CN108165637B - A kind of combination of genetic molecule label and its application for disease-resistant stichopus japonicus selection and breeding - Google Patents
A kind of combination of genetic molecule label and its application for disease-resistant stichopus japonicus selection and breeding Download PDFInfo
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Abstract
The invention discloses a kind of combinations of genetic molecule label and its application for disease-resistant stichopus japonicus selection and breeding.Belong to aquatic products genetic breeding field.The label is combined as 3 and infects relevant SNP marker SNP88, SNP112 and SNP126 with the anti-Vibrio splindidus of stichopus japonicus, and the preponderant genotype of 3 labels is respectively SNP88 (CC), SNP112 (AA), SNP126 (TT)；It includes (1) parent DNA extractions and parting to carry out breeding production operation step using the genetic marker；(2) Juvenile stage；(3) 3 parts of filial generation seed breeding.The present invention provides effective molecular labeling to infect this economic characters from the genetically raising anti-Vibrio splindidus of stichopus japonicus, assisted Selection is marked to stichopus japonicus with the genetic marker, carry out breeding for disease resistance, the ability that the anti-Vibrio splindidus of stichopus japonicus infects can be improved, the incidence for fundamentally reducing stichopus japonicus skin ulcer syndrome is of great significance to improving cultivation survival rate and culture efficiency.
Description
Technical field
The invention belongs to aquatic products genetic breeding fields, more particularly to a kind of genetic molecule mark for disease-resistant stichopus japonicus selection and breeding
Note combination 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.Wherein most outstanding is that disease caused by environmental carrying capacity is excessive and sea cucumber germplasm is degenerated takes place frequently, cultivates and survive
The low phenomenon of rate.Stichopus japonicus disease frequently occurs, and causes about 3,000,000,000 yuan of economic loss every year, and heavy losses are caused to stichopus japonicus industry,
Thus stichopus japonicus germplasm, which degenerates to have become, restricts the bottleneck that apostichopus japonicus culture develops in a healthy way, and on the one hand directly influences the volume increase of culturist
It increases income and industrial benefit, another aspect is also related to the sustainable development of the 5th cultivation tide.It is supported by improving stichopus japonicus itself
It is stichopus japonicus healthy aquaculture most efficient method that drag, which resists disease,.Therefore, genetic improvement is carried out to stichopus japonicus, cultivated with disease-resistant
Power waits by force the new varieties of merits, is the important guarantee that apostichopus japonicus culture industry develops in a healthy way.
During stichopus japonicus industry development, long-term epidemiological study the result shows that, influence the main of apostichopus japonicus culture industry
Kinds of Diseases have the side rot disease, rotten stomach trouble, the skin ulcer syndrome (also known as changing skin disease) etc. for changing plate disease and mature stage in seedling stage.It is domestic
The cause of disease for the culturing stichopus japonicus principal disease having found is based on bacterium, and wherein vibrios is the main pathogenic bacteria of stichopus japonicus disease, magnificent
Vibrios is to keep a full stand of seedings and cultivate phase major disease --- the principal causative of skin ulcer syndrome (changing skin disease) is former.The Beancurd sheet that the cause of disease causes
Syndrome causes massive losses to the apostichopus japonicus culture in China, and industry development is caused to be faced with unprecedented challenge.Therefore, it selects
The new varieties with the advantages character such as anti-Vibrio splindidus are educated for resisting industry risk, are regained cultivation dealer's confidence, are improved industry
Benefit has important practical significance.
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.
In order to develop the disease-resistant molecular labeling of stichopus japonicus, applicant constructs stichopus japonicus high density genetic linkage maps, utilizes variance
Analysis and composite interval mapping confrontation Vibrio splindidus infect character and carry out QTL positioning, select disease-resistant QTL sections and may be adapted to primer
The SNP site of design carries out SNP marker verification using designed primer in widened group, filters out disease resistance trait phase
Two times of off-position point, the preponderant genotype in site and advantage types, and productivity verification has been carried out in Breeding Practice, it finally obtains
It can be used for label combination and the preponderant genotype of the disease-resistant molecular marker assisted selection breeding of stichopus japonicus.
Stichopus japonicus marker assisted selection method is established using this method, and applies it to stichopus japonicus fine-variety breeding process, is trained
Disease-resistant stichopus japonicus new varieties are educated, the germplasm that effective solution apostichopus japonicus culture industry faces at present is degenerated and the bottleneck problems such as disease, and
It is strong to push apostichopus japonicus culture improved variety process.
Invention content
The genetic molecule label combination that the technical problem to be solved in the present invention is to provide a kind of for disease-resistant stichopus japonicus selection and breeding and
It is applied, and includes two times of types of preponderant genotype and advantage of molecular labeling primer and mark of correlation, and it is disease-resistant to use it for stichopus japonicus
The selection and breeding of material.
The present invention is realized by following technical solution：
A kind of genetic molecule for disease-resistant stichopus japonicus selection and breeding marks combination, wherein the combination of genetic molecule label comprising 3 with
The anti-Vibrio splindidus of stichopus japonicus infects relevant SNP marker SNP88, SNP112 and SNP126；The disease-resistant advantage base of this 3 genetic markers
Because type is respectively SNP88 (CC), SNP112 (AA), SNP126 (TT).
Further, the amplimer of 3 labels is respectively:SNP88F 5'-TTTTGTCCCAGAGCA TTATCCAA-3'
With SNP88R 5 '-TCCAGGAATGATATTTAGTGGTTGT-3 '；SNP112F 5'-ATTTTGGTTAGGGTTAAGAAGGCT-
3 ' and 5 '-GT TTCCAAACAACATATGCACTCC-3 ' of SNP112R；SNP126F 5'-GGATTGATCGGGG ACACACA-
3 ' and SNP126R 5 '-TGGGCTAAGTCACCAATCTGC-3 '.
Further, this 3 genetic marker augmentation detections are with system：1 μ L of DNA profiling (50ng/ μ L), 4.5 2 × ES of μ L
Taq Master Mix, each 0.5 μ L of upstream and downstream primer (10 μm of ol/L), 3.5 μ L ddH2O；The PCR of 3 genetic marker partings
Response procedures：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, annealing 30s (distinguish by SNP88, SNP112 and SNP126 annealing temperature
For：58.3 DEG C, 58.5 DEG C and 60 DEG C), 72 DEG C of extension 30s, totally 35 recycle；Last 72 DEG C of extensions 7min, 4 DEG C of preservations；3 something lost
The parting for passing label is carried out using high-resolution melting curve analysis technology (HRM) method.
The present invention also provides the method that a kind of application genetic marker combination carries out breeding, operating procedure includes (1) parent
DNA is extracted and parting；(2) Juvenile stage；(3) 3 parts of filial generation seed breeding.
Further, the parent DNA is extracted as extracting from candidate parent's pipe foot or quil tissue with invasive methods
DNA carries out parent in the genotype of 3 genetic marker sites of SNP88, SNP112, SNP126 using 3 primer pairs
Parting.
Further, in the Juvenile stage, two times of types of the advantage parent selected is in SNP88, SNP112, SNP126
3 loci gene types are the individual of (CC) (AA) (TT).
Further, the filial generation seed breeding, be with two times of types screening be SNP88, SNP112,
SNP1263 loci gene type is that the individual of (CC) (AA) (TT) is that parent's breeding population carries out filial generation seed breeding.
The advantageous effect of the present invention compared with prior art：
1, the present invention provides more efficiently and accurately, a simplicity for the molecular marking supplementary breeding of stichopus japonicus breeding for disease resistance work
Easy molecular genetic marker combination infects this disease resistance trait for the raising anti-Vibrio splindidus of stichopus japonicus and provides effective molecule mark
Remember breeding technique.It is tested in family, expansion breeding populations and breeding practice simultaneously using respective markers and combinations thereof
Card is marked assisted Selection to the Vibrio splindidus resistance of stichopus japonicus with label combination, can effectively alleviate in actual production
Stichopus japonicus skin ulcer syndrome (changing skin disease) problem, is of great significance to improving cultivation survival rate and 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 disease-resistant QTL；
Fig. 2 is that different genotype parent breeding filial generation fingerlings artificial attacks the cumulative life-incidence during poison.
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 of the genetic molecule label of disease-resistant stichopus japonicus selection and breeding
Stichopus japonicus family is built, the premunition of each family is assessed by challenge test, choosing has moderate premunition stichopus japonicus man
System, with 2 parents of the family and 142 sons on behalf of mapping population, by high-flux sequence platform, exploitation SLAF labels, profit
With HighMap software buildings stichopus japonicus high density genetic linkage maps.After high-flux sequence, 264,810 SLAF marks are developed altogether
Label, wherein 112,322, the SLAF labels of polymorphism.SLAF label parental means be sequenced depth be 23.67 ×, offspring mean survey
Sequence depth be 5.44 ×.Bioinformatic analysis is crossed in promoting menstruation, and the label that can be used for genetic map construction has 50,905, passes through
After mass filter, 4629, the polymorphism label that sequencing depth, integrity degree are high and do not detach partially is obtained.4629 will filtered out
A SLAF labels make linkage analysis, calculate recombination fraction and MLOD values between label two-by-two, obtain 23 linkage groups, total upper figure
4,002, it is positioned as icon note, upper figure rate 86.45%.As unit of linkage group, connected using the analysis of HighMap softwares
The linear array of Marker in group is locked, and estimates the genetic distance between adjacent Marker, structure genetic map is shown in Fig. 1.Wherein, in
Property collection of illustrative plates include 4002 labels, total figure is away from for 3223.84cM；Male collection of illustrative plates includes 2228 upper icons notes altogether, total figure away from for
3864.73cM；Female collection of illustrative plates includes 2307 upper icons notes altogether, and total figure is away from for 2075.84cM.
According to the measurement result of Vibrio splindidus artificial challenge's disease time of 142 filial generations, in conjunction with the figure of 23 linkage groups
Spectrum and typing data, this character of confrontation Vibrio splindidus dip dyeing ability carry out quantitative character association analysis.Using software MapQTL5
Internal Mapping methods carry out qtl analysis.The results show that there are 1 and trait associations area in LG19 linkage groups
Domain is contained 52 SLAF and is marked altogether.It can be used for designing the primer of SNP amplifications according to the sequencing sequence screening of this 52 SLAF labels
8 SNP markers of sequence, wherein including 3 SNP sites of SNP88, SNP112 and SNP126, this 3 sites are in linkage group
Relevant information be shown in Table 1.It can be seen that in the offspring individual used in structure family, SNP88, SNP SNP112 and SNP126 3
There are significant difference, each homozygous premunitions in site to be significantly higher than miscellaneous for the offspring individual premunition of SNP site different genotype
Zygote, i.e. 3 respective preponderant genotypes in site of SNP88, SNP112, SNP126 are respectively (CC), (AA), (TT).
13 SNP sites of table Multiple range test of different genotype and disease resistance trait in family is analyzed
Embodiment 2：Verification of the disease-resistant related SNP molecular labeling of stichopus japonicus in expanding group
According to the both sides flanking sequence for 3 SNP sites that high-flux sequence obtains, design SNP site augmentation detection is with drawing
Object, corresponding primer sequence are SNP88F 5 '-TTTTGTCCCAGAGCATTATCCAA-3 ' and SNP88R 5 '-
TCCAGGAATGATATTTAGTGGTTGT-3'；SNP112F 5 '-ATTTTGGTTAGGGTTAAGAAGGCT-3 ' and SNP112R
5'-GTTTCCAAACAACATATGCACTCC-3'；SNP126F 5 '-GGATTGATCGGGGACACACA-3 ' and SNP126R
5'-TGGGCTAAGTCACCAATCTGC-3'.Utilizing HRM small fragments method to this 3 in expanding group using corresponding sequence
The disease-resistant relevant SNP site of stichopus japonicus carries out parting and the correlated traits data for expanding group is combined to carry out QTL site verification.Institute
It is the August age seed cultivated in the extensive nursery of same batch and same breeding environment, premunition assay method with group is expanded
Poison is manually attacked for Vibrio splindidus and is infected (manually disseminates a concentration of 1.0 × 107CFU/ml, challenge viral dosage phase are 30d) it is a in the process
The time of body appearanceization skin symptom.This 3 genetic marker augmentation detections are with system：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；3 genetic marker partings
PCR response procedures：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, annealing 30s (SNP88, SNP112 and SNP126 annealing temperature
Degree is respectively：58.3 DEG C, 58.5 DEG C and 60 DEG C), 72 DEG C of extension 30s, totally 35 recycle；Last 72 DEG C of extensions 7min, 4 DEG C of guarantors
It deposits；The parting of 3 genetic markers is carried out using high-resolution melting curve analysis technology (HRM) method.To stichopus japonicus premunition
Shape and 3 label correlation analysis results (table 2) are as can be seen that SNP88, SNP126 and the significantly correlated (P of disease time<
0.05)；The site SNP112 and extremely significantly correlated (P of disease time<0.01).
23 SNP sites of table and premunition correlation analysis result
Note：Numerical value is the probability value of premunition (disease time) and SNP site association analysis in table, and " * " indicates P<0.05,
" * * " indicates P<0.01.
It pair analyzes and to tie with the Multiple range test of the different genotype Yu premunition (disease time) of 3 SNP sites this character
Fruit is shown in Table 3.SNP88, SNP112, SNP126 genotype respectively (CC), (AA), individual of (TT) are malicious real in attacking for Vibrio splindidus
The average value of disease time in testing is significantly higher than (CT), (AG), (TG) genotype (P<0.05), also just illustrate SNP88,
The preponderant genotype in 3 sites SNP112, SNP126 is respectively (CC), (AA), (TT).
Multiple range test analysis of the 33 SNP site different genotypes of table in association character
Two times of types are built to 3 SNP sites respectively, compares two times of types and divides with the significance of difference result being associated between character
It is not shown in Table 4.3 relevant SNP sites of premunition detect 6 kinds of two times of types, wherein K1 (CC) (AA) altogether in detecting group
(TT) morbidity number of days highest, all individuals of the genotype are not fallen ill during entirely attacking poison, and K2 (CC) (AA) (TG) takes second place,
But all it is significantly higher than other two times of types, shows to show as (CC) (AA) (TT) base in 3 sites SNP88, SNP112, SNP126
Because the individual of two times of types is optimal two times of types.
The association analysis of table 5 SNP bis- times of types and stichopus japonicus premunition
Embodiment 3：Application of the genetic molecule label combination of disease-resistant stichopus japonicus selection and breeding in the production of stichopus japonicus select index
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 site SNP88, SNP112, SNP126 carries out parting.Primer sequence used is SNP88F 5 '-
TTTTGTCCCAGAGCATTATCCAA-3 ' and SNP88R5 '-TCCAGGAATGATATTTAGTGGTTGT-3 '；SNP112F 5'-
ATTTTGGTTAGGGTTAAGAAGGCT-3 ' and SNP112R 5 '-GTTTCCAAACAACATATGCACTCC-3 '；SNP126F
5 '-GGATTGATCGGGGACACACA-3 ' and SNP126R 5 '-TGGGCTAAGTCACCAATCTGC-3 '.This 3 genetic markers
Augmentation detection is with system：1 μ L of DNA profiling (50ng/ μ L), 4.5 μ L 2 × ES Taq Master Mix, upstream and downstream primer are each
0.5 μ L (10 μm of ol/L), 3.5 μ L ddH2O；The PCR response procedures of 3 genetic marker partings：95 DEG C of pre-degeneration 5min；94
DEG C denaturation 30s, (SNP88, SNP112 and SNP126 annealing temperature is respectively annealing 30s：58.3 DEG C, 58.5 DEG C and 60 DEG C), 72
DEG C extend 30s, totally 35 cycle；Last 72 DEG C of extensions 7min, 4 DEG C of preservations.According to genotyping result, choose genotype SNP88,
3 sites SNP112, SNP126 show as two times of types of (CC) (AA) (TT) gene individual 10 (5 female 5 male) and SNP88,
The individual 10 (5 female 5 is male) that 3 sites SNP112, SNP126 show as two times of types of (CT) (AG) (TG) gene carries out filial generation seedling
Kind breeding.2 batch seed same period nursery, seed rearing ensure the consistency of aquaculture management and breeding environment, are cultivating in the process
When to August ages, 200 seeds are respectively chosen from the filial generation cultivated at random, malicious infection experiment is manually attacked with Vibrio splindidus,
Experiment condition is Vibrio splindidus a concentration of 1.0 × 107CFU/ml, challenge viral dosage phase are 30d.Count tired during manually attacking poison
Product incidence is shown in Fig. 2, it can be seen that bright in high concentration with the filial generation seed of the individual breeding of two times of types of (CC) (AA) (TT) gene
Rotten vibrios manually attacks the son for the initial onset time under malicious infectious condition being later than the individual breeding of two times of types of (CT) (AG) (TG) gene
Generation, and its cumulative morbidity in entirely attacking malicious infection processs is also significantly lower than the individual of (CT) (AG) (TG) two times of types of gene
The filial generation of breeding, it was demonstrated that there is stronger premunition with the filial generation seed of the individual breeding of two times of types of (CC) (AA) (TT) gene.
Sequence table
<110>Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
<120>A kind of combination of genetic molecule label and its application for disease-resistant stichopus japonicus selection and breeding
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>The primer sequence SNP88F (artifical) of engineer
<400> 1
ttttgtccca gagcattatc caa 23
<210> 2
<211> 25
<212> DNA
<213>The primer sequence SNP88R (artifical) of engineer
<400> 2
tccaggaatg atatttagtg gttgt 25
<210> 3
<211> 24
<212> DNA
<213>The primer sequence SNP112F (artifical) of engineer
<400> 3
attttggtta gggttaagaa ggct 24
<210> 4
<211> 24
<212> DNA
<213>The primer sequence SNP112R (artifical) of engineer
<400> 4
gtttccaaac aacatatgca ctcc 24
<210> 5
<211> 20
<212> DNA
<213>The primer sequence SNP126F (artifical) of engineer
<400> 5
ggattgatcg gggacacaca 20
<210> 6
<211> 21
<212> DNA
<213>The primer sequence SNP126R (artifical) of engineer
<400> 6
tgggctaagt caccaatctg c 21
Claims (4)
1. a kind of SNP marker for disease-resistant stichopus japonicus selection and breeding combines, it is characterised in that the SNP marker combination includes 3 and thorn
Join anti-Vibrio splindidus and infects relevant SNP marker SNP88, SNP112 and SNP126；The disease-resistant protogene of this 3 SNP markers
Type is respectively the sites the SNP88 site CC, SNP112 site AA, SNP126 TT；The amplimer of 3 SNP markers is respectively：
SNP88F 5 '-TTTTGTCCCAGAGCATTATCCAA-3 ' and SNP88R 5 '-
TCCAGGAATGATATTTAGTGGTTGT-3'；SNP112F 5 '-ATTTTGGTTAGGGTTAAGAAGGCT-3 ' and SNP112R
5'-GTTTCCAAACAACATATGCACTCC-3'；SNP126F 5 '-GGATTGATCGGGGACACACA -3 ' and SNP126R
5’-TGGGCTAAGTCACCAATCTGC-3’。
2. a kind of combining the method for carrying out anti-Vibrio splindidus stichopus japonicus breeding using SNP marker described in claim 1, it is characterised in that
The operating procedure of the method includes（1）Parent DNA extractions and parting；（2）Juvenile stage；（3）3 portions of filial generation seed breeding
Point；In the Juvenile stage, two times of types of the advantage parent selected is in 3 locus genes of SNP88, SNP112, SNP126
Type is the individual of CC, AA and TT.
3. according to the method described in claim 2, it is characterized in that the parent DNA is extracted as with the certainly candidate parent of invasive methods
DNA is extracted at pipe foot or quil tissue, using 3 primer pairs of SNP marker to parent in SNP88, SNP112, SNP126 3
The genotype in a SNP marker site carries out parting.
4. according to the method described in claim 2, it is characterized in that the filial generation seed breeding, be with screen two
It is that parent's breeding population carries out that times type, which is in the individual that 3 loci gene types of SNP88, SNP112, SNP126 are CC, AA and TT,
Filial generation seed breeding.
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| Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Coelomocytes of Sea Cucumber (Apostichopus japonicus) after Vibrio splendidus Challenge;Qiong Gao等;《Int. J. Mol. Sci.》;20150715;第16卷;16347-16377 * |
| Transcriptome analysis of tube foot and large scale marker discovery in sea cucumber, Apostichopus japonicus;Xiaoxu Zhou等;《Comparative Biochemistry and Physiology, Part D》;20160804;第20卷;41–49 * |
| 刺参体腔细胞转录组测序与抗灿烂弧菌感染相关免疫基因的筛选及表达研究;高琼;《中国学位论文全文数据库》;20160330;全文 * |
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