SNP markers and dominant genotypes which can be used for assisting the selective breeding of the growth traits of stichopus japonicus are screened out from sif gene exon regions by a Sanger sequencing method. By using the marker to establish a stichopus japonicus molecular marker assisted breeding method, a new stichopus japonicus variety with a high growth speed and excellent phenotype is bred, and a solution is provided for solving the problems of stichopus japonicus original stock, lack of improved variety and the like.
Example 1: obtaining SNP molecular markers related to stichopus japonicus growth traits
60 Stichopus japonicus were randomly selected as experimental materials from the Dongke group I (variety registration number: GS-01-015-2017) and the general group, respectively, and the stichopus japonicus body wall tissues were subjected to DNA extraction according to the DNA extraction kit (brand: taKaRa) specification, and the concentration was measured using a Nanodrop 2000 ultraviolet-visible spectrophotometer and the DNA quality was evaluated by 1% agarose gel electrophoresis.
The exon regions of sif genes were examined according to Sanger sequencing to identify 26 SNP loci altogether, and in the two populations, multiple comparison analyses were performed for different genotypes and growth traits at the loci, the results are shown in Table 1, wherein 3 loci were significantly different in the two populations. Based on the position of the transcription initiation site, these 3 SNPs were designated as e2-2.1538T > C, e2-4.1574T > C, e 9-2.283T > C, respectively, with the corresponding sites located at 1538, 1574 and 29283 of the sif gene, respectively. The 3 SNP loci all detect the (TT), (CT) and (CC) 3 genotypes. The novel stichopus japonicus of Dongke has the advantage of high growth speed, so that the growth speed of (TT) and (CT) type individuals is higher than that of (CC) type individuals, the growth speed of (TT) and (CT) type individuals at e2-4.1574T > C is higher than that of (CC) type individuals, and the growth speed of (CT) type individuals at e 9-2.283T > C is higher Than That of (TT) and (CC) type individuals at molecular markers e2-2.1538T > C.
Table 1sif 3 significantly different SNPs identified in the exon regions of the genes
Example 2: application of molecular marker in growth trait assisted breeding
Designing SNP locus amplification detection primers according to 3 SNP loci obtained by a sanger sequencing method, wherein the flanking sequences at two sides of the SNP locus amplification detection primers are respectively provided with AJ_e2-2_F5'-TCTCCAAGAATGGGGACCG-3' and AJ_e2-2_R5'-CGCAGATCAGGACCAACTGT-3'; AJ_e2-4_F5'-ACATAA CCGATGGAGCGACA-3' and AJ_e2-4_R5'-TTCCGGGTCAAAGGTCGTG-3'; AJ_e9-2_F5'-AGCACAGCGTTTGGAGATTC-3' and AJ_e9-2_R5'-CCGTGTTGT CTCGTAAATGCC-3'.
The SNP molecular marker amplification detection system comprises: 50ng/ul 1ul of DNA template, 7.5 ul of Premix Taq enzyme, 0.5 ul of forward and reverse primers, 5.5 ul of RNase-FREE WATER, respectively; the PCR reaction procedure was: pre-denaturation at 95℃for 5min, denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, total of 35 cycles, extension at 72℃for 7min and preservation at 4 ℃.
The PCR amplified sequence of the detection primer is gray, namely a molecular marker locus, and specifically comprises the following components:
CCAACCAGAGAAGAAACACAAGTGGCTTCAGATTCTTTACAGTTGTTGCTGAGCG
TTGAATTACCGACAGGTCTGCAGACCGTGGACGATCAAGGCAACACGTCCACCAA
TCACAGCCCTGCGATGGTGAACGGACGCAAAGATGGCTCCCGTCCGGGTCAGACG
GGCCTCTTCTCTACCCTGGCCCTGCCGGACAAGAAAACAGATTCCGTCCCCGTCTC
TGGCAGCGTCCGAAAGGCCATCGCAAACCGCAAAGATCAGCGCAAGTTGAAATAT
GCGACATACTCCGGAAAGAACCGCGAGGCCCTGCAGAGGTCTTACTACGAAAACA
TGGATACGGAGGACGGTAAATCGGAGGACTTTGACGACTCCACCCTTAAAGCATC
CAAGTCGAAACCGTCCAGGCACGGAAGCCACCCCGAGCTGGATATTGCCTCCAGC
AGTCCGAGTGCCGACGAGCACCATGAATTGGTCAGACAGCTAAGTGGGAGCCCGG
GGAGGGGCAGCGGGTATTTAAGCGCATCTCAAATGGACGCGGGCATAAACAATGG
TGTCTTGAAGAGCGCCAAGTTCGTGGCAGCGAACGCCCAAAATTTCAAGACGGCA
AACAGCGTCTCCCAGATCACCCCTACGACACCCGAAAAGGAATTAAAAAATGACA
GAAAGTCTGGCATTAGGACTCGACTGAAGGTGGAGGGGCAGTCTTTCTTCGGTCG
TGGGAACAAGACGAAGACGAAAAACAAAGCCAAGAGGTCTACCAGTTTCAGCGA
GAAATTCTTCACCCGGGTCCACGTGAAGGATAACGCCAACAAGAACAGTCTGGAC
GAGGCCCAGCGGGTACTGGAAGAAACGAACTCGCTCTTACAGGCCTACGAGGTTC
ACCTGCAGATGCAGACACTCGACGAAACGATAGACGGTCTACCTTCGACCGGAGA
CGACCGATCGGTGGCGTCCAAGTCGTCGTGTGGGACCGCATCGGATATACCGAAAT
GGAAGAAGCGTCTCTCAGAGGGCATCACCAGTGGCAGCGAGTGCAGTAGCTTAAG
TCACCATGGCGACGGTAGTACTTTGACCTCAAGTGAGACCATGTCCAAAGGGGAT
CAGGATCTACAGGAGTTCTTCTACCACGATGAGGAGGACATGGAAGAGGAGGAGG
ATGGTGAGGAGGATGGAGACAGCCATTCTCTCAGCTCACACCACACACACGCTTC
CATGTTCCAACAGAGGGGCGCCATCAGGAAGGCCGGTTGGCTGCACGTCAAGAGC
ATGCTCGTACAGAGGAAGAAACGGGTGGAACGACCAGGCAAGAGGTCCTGGAAG
AAATATTGGGTCTGTTTGAAAGGAACAATGCTGTTATTCCATGCACCCTCAGACAG
AGAAACCGATGAGAGCAGTGAACCTAG
ACATATCCTGGTGGTTGAGAACGGCCTCGCCCAGGCGGTCCCAGAGCATCCCAAG
CGAGAGAACATCTTCTGTTTGAGCACAGCGTTTGGAGATTCCTATTTCTTACAAGC
ACCTAATCAGATTGAGCTCGAAAATTGGACCATTGCAATACATTCAGCCTGTTCCTCG
GCATTTACGAGACAACACGGAAAAGAGGAAACTTTACGTCTTCTACGATTAGAAACACACAAA
CTGGAGGACAAGATTGAACTGGAAGACAAAGAAGATGGCCGAGCTGCAGCTGAGCAC
GGTTTCGGATCCAAAGAATCGACAAGCTATAGCATCTCAGGTAGAGACAATGCCCA
CGATAATTGATTGTTATGATGCATTGTGTGACTCAAGTACAGGAAAACGACTTGCAT
GTTACTCTGTTTGTTTTTGTGTCGTCAGAGTTGGATTAGAAGCCTGGGAATTATTCC
ATGTCGTACAATGA
In the stichopus japonicus seedling stage, randomly selecting parent stichopus japonicus, carrying out genotyping on the SNP locus by PCR amplification, and selecting individuals with genotypes at e2-2.1538T > C (TT) and (CT), e2-4.1574T > C (TT) and (CT) and e9-2.29283T > C (CT) according to the genotyping result.
Claims (2)
1. The application of a primer or a kit for SNP molecular marker genotyping in screening, identifying or breeding stichopus japonicus with fast growth speed is characterized in that the molecular marker is an exon region of a gene sif in a stichopus japonicus genome No. 18 linkage group, and the nucleotide sequence of the exon region is shown as a Seq ID No. 1; one or more of a T > C mutation at 1538, a T > C mutation at 1574, a T > C mutation at 29283, starting at the transcription initiation site of the gene;
At the molecular marker e 2-2.1538T > C, TT and CT type individuals grow faster than CC type individuals at e 2-4.1574T > C, and CT type individuals grow faster than TT and CC type individuals at e 9-2.283T > C;
the kit comprises primers, wherein the primers are as follows:
AJ_e2-2_F:CTCTCCAAGAATGGGGACCG；
AJ_e2-2_R: CGCAGATCAGGACCAACTGT；
AJ_e2-4_F:ACATAACCGATGGAGCGACA；
AJ_e2-4_R:TTCCGGGTCAAAGGTCGTG；
AJ_e9-2_F:AGCACAGCGTTTGGAGATTC；
AJ_e9-2_R:CCGTGTTGTCTCGTAAATGCC。
2. a method for breeding stichopus japonicus strain with high growth speed is characterized in that:
(1) Amplifying the growth-related sif gene of claim 1 in an individual of stichopus japonicus to be detected using the primer of claim 1;
(2) Molecular markers are used for breeding stichopus japonicus with high growth speed and good quality at one or more sites among T > C mutation at 1538, T > C mutation at 1574 and T > C mutation at 29283 which are started at the transcription initiation site of the gene;
(3) The individuals selected according to the steps are used as seed ginseng for breeding, and stichopus japonicus strains with excellent phenotype and high growth speed are cultivated.
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| CN118460736A (en) * | 2024-07-09 | 2024-08-09 | 中国科学院海洋研究所 | Apostichopus japonicus multi-thorn character SNP molecular marker combination and application thereof |
| CN118562976B (en) * | 2024-07-11 | 2024-11-08 | 中国科学院海洋研究所 | Apostichopus japonicus growth trait SNP molecular marker combination based on whole genome association analysis and application |
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| CN107937569A (en) * | 2018-01-16 | 2018-04-20 | 中国水产科学研究院黄海水产研究所 | A kind of molecular labeling and its application for stichopus japonicus growth traits assisted selection |
| CN113789391A (en) * | 2021-07-07 | 2021-12-14 | 中国海洋大学 | Apostichopus japonicus breeding whole genome 50K SNP chip and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107937569A (en) * | 2018-01-16 | 2018-04-20 | 中国水产科学研究院黄海水产研究所 | A kind of molecular labeling and its application for stichopus japonicus growth traits assisted selection |
| CN113789391A (en) * | 2021-07-07 | 2021-12-14 | 中国海洋大学 | Apostichopus japonicus breeding whole genome 50K SNP chip and application |
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