CN107937569B - A kind of molecular labeling and its application for stichopus japonicus growth traits assisted selection - Google Patents

A kind of molecular labeling and its application for stichopus japonicus growth traits assisted selection Download PDF

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CN107937569B
CN107937569B CN201810039145.0A CN201810039145A CN107937569B CN 107937569 B CN107937569 B CN 107937569B CN 201810039145 A CN201810039145 A CN 201810039145A CN 107937569 B CN107937569 B CN 107937569B
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stichopus japonicus
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CN107937569A (en
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廖梅杰
李彬
王印庚
荣小军
刘安然
陈贵平
范瑞用
张正
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Abstract

The invention discloses a kind of molecular labeling for stichopus japonicus growth traits assisted selection and its applications.Belong to molecular mark field, which marks for SNP40, and the primer pair of the label is SNP40F:5 '-GGGTGGACTTGGAGCAGAAG-3 ' and SNP40R:The step of 5 '-ACCAACGTCCTCTCTGGTTATCA-3 ', preponderant genotype of the label for growth traits assist-breeding is CC, and the present invention also discloses above-mentioned marker-assisted breedings.The present invention provides effective molecular labeling to grow this economic characters from genetically raising stichopus japonicus, assisted Selection is marked to stichopus japonicus with the genetic marker, carry out hereditary and selection, the speed of growth of juvenile stichopus can be effectively improved, is of great significance to improving culture efficiency.

Description

A kind of molecular labeling and its application for stichopus japonicus growth traits assisted selection
Technical field
The invention belongs to molecular mark fields, and stichopus japonicus growth traits assisted Selection is used for more particularly to one kind The molecular labeling of breeding and its application.
Background technology
Stichopus japonicus belongs to Echinodermata Echinodermata, Holothuroidea Holothuroidea, is that northern China is important One of precious marine product.In recent years, China's apostichopus japonicus culture is able to tremendous development, and scale expands swift and violent, the direct economy output value about 30,000,000,000 Member is the single maximum species of the output value in China's sea-farming industry, is adjustment and the fisherman's employment of inshore fishing economic structure And increasing both production and income provides important channel.But with the continuous expansion of stichopus japonicus industry size, germplasm degenerates, is slow-growing, foster Grow a series of restrictions such as the period is long, resist environmental change energy force difference, disease takes place frequently or the potential bottleneck problem for restricting industry development Also increasingly prominent.Genetic improvement is carried out to stichopus japonicus, cultivates the new varieties for waiting merits soon with the speed of growth, is that stichopus japonicus is supported Grow the important guarantee of industry sound development.
The fine-variety breeding work of stichopus japonicus relative to other economic aquatic biologicals carry out later, current selection and breeding mode still with Based on selection and use and crossbreeding, other such as marker assisted selections, genome selection and use core breeding technique are studied still It is in the junior stage.
As stichopus japonicus studies the increase (Sun Guohua etc., 2011) of temperature, stichopus japonicus character related SNP is excavated at present more next More, Gao etc. (2013) develops 26 relevant SNP sites of defense mechanism with stichopus japonicus;Dong etc. (2016) is to wild It is explained with 51 associated SNP positions have been excavated using HRM in the stichopus japonicus genetic diversity of artificial breeding and the research of population structure The reason of genetic diversity reduces;Li et al. (2016) has been cloned stichopus japonicus myostain genes and has been found that related to stichopus japonicus dry weight 3 SNP sites;Dong Yu etc. (2016) is found that 10 growth traits are significantly correlated in the association analysis to SNP and growth Site.But these labels do not carry out expansion verification and the productivity verification of next step, the feasibility and accuracy of application Need further to be verified.
In order to develop the stichopus japonicus growth traits related molecular marker that can be used for practical selection and breeding production practices, applicant constructs Stichopus japonicus high density genetic linkage maps carry out QTL positioning, selective body using variance analysis and composite interval mapping to weight character Weight QTL sections and the SNP site that may be adapted to design of primers, SNP marker is carried out using designed primer in widened group Verification, filters out growth traits related locus and the preponderant genotype in site, and productivity verification has been carried out in Breeding Practice, The label and its preponderant genotype that can be used for stichopus japonicus growth traits molecular marker assisted selection breeding are finally obtained.Utilize the mark Note establishes stichopus japonicus marker assisted selection method, and applies it to stichopus japonicus fine-variety breeding process, cultivates the fast thorn of the speed of growth Join new varieties, by effective germplasm degenerate problem for solving apostichopus japonicus culture industry and facing at present, and effectively pushes apostichopus japonicus culture breeding Change process.
Invention content
The technical problem to be solved in the present invention is to provide a kind of molecule marks for stichopus japonicus growth traits assisted selection Note and its application, include the preponderant genotype and detection method of molecular labeling primer and mark of correlation, and it is fast to use it for growth The selection and breeding of stichopus japonicus material.
The present invention is realized by following technical solution:
A kind of molecular labeling for stichopus japonicus growth traits assisted selection, which marks for SNP40, described SNP40 labels are (CC) for the preponderant genotype of growth traits assist-breeding.
Further, the primer pair of the label is SNP40F:5 '-GGGTGGACTTGGAGCAGAAG-3 ' and SNP40R: 5’-ACCAACGTCCTCTCTGGTTATCA-3’。
Further, which is with system:DNA profiling 50ng/ μ L1 μ L, 4.5 μ 2 × ES of L Taq Master Mix, each 0.5 μ L of upstream and downstream primer, 10 μm of ol/L, 3.5 μ L ddH2O;Label parting be with PCR response procedures:95 DEG C pre-degeneration 5min;94 DEG C of denaturation 30s, 60.5 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 recycle;Last 72 DEG C of extensions 7min, 4 DEG C of preservations;Parting is carried out using high-resolution melting curve analysis technology (HRM) method.
Further, it includes (1) parent DNA extractions and parting to carry out stichopus japonicus growth traits assistant breeding using the label;(2) Juvenile stage;(3) 3 parts of filial generation seed breeding.
Further, the parent DNA extractions described in previous step and parting are with the certainly candidate parent's pipe foot of invasive methods or spine It pierces and extracts DNA at tissue, parting is carried out to the genotype of parent's corresponding site with SNP40 labels.
Further, in the Juvenile stage, the advantage parent selected is in the individual that the loci gene type is (CC).Into One step, the filial generation seed breeding are to state the parent selected with step (2) to carry out filial generation seed breeding for parent's breeding population.
The advantageous effect of the present invention compared with prior art:
1, the present invention for stichopus japonicus growth traits breeding work molecular marking supplementary breeding provide one more efficiently and accurately, Easy-to-use molecular genetic marker, for the raising stichopus japonicus speed of growth, this important character provides effective molecular marker breeding Means.It is verified in family, expansion development group and breeding practice simultaneously using respective markers and combinations thereof, with this Assisted Selection is marked to stichopus japonicus growth in label combination, can effectively alleviate the problems such as speed of growth in actual production is slow, It is of great significance to improving culture efficiency.
2, it is combined using the molecular labeling and carries out molecule selection and breeding, with clearly defined objective, efficiency of selection is high, and Breeding Traits are steady after selection and breeding It is fixed.
3, detection method is easy to operate, and expense is more cheap, and accuracy is high, and can realize that automated proceduresization are examined It surveys, will play a great role in stichopus japonicus breeding for disease resistance.
Description of the drawings
Fig. 1 is stichopus japonicus high density genetic linkage maps and weight QTL;
Fig. 2 is the growing state that different genotype parent breeds filial generation seed.
Specific implementation mode
Describe the technology contents of the present invention in detail below by embodiment, but protection scope of the present invention is not appointed by embodiment What formal limitation.
Embodiment 1:The acquisition and its verification in family progeny that stichopus japonicus growth traits selection and breeding genetic molecule marks
Stichopus japonicus family is built, it is flat by high-flux sequence with 2 parents of the family and 142 sons on behalf of mapping population Platform, exploitation SLAF labels, utilizes HighMap software buildings stichopus japonicus high density genetic linkage maps.After high-flux sequence, open altogether Send out 264,810 SLAF labels, wherein 112,322, the SLAF labels of polymorphism.Depth is sequenced in SLAF label parental means 23.67 ×, offspring mean be sequenced depth be 5.44 ×.Bioinformatic analysis is crossed in promoting menstruation, can be used for genetic map construction Label has 50,905, after mass filter, obtains the polymorphism label that sequencing depth, integrity degree are high and do not detach partially 4629.By filter out 4629 SLAF labels, make linkage analysis, calculate recombination fraction and MLOD values between label two-by-two, 23 linkage groups are obtained, altogether figure 4 above, 002, is positioned as icon note, upper figure rate 86.45%.As unit of linkage group, use The analysis of HighMap softwares obtains the linear array of Marker in linkage group, and estimates the genetic distance between adjacent Marker, builds Dense genetic map is shown in Fig. 2.Wherein, neutral collection of illustrative plates includes 4002 labels, and total figure is away from for 3223.84cM;Male collection of illustrative plates is total Including 2228 upper icons notes, total figure is away from for 3864.73cM;Female collection of illustrative plates includes 2307 upper icons notes altogether, total figure away from for 2075.84cM。
According to the measurement result of 142 filial generation weight, in conjunction with the collection of illustrative plates and typing data of 23 linkage groups, to weight this Character carries out quantitative character association analysis.Qtl analysis is carried out using the Internal Mapping methods of software MapQTL5.Knot Fruit shows that there are 1 and trait-associated regions in LG22 linkage groups, contains 4 SLAF altogether and marks.According to this 4 SLAF marks The sequencing sequence screening of note can be used for designing 3 SNP markers of the primer sequence of SNP amplifications, wherein comprising being located at The sites SNP40 on marker26440, to the site, the analysis of the Multiple range test of different genotype and weight character is shown in family Table 1.It can be seen that in the offspring individual used in structure family, SNP40 site primers to (CT) and (CC) 2 kinds of genotype are different There are significant difference, the seed weight of (CC) genotype to be significantly higher than (CT) genotype individuals body for the offspring individual weight of genotype Weight shows that the preponderant genotype in the site is respectively (CC).
1 sites SNP40 of table Multiple range test of different genotype and weight character in family is analyzed
Embodiment 2:Stichopus japonicus grows verification of the related SNP molecular labeling in expanding group
According to the both sides flanking sequence in the sites SNP40 that high-flux sequence obtains, design SNP site augmentation detection is with drawing Object, corresponding primer sequence are SNP40F:5 '-GGGTGGACTTGGAGCAGAAG-3 ' and SNP40R:5'- ACCAACGTCCTCTCTGGTTATCA-3'.Utilize HRM small fragments method to this SNP in expanding group using corresponding sequence Point carries out parting and the correlated traits data for expanding group is combined to carry out QTL site verification.Expansion group used is with a batch The 10 monthly age seeds cultivated in secondary extensive nursery and same breeding environment, randomly select 96 individuals, determine the body of individual Weight.The genotype detection of individual is carried out using HRM methods, the augmentation detection of the label is with system:1 μ L (50ng/ μ of DNA profiling L), 4.5 μ L 2 × ES Taq Master Mix, each 0.5 μ L of upstream and downstream primer (10 μm of ol/L), 3.5 μ L ddH2O;PCR reacts Program:95 DEG C of pre-degeneration 5min;94 DEG C of denaturation 30s, 60.5 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 recycle;Last 72 DEG C Extend 7min, 4 DEG C of preservations;The parting of label is carried out using high-resolution melting curve analysis technology (HRM) method.To expanding group Body juvenile stichopus weight character obtains with the label correlation analysis result, the SNP40 and extremely significantly correlated (P of weight character< 0.01)。
2 are shown in Table to the Multiple range test analysis result of the different genotype Yu weight in the sites SNP40 this character.It can be seen that Genotype is that the seed average weight of (CT) is substantially less than (CC) type seed, shows that the preponderant genotype in the site is respectively (CC)。
The Multiple range test analysis of different genotype in association character in expanding group of 2 sites SNP40 of table
Embodiment 3:Application of the molecular labeling of growth traits assisted selection in stichopus japonicus breeding production
In stichopus japonicus spring nursery stage, 100 parent participations are randomly choosed from the parent participation cultivated, and utilize HRM technologies pair 100 Genotype of the parent participation in the sites SNP40 carries out parting.Primer sequence used is SNP40F:5'- GGGTGGACTTGGAGCAGAAG-3 ' and SNP40R:5 '-ACCAACGTCCTCTCTGGTTATCA-3 ', augmentation detection system For:1 μ L of DNA profiling (50ng/ μ L), 4.5 μ L 2 × ES Taq Master Mix, each 0.5 μ L of upstream and downstream primer (10 μm of ol/ L), 3.5 μ L ddH2O;PCR response procedures:95 DEG C of pre-degeneration 5min;94 DEG C of denaturation 30s, 60.5 DEG C of annealing 30s, 72 DEG C extend 30s, totally 35 recycle;Last 72 DEG C of extensions 7min, 4 DEG C of preservations;The parting of label uses high-resolution melting curve analysis skill Art (HRM) method carries out.According to genotyping result, choose genotype the site show as (CC) individual 10 (5 female 5 hero) and (CT) individual 10 (5 female 5 is male) carries out filial generation seed breeding.2 batch seed same period nursery, seed rearing ensure to support in the process The consistency of management and breeding environment is grown, randomly selects seed 500 at 5 monthly age of seed, 6 monthly ages, 7 monthly ages and August age respectively Head carries out body weight determination, draws the body weight increase curve graph of different seeds (see Fig. 2).As seen from Figure 2, it is by genotype (CC) average weight of the filial generation of parent's breeding during seed rearing is significantly higher than the filial generation seedling bred by (CT) type parent Kind.It proves with the filial generation seed speed of growth for the individual breeding that the sites SNP40 are (CC) faster.
Sequence table
<110>Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
<120>A kind of molecular labeling and its application for stichopus japonicus growth traits assisted selection
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>The primer sequence SNP40F (artifical) of engineer
<400> 1
gggtggactt ggagcagaag 20
<210> 2
<211> 23
<212> DNA
<213>The primer sequence SNP40R (artifical) of engineer
<400> 2
accaacgtcc tctctggtta tca 23

Claims (5)

1. a kind of molecular labeling for stichopus japonicus growth traits assisted selection, it is characterised in that the molecular labeling is SNP40 is marked, and preponderant genotype of the SNP40 labels for growth traits assist-breeding is CC;
The primer pair for expanding the molecular labeling is SNP40F:5 '-GGGTGGACTTGGAGCAGAAG-3 ' and SNP40R:5'- ACCAACGTCCTCTCTGGTTATCA-3’。
2. molecular labeling according to claim 1, it is characterised in that the label augmentation detection is with system:DNA profiling 50ng/ μ L1 μ L, 4.5 μ L 2 × ES Taq Master Mix, each 0.5 μ L of upstream and downstream primer, 10 μm of ol/L, 3.5 μ L ddH2O; Label parting be with PCR response procedures:95 DEG C of pre-degeneration 5min;94 DEG C of denaturation 30s, 60.5 DEG C of annealing 30s, 72 DEG C of extension 30s, Totally 35 cycles;Last 72 DEG C of extensions 7min, 4 DEG C of preservations;Parting is carried out using high-resolution melting curve analysis technical method.
3. using the method that label carries out stichopus japonicus growth traits assistant breeding described in claim 1, step includes (1) parent DNA Extraction and parting;(2) Juvenile stage;(3) 3 parts of filial generation seed breeding;In the Juvenile stage, the advantage parent of selection For the individual for being CC in SNP40 marker site genotype.
4. according to the method described in claim 3, it is characterized in that the described parent DNA extractions and parting be with invasive methods from DNA is extracted at candidate parent's pipe foot or quil tissue, and the genotype of parent's corresponding site is divided with SNP40 labels Type.
5. according to the method described in claim 3, it is characterized in that the filial generation seed breeding, is stated with step (2) and is selected Parent is that parent's breeding population carries out filial generation seed breeding.
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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
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

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