AU2021104302A4 - Marker primer combination for molecular identification of quantitative traits of spines of apostichopus japonicus and use thereof - Google Patents
Marker primer combination for molecular identification of quantitative traits of spines of apostichopus japonicus and use thereof Download PDFInfo
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- 241000965254 Apostichopus japonicus Species 0.000 title claims abstract description 72
- 239000003550 marker Substances 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 claims abstract description 14
- 238000003205 genotyping method Methods 0.000 claims abstract description 11
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims abstract description 8
- 238000012360 testing method Methods 0.000 claims abstract description 3
- 230000002068 genetic effect Effects 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 23
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- 238000012163 sequencing technique Methods 0.000 description 8
- 239000003147 molecular marker Substances 0.000 description 7
- 241000028318 Parapodium Species 0.000 description 6
- 238000001514 detection method Methods 0.000 description 4
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- 241001465754 Metazoa Species 0.000 description 3
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- 238000007400 DNA extraction Methods 0.000 description 1
- 241000237516 Mizuhopecten yessoensis Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000003975 animal breeding Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
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- 238000012165 high-throughput sequencing Methods 0.000 description 1
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- 230000000877 morphologic effect Effects 0.000 description 1
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Abstract
OF THE DISCLOSURE
The present disclosure relates to a marker primer combination for molecular identification
of quantitative trait of spines of Apostichopus japonicus and use thereof, and belongs to the
field of molecular biology. The primer combinations are set forth in SEQ ID NO:1 to SEQ ID
NO:15. The method is specifically as follows: extracting DNA from the A. japonicus sample by
sodium dodecyl sulfate (SDS) method, using a KASP combination marker to conduct SNP
genotyping on the test sample, and detecting specific marker loci of spiney or less spiney A.
japonicus; the molecular identification standard for the spiney A. japonicus is that: each
individual has at least three markers covered. The method can determine the spine number of A.
japonicus from a genetic point of view.
17885566_1 (GHMatters) P116743.AU
Description
[01] The present disclosure belongs to the field of molecular biology, and particularly relates to a marker primer combination for molecular identification of quantitative trait of spines of Apostichopusjaponicus(A. japonicus) and use thereof.
[02] Molecular markers refer to DNA sequences or proteins that are heritable and detectable; molecular marker of Mizuhopecten yessoensis refers to a specific DNA fragment that reflects certain differences in genome between individuals and populations. Compared with morphological markers, biochemical markers, and cytological markers used in conventional breeding, DNA molecular markers have outstanding superiority, and are not influenced by the development and environment of the biological individuals. Through the characteristic of tight linkage of the molecular marker to a gene determining the target trait, molecular marker-assisted breeding uses molecular markers to detect the presence or absence of a target gene to achieve the selection of a target trait. The molecular marker-assisted breeding can serve as a supplementary means in the process of the breeding such as the identification of the parental relationship, the transfer of quantitative traits and recessive traits in backcross breeding, the selection of hybrid progenies, the prediction of heterosis, and the identification of varietal purity. The molecular marker-assisted breeding is of great importance in the genetic breeding of aquatic animals, which can clarify trait characteristics of progenies obtained by population breeding, and effectively avoid missing the target trait by detecting molecular markers; meanwhile, molecular marker information can further be used to analyze the levels of genetic variation of progenies and guide the population breeding. Herein, single nucleotide polymorphism (SNP) markers play an important role in the genetic breeding due to their low cost and high efficiency.
[03] In aquatic economic animal breeding technology, selection of individuals with different genetic traits by using molecular markers is of guiding significance in parents selection before breeding season. Due to the rapidness and high efficiency, the molecular marker-assisted breeding technology is suitable for aquatic animals with high fecundity and spermiation, can be directly used in the production process of parents selection, and is widely used in the prevalent variety cultivation of aquatic animals. A. japonicus is a common rare sea treasure in North
1 17885566_1 (GHMatters) P116743.AU
China. Sensory evaluation criteria for high-quality A. japonicus can be summarized as "black color, glutinous flesh, and spiny". An important breeding feature of new varieties A. japonicus, Shuiyuan No. 1 and A. japonicus, Anyuan No. 1 approved in China is six rows of spines, and both varieties have more than 45 spines, significantly higher than that of common A. japonicus. A. japonicus with spiney trait is favored by the market, and breeding of spiney A. japonicus may bring remarkable economic benefits. The spiney trait is determined by genetic factors and influenced by a plurality of environmental factors (for example, temperature is one of the factors, and there is a difference in spine number among A. japonicus in the same line under different temperature environments); use of spine phenotype alone for parents selection during breeding may result in missed selection or wrong selection of parents of the spiney A. japonicus. The present disclosure provides a marker primer combination for molecular identification of spines quantitative trait of A. japonicus and a using method, wherein parents are selected from the perspective of gene, and the spine number is determined and identified genetically, thus realizing early selection of economic traits and making the parents selection more accurate and economical.
[04] The technical problems to be solved in the present disclosure is to provide a marker primer combination for molecular identification of quantitative trait of spines of A. japonicus and use thereof, and the marker primer combination can determine the spine number of A. japonicus from a genetic point of view.
[05] The present disclosure is achieved by the following technical solutions:
[06] a marker primer combination for detecting quantitative trait of spines of A. japonicus, wherein the marker primer combination is shown in Table 1;
[07] Table 1 KASP marker combination for detecting the spine number of A. japonicus
No. less spiney SNP locus spiney SNP locus Forward primers 1 and 2 Reverse primer
5' GCTTTGGTGGAGTTCTTT 3' (SEQ ID NO:1) 5' TGACTGACCGACTGAGAAG 3' 5' GGTCGGTATTATTTGTGAA 3' (SEQ ID NO:3) (SEQ ID NO:2) 5' GAGAATGGTATCCCAGCAA 3' (SEQ ID NO:4) 5' AAAGCATCCCGTGTCTG 3' RC-002 [A/G] [A/A] 5' AACCAAGTACAATGTAGGACC (SEQ ID NO:6) 3' (SEQ ID NO:5)
2 17885566_1 (GHMatters) P116743.AU
5' GAGTTACCCCTCTTTCACC 3' (SEQ ID NO:7) 5' AACGGTGTTATTGTCATTCTT 3' 5' CACCATCTGGCAATAAAT 3' (SEQ ID NO:9) (SEQ ID NO:8) 5' CTTCCCCACCACCACTT 3' (SEQIDNO:10) 5' CCACCTAACAACCAAACAA 3' 5' AAGTAAACCCGTCCATCC 3' (SEQ ID NO:12) (SEQ ID NO:11) 5' TAAAGGGCAACCAAAAG 3' (SEQ ID NO:13) 5' AAGACGAATACCAAAGAAAC 3' 5' AAGTCCCTTAGCCAGTTC 3' (SEQ ID NO:15) (SEQ ID NO:14)
[08] *Hatched loci are specific genotypes of spiney A. japonicus.
[09] The present disclosure further provides a method for identifying the parapodium number of A. japonicus using the above primer combination, wherein the method is specifically as follows: extracting DNA from an A. japonicus sample by sodium dodecyl sulfate (SDS) method, using a KASP marker combination to conduct SNP genotyping on the test sample, and detecting specific marker loci of spiney or less spiney A. japonicus. A determination criterion is that: it is not necessary to detect all individuals carrying spiney A. japonicus markers in practical production, but in order to ensure detection accuracy, at least three markers in which there is no less-spiney marker should be covered in each individual.
[10] The technical solutions of the present disclosure will be further described with reference to the examples, but the protection scope of the present disclosure is not limited by the examples in any manner.
[11] EXAMPLE 1: Screening of markers
[12] A complete genome sequence of A. japonicus was used as a reference in the study, 215 A. japonicus individuals from eight different representative regions were subjected to whole-genome re-sequencing, respectively, and SNP marker loci related to the parapodium number of A. japonicus were obtained by comparing with the reference genome; accordingly, KASP primers were designed, and genotyped, and then verified in the A. japonicus population with different parapodium numbers; finally, an SNP primer sequence combination for identifying the parapodium number of A. japonicus was obtained.
[13] A specific method was as follows:
[14] 1. Acquisition of markers
[15] 1.1 DNA sample extraction 3 17885566_1 (GHMatters) P116743.AU
[16] A total of 215 representative and healthy A. japonicus were selected, including A. japonicus that from Liaoning (main A. japonicus producing areas in China), Shandong, and Russia (Vladivostok) and new variety A. japonicus, Shuiyuan No. 1. Wherein, there were 210 A. japonicus selected from sea areas of Lishun (at the junction between the Yellow Sea and the Bohai Sea), Xixiaomo (high temperature resistant), Shandong (low latitude), Huanglongwei (offshore waters of the Yellow Sea), Baxiao Island (open seas of the Yellow Sea), Pingdao Island (artificial rearing), and Russia (Vladivostok) (with 30 in each sea area), and there were five A. japonicus, Shuiyuan No. 1. Genomic DNAs were extracted from the above A. japonicus by using the SDS method, and DNA concentrations were greater thanl00 ng/[l.
[17] 1.2 Genome re-sequencing
[18] Sample quality inspection, library construction, library quality inspection, and library sequencing were implemented in accordance with the protocol standard (second-generation high-throughput sequencing provided by Illumina). Qualified DNA samples were randomly broken into 350 bp fragments by Covaris Crusher, and the whole library preparation was completed by end repair, ployA-tail addition, sequencing adapter addition, purification, and 10 cycles of amplification were in a PCR system. The qualified libraries were subjected to whole-genome re-sequencing on the Illumina platform; every four rows of reads of original double-ended sequences obtained by sequencing were used as a unit to count the number of sequences, and the quality was evaluated. The evaluation principles were as follows:
[19] (1) removing an adapter;
[20] (2) removing reads that contains N (N indicates failure to determine the base information) greater than 5%;
[21] (3) removing low-quality reads (bases with quality value of Q < 10 accounts for more than 20% of the reads); and
[22] (4) calculating the original sequencing quantity, the effective sequencing quantity, and the content of Q20, Q30, and GC, and evaluating comprehensively.
[23] Filtered Clean Reads were compared with the reference genome sequence in BWA software. According to the location results of the Clean Reads in the reference genome, SNPs and InDels were detected by using GATK software; according to the principles of MAF > 0.05 and data integrity > 0.8, variable loci were subjected to quality filtering, and SNP loci with two-allelic polymorphism were preserved.
[24] 1.3 Correlation analysis
[25] Combined with the data of spines of A. japonicus, the SNP loci were correlated by a generalized linear model (GLM) in plink software, correlated p-value was obtained by calculating each SNP locus, and a significance threshold was obtained by Bonferroni correction. 4 17885566_1 (GHMatters) P116743.AU
A Manhattan plot was plotted by using an emmax model (tested by QQ-plot); and 0.95 and 0.99 were set as thresholds, a string of loci above the thresholds were SNP loci related to the trait of spine number.
[26] 1.4 Genotyping and screening of specific SNP loci
[27] According to the comparison results of samples with the reference genome, variable loci with differences among samples were summarized, and 50 high-quality SNP loci were selected therefrom for subsequent KASP verification. Screening principles were as follows: (1) the locus had different dominant genotypes in samples of A. japonicus with different parapodium numbers (for example, AA or AT genotype was dominant in a sample with few parapodia, and inferior in a sample with more parapodia); (2) adjacent variable loci could not be too close, with a distance of more than 50 bp; and (3) the forward and reverse sequences at the locus are unique in the genome, and there are few repetitive sequences. 200 bp of the forward and reverse sequences at high-quality SNP loci were extracted to develop markers.
[28] 1.5 KASP primer design
[29] Fifty pairs of 3'-terminal-specific PCR amplification primer were designed by Primer 5 software according to the SNP loci, and each sequence needed three primers, i.e., two specific forward primers and one universal reverse primer. Primer design principles were as follows: (1) the primer itself had no hairpin structure, and no primer dimer was formed between the forward and reverse primers; and (2) the GC content of the primer was 40-60%, and the Tm value was -65 0 C.
[30] 2. Verification of KASP markers
[31] 2.1 DNA extraction and marker detection of the verification population
[32] Ninety-two A. japonicus from different batches (60 were those from marked sources populations, 30 were those from different batches, and the difference in parapodium number was >10) were randomly selected; DNAs were extracted by the SDS method; markers were detected on the Douglas Array Tape® Platform; fluorescence quantitative PCR was conducted in a SOELLEX high-throughput PCR water bath environment, TB GreenTM Premix Ex TaqTM i was used as a reagent, and a 20 pl mixture system was formulated to conduct specific amplification according to a two-step reaction program of "degeneration-annealing extension".
[33] 2.2 Genotyping of KASP markers
[34] Two fluorophore signals, FAM and VIC, were read on an ARAYA fluorescent reader, results were imported to a database, and samples were subjected to SNP genotyping in Douglas Scientific Dashboard according to the principles of definite genotyping, no template control (NTC), and no specific amplification. The genotyping results were analyzed by using genotype reading software (SNP viewer); the genotyping was considered to be successful once KASP 5 17885566_1 (GHMatters) P116743.AU markers in samples were read into heterozygous genotypes. In a genotyping map, X:X represents a homozygous genotype, including A:A, T:T, C:C, G:G, and -:- (deletion); X:Y represents a heterozygous genotype, including A:G, C:T, and A:- (partial deletion); "?" represents no signal or a weak signal; "Uncallable" represents the presence of a signal but no definite genotyping. Ten successfully genotyped KASP markers were obtained finally.
[35] Markers that the spiney genotype were homozygous were eliminated from 50 KASP markers, and five markers with the highest detection rate were screened out(see Table 1).
[36] 3. Identification and use of spiney A. japonicus
[37] Among 60 A. japonicus individuals from the original batch (i.e., marked source population) and 32 A. japonicus individuals bred from different batches, the KASP markers obtained were verified. The results showed that:
[38] (1) For the A. japonicus individuals from the original batch (30 spiney A. japonicus and less spiney A. japonicus from 8 populations, wherein spiney individuals had more than 45 spines on average, and less spiney individuals had fewer than 35 spines), the marker coverage rate of spiney A. japonicus was 100%, and each spiney A. japonicus sample had a marker coverage rate of 3.5 markers on average.
[39] (2) Population from different batches was selected, with 32 A. japonicus individuals contained, wherein, spiney A. japonicus individuals were 20, with 48-65 spines, and less spiney A. japonicus individuals were 12, with 28-32 spines, the marker coverage rate of spiney A. japonicus was 85%, each spiney A. japonicus sample had a marker coverage rate of 2.5 markers on average, and no confounding marker was detected in spiney and less spiney A. japonicus individuals.
[40] (3) Example of early selection: Two cultured A. japonicus populations were selected; at the age of 0.5, 30 A. japonicus individuals were randomly selected from each population for breeding; by the age of 2 (able to propagate progenies), the average spine number was 36 and 28, respectively. Meanwhile, the above two A. japonicus populations were dealt with marker detection at the age of 0.5, and 30 A. japonicus populations that each with at least three spiney markers and no less spiney marker were selected for culture; by the age of 2 (able to propagate progenies), the average spine number was 62 and 46, respectively.
[41] (4) When being used in the selective breeding of spiney A. japonicus, the markers were used to select seed A. japonicus with at least three spiney markers (and no less spiney marker) for subsequent propagation and breeding, so as to breed new spiney A. japonicus varieties.
[42] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in
Australia or any other country. 6 17885566_1 (GHMatters) P116743.AU
[43] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of
the invention.
7 17885566_1 (GHMatters) P116743.AU
SEQUENCE LISTING 19 Jul 2021
<110> Dalian Ocean University <120> MARKER PRIMER COMBINATION FOR MOLECULAR IDENTIFICATION OF QUANTITATIVE TRAITS OF SPINES OF APOSTICHOPUS JAPONICUS AND USE THEREOF
<130> GWP202105436
<160> 15 2021104302
<170> PatentIn version 3.5
<210> 1 <211> 18 <212> DNA <213> Artificial Sequence
<220> <223> forward primer 1 of KASP marker combination RC‐001
<400> 1 gctttggtgg agttcttt 18
<210> 2 <211> 19 <212> DNA <213> Artificial Sequence
<220> <223> forward primer 2 of KASP marker combination RC‐001
<400> 2 ggtcggtatt atttgtgaa 19
<210> 3 <211> 19 <212> DNA <213> Artificial Sequence
<220> <223> reverse primer of KASP marker combination RC‐001
<400> 3 tgactgaccg actgagaag 19
<210> 4 <211> 19 <212> DNA <213> Artificial Sequence
<220> <223> forward primer 1 of KASP marker combination RC‐002
<400> 4 gagaatggta tcccagcaa 19
Page 1
<210> 5 <211> 21 <212> DNA <213> Artificial Sequence
<220> <223> forward primer 2 of KASP marker combination RC‐002
<400> 5 aaccaagtac aatgtaggac c 21 2021104302
<210> 6 <211> 17 <212> DNA <213> Artificial Sequence
<220> <223> reverse primer of KASP marker combination RC‐002
<400> 6 aaagcatccc gtgtctg 17
<210> 7 <211> 19 <212> DNA <213> Artificial Sequence
<220> <223> forward primer 1 of KASP marker combination RC‐003
<400> 7 gagttacccc tctttcacc 19
<210> 8 <211> 18 <212> DNA <213> Artificial Sequence
<220> <223> forward primer 2 of KASP marker combination RC‐003
<400> 8 caccatctgg caataaat 18
<210> 9 <211> 21 <212> DNA <213> Artificial Sequence
<220> <223> reverse primer of KASP marker combination RC‐003
<400> 9 aacggtgtta ttgtcattct t 21
Page 2
<210> 10 19 Jul 2021
<211> 17 <212> DNA <213> Artificial Sequence
<220> <223> forward primer 1 of KASP marker combination RC‐004
<400> 10 cttccccacc accactt 17 2021104302
<210> 11 <211> 18 <212> DNA <213> Artificial Sequence
<220> <223> forward primer 2 of KASP marker combination RC‐004
<400> 11 aagtaaaccc gtccatcc 18
<210> 12 <211> 19 <212> DNA <213> Artificial Sequence
<220> <223> reverse primer of KASP marker combination RC‐004
<400> 12 ccacctaaca accaaacaa 19
<210> 13 <211> 17 <212> DNA <213> Artificial Sequence
<220> <223> forward primer 1 of KASP marker combination RC‐005
<400> 13 taaagggcaa ccaaaag 17
<210> 14 <211> 18 <212> DNA <213> Artificial Sequence
<220> <223> forward primer 2 of KASP marker combination RC‐005
<400> 14 aagtccctta gccagttc 18
<210> 15
Page 3
<211> 20 19 Jul 2021
<212> DNA <213> Artificial Sequence
<220> <223> reverse primer of KASP marker combination RC‐005
<400> 15 aagacgaata ccaaagaaac 20 2021104302
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Claims (2)
1. A marker primer combination for molecular identification of quantitative trait of spines of Apostichopusjaponicus,wherein the marker primer combination is shown in Table 1 below: Table 1 KASP marker combination for detecting the spine number of Apostichopusjaponicus No. less spiney SNP locus spiney SNP locus Forward primers 1 and 2 Reverse primer
5' GCTTTGGTGGAGTTCTTT 3' RC-001 [T/G] [GIG] (SEQ ID NO:1) 5' TGACTGACCGACTGAGAAG 3' 5' GGTCGGTATTATTTGTGAA 3' (SEQ ID NO:3) (SEQ ID NO:2) 5' GAGAATGGTATCCCAGCAA 3' (SEQ ID NO:4) 5' AAAGCATCCCGTGTCTG 3' RC-002 [A/G] [A/A] (SQDO4 5' AACCAAGTACAATGTAGGACC 3' (SEQ ID NO:6) (SEQ ID NO:5) 5' GAGTTACCCCTCTTTCACC 3' (SEQ ID NO:7) 5' AACGGTGTTATTGTCATTCTT 3' RC-003 [GIG] [A/A] (SQDO7 5' CACCATCTGGCAATAAAT 3' (SEQ ID NO:9) (SEQ ID NO:8) 5' CTTCCCCACCACCACTT 3' (SEQIDNO:10) 5' CCACCTAACAACCAAACAA 3' 5' AAGTAAACCCGTCCATCC 3' (SEQ ID NO:12) (SEQ ID NO:11)
5' TAAAGGGCAACCAAAAG 3' (SEQ ID NO:13) 5' AAGACGAATACCAAAGAAAC 3' 5' AAGTCCCTTAGCCAGTTC 3' (SEQ ID NO:15) (SEQ ID NO:14)
wherein hatched loci are specific genotypes of spiney Apostichopusjaponicus.
2. A method for molecular identification of quantitative trait of spines of Apostichopus japonicus by using the primer combination according to claim 1, wherein the method is specifically as follows: extracting DNA from the Apostichopus japonicus sample by sodium dodecyl sulfate (SDS) method, using a KASP combination marker to conduct SNP genotyping on the test sample, and detecting specific marker loci of spiney or less spiney Apostichopus japonicus;wherein the molecular identification standard for the spiney Apostichopusjaponicus is that: each individual has at least three markers covered, and no less-spiney marker.
8 17885566_1 (GHMatters) P116743.AU
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| CN202110175759.3A CN113151487A (en) | 2021-02-09 | 2021-02-09 | Molecular identification marker primer combination for quantitative character of stichopus japonicus and thorn and application method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113151487A (en) * | 2021-02-09 | 2021-07-23 | 大连海洋大学 | Molecular identification marker primer combination for quantitative character of stichopus japonicus and thorn and application method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114525362B (en) * | 2022-03-18 | 2023-05-23 | 大连海洋大学 | Primer combination for identifying Apostichopus japonicus population of Anyuan No. 1 and application thereof |
| CN115948577B (en) * | 2023-01-09 | 2023-08-22 | 山东省渔业发展和资源养护总站 | Breeding method of high-temperature-resistant sea cucumber |
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| CN107937569B (en) * | 2018-01-16 | 2018-10-30 | 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 |
| CN112080570A (en) * | 2020-10-26 | 2020-12-15 | 大连海洋大学 | KASP labeled primer combination for identifying hybrid stichopus japonicus in Zhongrussia and application thereof |
| CN112251533B (en) * | 2020-11-09 | 2022-08-23 | 浙江省农业科学院 | SNP molecular marker closely linked with main effect QTL site of eggplant fruit length and application |
| CN113151487A (en) * | 2021-02-09 | 2021-07-23 | 大连海洋大学 | Molecular identification marker primer combination for quantitative character of stichopus japonicus and thorn and application method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113151487A (en) * | 2021-02-09 | 2021-07-23 | 大连海洋大学 | Molecular identification marker primer combination for quantitative character of stichopus japonicus and thorn and application method thereof |
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