CN101892319A - Germany mirror carp stress resistance gene label and core selective breeding group building method thereof - Google Patents
Germany mirror carp stress resistance gene label and core selective breeding group building method thereof Download PDFInfo
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- CN101892319A CN101892319A CN 201010248328 CN201010248328A CN101892319A CN 101892319 A CN101892319 A CN 101892319A CN 201010248328 CN201010248328 CN 201010248328 CN 201010248328 A CN201010248328 A CN 201010248328A CN 101892319 A CN101892319 A CN 101892319A
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Abstract
The invention discloses a Germany mirror carp stress resistance gene label and a core selective breeding group building method thereof. The sequences of the Germany mirror carp stress resistance gene label are HLJI-9 amplification sequence of MHCclass I and the HLJB amplification sequence of MHCclass II B. The method comprises the following steps of: electronically labeling the selective breeding group by adopting the method of combining gene label and electronic label; labeling a basic group which only appears in a group with poor stress resistance as the gene label relevant to stress resistance according to a gene label detection result; screening individuals which do not carry the stress resistance gene labels; and building a core selective breeding group. The core selective breeding group built by the invention can solve the problem that Germany mirror carps die in a large scale because of diseases in high-temperature seasons if cultured in a high-density net cage. The invention has the important meaning and value for the selective breeding of the resistant variety of fish.
Description
Technical field
The present invention relates to fish adversity gene mark and core selective breeding colony construction process thereof.
Background technology
Germany's mirror carp original seed was introduced by former federal republic of germany in 1984, and this fish is the main breed variety in European Region pond, and characteristics are that artificial domestication degree height, growth are fast, feed conversion rate is high.But the high-density rich water cultivating condition of its incompatibility China, anti-adversity ability is low.Aquatic products institute in Heilungkiang has carried out systematic breeding to it under the subsidy of the Ministry of Agriculture and national Economic and Trade Commission, has passed through evaluation to the breeding line in 4 generations of nineteen ninety-five seed selection to the, and anti-adversity ability improves, and the speed of growth is than original seed fast 25%.The breeding that Germany's mirror carp breeding line is promoted in the whole nation for suiting by the authorization of the former breeding of national aquatic products validation board.At present, Germany's mirror carp breeding line is as an important carp culture kind, have that build is good, meat flavour is delicious, contain meat rate height, feeding habits are assorted, low temperature resistant, the fast advantages such as (the fast 20%-30% of more common carp) of easily fishing for, grow, at the many cultivating pools of China, particularly liked by culturist and human consumer this fish of northern area, its cultured output and economic benefit increase year by year.Yet, compare with China original inhabitants carp, its anti-adversity ability is still relatively poor, especially along with the deterioration and the high-density breeding demand of breeding environment in recent years, since two thousand German mirror carp breeding line is ill at the high temperature season of high-density cage culture, and large-scale death incident (mortality ratio is up to 90%) takes place, and indigenous carp is uninfluenced, Germany's mirror carp aquaculture has been subjected to enormous economic loss, has had a strong impact on the health of German mirror carp industry, stable and sustainable development.At present, do not see the report of relevant German mirror carp anti contravariance related gene mark, do not see the report that the degeneration-resistant improved seeds of relevant fish are cultivated yet.
Summary of the invention
The invention provides German mirror carp adversity gene mark and core selective breeding colony construction process thereof.
The present invention Germany mirror carp adversity gene flag sequence is the HLJI-9 extension increasing sequence of MHC class I and the HLJB extension increasing sequence of MHC class IIB, and wherein the HLJI-9 extension increasing sequence of MHC class I is 5'-TGATGGCACCAAGGAGGATACTATCAG
TTTGGTTACGATGGAGAGGACTTCCTCAGTCTGGATAAGAGCTCCCTCACCTGGACT GCTGCCAATCCTCAAGCTGTCATTACCA-3', the 28th base T is the adversity gene site; The HLJB extension increasing sequence of MHC class IIB is 5'-TCCTGTATGGTTGAGCATGCCAGCTTCAGTAAACCCATGATTTATGACTGGGG TAAGATGAGACACGCAACAGATGTCATATTATAACTGTGTTTGTGGTAAGCATAAT TTGACATATGACATATGACATACTCCACAGATCCGTCTCTCCCTGAGTCTGAGAGG AATAAAATTGCTATTGGAGCATTTGGTCTGGTGCTGGGAATCATTATT
GCAGCTGCTGGACTCATTTACA-3', the 214th bases G is the adversity gene site.
The present invention Germany mirror carp core selective breeding colony construction process as: the German mirror carp parent fish population applying electronic mark that will carry out seed selection carries out mark, in conjunction with screening two anti contravariance related gene sites, according to two pairs of primers selecting to have degeneration-resistant site increase, order-checking and degeneration-resistant site base analysis, with the base that only in resistance difference colony, occurs as the anti contravariance related gene mark, the individuality of these adversity gene marks is carried in rejecting, does not carry the individual core selective breeding colony that forms of these genetic markers.
The present invention is by German mirror carp adversity gene mark, set up the degeneration-resistant core selective breeding of German mirror carp colony, the property of the present invention is directed to is strong, be not subjected to the influence of body growth etap and envrionment conditions, making up core selective breeding colony selects accurately, can quicken breeding process, improve breeding efficiency, thereby effectively solve the ill problem that causes extensive death of German mirror carp high temperature season.The present invention has opened up a molecular breeding technological approaches for the degeneration-resistant molecular breeding seed selection of fish, and cultivation has great importance and is worth to the fish anti-adversity.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment Germany mirror carp adversity gene flag sequence is the HLJI-9 extension increasing sequence of MHC class I and the HLJB extension increasing sequence of MHC class IIB, and wherein the HLJI-9 extension increasing sequence of MHC class I is 5'-TGATGGCACCAAGGAGGATACTATCAG
TTTGGTTACGATGGAGAGGACTTCCTCAGTCTGGATAAGAGCTCCCTCACCTGGACT GCTGCCAATCCTCAAGCTGTCATTACCA-3', the 28th base T is the adversity gene site; The HLJB extension increasing sequence of MHC class IIB is 5'-TCCTGTATGGTTGAGCATGCCAGCTTCAGTAAACCCATGATTTATGACTGGGG TAAGATGAGACACGCAACAGATGTCATATTATAACTGTGTTTGTGGTAAGCATAAT TTGACATATGACATATGACATACTCCACAGATCCGTCTCTCCCTGAGTCTGAGAGG AATAAAATTGCTATTGGAGCATTTGGTCTGGTGCTGGGAATCATTATT
GCAGCTGCTGGACTCATTTACA-3', the 214th bases G is the adversity gene site.
German mirror carp adversity gene flag sequence obtains by the following method in the present embodiment: one, the acquisition of German mirror carp strong stress resistance and resistance difference colony sample: the German mirror carp of high-density cage culture, at high temperature season, begin to take place large-scale death incident, gather the colony sample of dead individual fin ray as the resistance difference, treat after the high temperature season, gather the individual fin ray of surviving as colony's sample of strong stress resistance, respectively with colony's sample retention of colony's sample of resistance difference and strong stress resistance in volumetric concentration is 75% alcohol; Two, German mirror carp MHC class I and MHC class IIB gene genetic polymorphism: according to the known MHC class I of the close fish of other sibship and the comparison result of MHC class IIB gene extron and intron, use oligo 6.0 softwares and carry out design of primers, wherein G+C content is generally 40-60%, avoid as far as possible primer secondary structure or secondary structure be controlled at 3 continuous bases with interior in order to avoid form primer dimer, for the relatively low sequence of conservative property, the design degenerate primer; Then again the primer that designs is screened, wherein the primer screening method is: the pcr amplification reaction system is 15 μ L:10 x PCR buffer, 1.5 μ L, 10mM dNTP 0.3 μ L, TaqDNA polysaccharase 0.6U, the genomic dna template is 50ng, each 0.15 μ M of upstream and downstream primer concentration, and the sterilization distilled water is supplied volume, the genomic dna template adopts the mixture of 5 individualities earlier, and the primer of preliminary screening is verified through 10 individualities again; Preliminary screening goes out 5 pairs of expanding effects primer preferably, and after the empirical tests, that the 3 pairs of primers can amplify is stable, bands of a spectrum clearly; In the step 2 primer HLJI-9 at be the exon 3 sequence, primer HLJB at be exon 3 to exon 4 sites, primer HLJB5 at be the exon 3 sequence.Extract the genomic dna of German mirror carp MHC class I and MHC class IIB respectively, then carry out pcr amplification respectively, use electrophoresis detection then, the purpose band is reclaimed, the secondary amplification, amplified production is directly checked order by Shanghai biotechnology company limited, the sequence SNP information that records is directly read by software Mutation Surveyor, each SNP site gene frequency, observation and expectation heterozygosity, Hardy-Weinberg balance and linkage disequilibrium are added up by software POPGENE version 1.32, comparison application software Clustal W between the sequence, wherein, in the HLJI-9 site, identifying gene frequency altogether is 10 different genotype of 1.89%-41.51%, the 66th (A/C) on the exon 3 sequence wherein, 67 (T/G), there is polymorphism on 72 (A/T) and 102 (T/C) base site and all can changes coded amino acid, the observation heterozygosity is 0.0377-0.3774, average out to 0.1745, all sites all is under the Hardy-Weinberg balance, has linkage disequilibrium situation (P＜0.05) between the 66th and 72 bases; In the HLJB site, through comparison, with cyca-DAB3*01/ cyca-DAB4*01 gene similarity be 87.33%-91.33%, identifying gene frequency altogether is 28 different genotype of 2.94%-11.76%, intron sequences length is 85-87bp, the 350th (T/C) in the exon 3 sequence, 374 (T/C) and exon 4 the 36th (G/T), 71 (C/T), 74 (C/T), there is polymorphism in 75 (T/G) base on the site, two sites can change coded amino acid, exon sequence observation heterozygosity is 0.1176-0.4706, average out to 0.3480, all sites all are under the Hardy-Weinberg balance; Between exon 4 the 71st and 74 bases, there is linkage disequilibrium situation (P＜0.05); In the HLJB5 site, extension increasing sequence can be divided into two locus, first identifies 9 different genotype that gene frequency is 3.85%-26.92% altogether, wherein there is polymorphism on the site in the 93rd (T/G), 104 (G/A), 170 (G/A), 195 (G/C), 197 (G/C) base on the exon sequence, wherein 3 sites also all can change coded amino acid, the observation heterozygosity is 0.0000-0.6154, average out to 0.2308,97 and 197 exist Hardy-Weinberg imbalance (P＜0.001) in the site; Between the 195th and 197 bases, there is linkage disequilibrium situation (P＜0.05); In the HLJB5 site, second locus identifies 11 different genotype that gene frequency is 3.85%-23.08% altogether, wherein there is polymorphism on the site in the 125th (A/T), 127 (A/G), 139 (G/C), 176 (A/T), 180 (C/T), 198 (A/G) base on the exon sequence, wherein 6 sites can change coded amino acid, the observation heterozygosity is 0.0769-0.8462, average out to 0.3333; Site 180 is in the Hardy-Weinberg imbalance; Between the 125th and 176 bases, there is linkage disequilibrium situation (P＜0.05); Three, the screening of Germany's mirror carp anti contravariance related gene mark: according to each distribution of polymorphic base in the individuality of resistance difference and strong stress resistance, use SPSS 13.0 softwares and carry out the degeneration-resistant correlation analysis of each polymorphic site, by analysis, in MHC class I sequence exon 3 the 72nd base, through chi square test, remarkable (the Pearson c2=4.046 of A/T base distributional difference in strong stress resistance and resistance difference individuality, df=1, P=0.044), T base frequency of occurrences in the strong stress resistance individuality is 28%, the frequency of occurrences is 64.3% in resistance difference individuality, in MHC class IIB sequence exon 4 the 75th base site, through chi square test, remarkable (the Pearson c2=20.526 of T/G base distributional difference in strong stress resistance and resistance difference individuality, df=1, P=0.000), G base frequency of occurrences in the strong stress resistance individuality is 0.0%, the frequency of occurrences is 71.4% in dead individuality, thereby obtained the anti contravariance related gene flag sequence, and determined the adversity gene site, the PCR primer HLJI-9 of German mirror carp MHC class I gene in the step 2 wherein: upstream primer 5'-TGTTCACTCAGTCCAGCAGA-3', downstream primer 5'-TTGGTAATGACAGCTTGAGG-3', PCR reaction conditions: 95 ℃ of sex change 5min, 95 ℃ of sex change 30 s, 58.2 ℃ annealing 30s, 72 ℃ are extended 30s, totally 25 circulations, 72 ℃ are extended 5min, 4 ℃ of insulations again; The PCR primer of Germany mirror carp MHC class IIB gene is HLJB and HLJB5, HLJB upstream primer 5'-CACTCT/AGAGCTGGAGTACAC-3', downstream primer is 5'-GTAAATGAGTCCAGCAGCTG-3', PCR reaction conditions: 95 ℃ of sex change 5min, 95 ℃ of sex change 30 s, 56.0 ℃ annealing 30s, 72 ℃ are extended 30s, totally 25 circulations, 72 ℃ are extended 5min again, 4 ℃ of insulations, HLJB5 upstream primer 5'-A/GTTAC/AA/GCTCAGG/TTC/TAGC/TGAG/AG-3', HLJB5 downstream primer 5'-TCTTCTCTCCAGATTTGGGGG-3', PCR reaction conditions: 95 ℃ of sex change 5min, 95 ℃ of sex change 30 s, 59.5 ℃ annealing 30s, 72 ℃ are extended 30s, totally 25 circulations, 72 ℃ are extended 5min, 4 ℃ of insulations again; The method of extracting DNA in the step 2 is: respectively German mirror carp is gathered fin ray and take out from alcohol, clean, shred with distilled water, at Digestive system (10mmol/L Tris-HCl pH 8.0,100mmol/L EDTA (pH8.0), 0.5% SDS, 100 μ g/mL Proteinase Ks) 55 ℃ of thoroughly digestion in, use saturated phenol, phenol/chloroform, chloroform/primary isoamyl alcohol extrct albumen, dialysis, ethanol sedimentation more respectively, be dissolved among the 1/10TE then, and its concentration transferred to 50 ng/ μ L ,-20 ℃ of preservations are standby; Carp MHC class I in the step 2 and MHC class IIB gene have obtained the clone.
Step 3 is verified: to the individuality of surviving has carried out the particular bases check and analysis through resistance is dead, in MHC class I gene, in 30 population of individuals, the T base frequency of occurrences is 0%; In 500 population of individuals, the T base frequency of occurrences 39.6%.In MHC class II gene, in 30 population of individuals, the G base frequency of occurrences is 2.8%; In 500 population of individuals, the G base frequency of occurrences 2.9%.These two kinds of bases occurrence probability in the survival individuality is all lower, and is consistent with The selection result, verified and degeneration-resistant dependency.
Embodiment two: present embodiment Germany mirror carp core selective breeding colony construction process as: the German mirror carp parent fish population applying electronic mark that will carry out seed selection carries out mark, in conjunction with screening two anti contravariance related gene sites, according to two pairs of primers selecting to have degeneration-resistant site increase, order-checking and degeneration-resistant site base analysis, with the base that only in resistance difference colony, occurs as the anti contravariance related gene mark, the individuality of these adversity gene marks is carried in rejecting, does not carry the individual core selective breeding colony that forms of these genetic markers.
The described German mirror carp adversity gene flag sequence of present embodiment is the HLJI-9 extension increasing sequence of MHC class I and the HLJB extension increasing sequence of MHC class IIB, and wherein the HLJI-9 extension increasing sequence of MHC class I is 5'-TGATGGCACCAAGGAGGATACTATCAG
TTTGGTTACGATGGAGAGGACTTCCTCAGTCTGGATAAGAGCTCCCTCACCTGGACT GCTGCCAATCCTCAAGCTGTCATTACCA-3', the 28th base T is the adversity gene site; The HLJB extension increasing sequence of MHC class IIB is 5'-TCCTGTATGGTTGAGCATGCCAGCTTCAGTAAACCCATGATTTATGACTGGGG TAAGATGAGACACGCAACAGATGTCATATTATAACTGTGTTTGTGGTAAGCATAAT TTGACATATGACATATGACATACTCCACAGATCCGTCTCTCCCTGAGTCTGAGAGG AATAAAATTGCTATTGGAGCATTTGGTCTGGTGCTGGGAATCATTATT
GCAGCTGCTGGACTCATTTACA-3', the 214th bases G is the adversity gene site.
Present embodiment is by German mirror carp adversity gene mark, set up the degeneration-resistant core selective breeding of German mirror carp colony, the property of the present invention is directed to is strong, be not subjected to the influence of body growth etap and envrionment conditions, making up core selective breeding colony selects accurately, can quicken breeding process, improve breeding efficiency, thereby effectively solve the ill problem that causes extensive death of German mirror carp high temperature season.Present embodiment is that a molecular breeding technological approaches has been opened up in the degeneration-resistant molecular breeding seed selection of fish, and cultivation has great importance and is worth to the fish anti-adversity.
Claims (2)
1. German mirror carp adversity gene mark, it is characterized in that German mirror carp adversity gene flag sequence is the HLJI-9 extension increasing sequence of MHC class I and the HLJB extension increasing sequence of MHC class IIB, wherein the HLJI-9 extension increasing sequence of MHC class I is 5'-TGATGGCACCAAGGAGGATACTATCAG
TTTGGTTACGATGGAGAGGACTTCCTCAGTCTGGATAAGAGCTCCCTCACCTGGACT GCTGCCAATCCTCAAGCTGTCATTACCA-3', the 28th base T is the adversity gene site; The HLJB extension increasing sequence of MHC class IIB is 5'-TCCTGTATGGTTGAGCATGCCAGCTTCAGTAAACCCATGATTTATGACTGGGG TAAGATGAGACACGCAACAGATGTCATATTATAACTGTGTTTGTGGTAAGCATAAT TTGACATATGACATATGACATACTCCACAGATCCGTCTCTCCCTGAGTCTGAGAGG AATAAAATTGCTATTGGAGCATTTGGTCTGGTGCTGGGAATCATTATT
GCAGCTGCTGGACTCATTTACA-3', the 214th bases G is the adversity gene site.
2. German mirror carp core selective breeding colony construction process, it is characterized in that German mirror carp core selective breeding colony construction process is as follows: the German mirror carp parent fish population applying electronic mark that will carry out seed selection carries out mark, in conjunction with screening two anti contravariance related gene sites, increase according to two pairs of primers selecting to have degeneration-resistant site, order-checking and degeneration-resistant site base analysis, with the base that only in resistance difference colony, occurs as the anti contravariance related gene mark, the individuality of these adversity gene marks is carried in rejecting, does not carry the individual core selective breeding colony that forms of these genetic markers.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108796095A (en) * | 2018-07-03 | 2018-11-13 | 中国水产科学研究院黑龙江水产研究所 | A kind of selection improving carp feed efficiency |
| CN112813171A (en) * | 2020-12-17 | 2021-05-18 | 水利部中国科学院水工程生态研究所 | MHC gene primer for cupreous rotundifolia fish and application thereof |
| CN113265470A (en) * | 2021-05-09 | 2021-08-17 | 湖州师范学院 | SNP (single nucleotide polymorphism) locus related to adverse resistance of Chinese lateolabrax japonicus and application thereof |
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| CN1958807A (en) * | 2005-10-31 | 2007-05-09 | 中国水产科学研究院黑龙江水产研究所 | Molecule marker in use for identifying cold resistance of common carp |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108796095A (en) * | 2018-07-03 | 2018-11-13 | 中国水产科学研究院黑龙江水产研究所 | A kind of selection improving carp feed efficiency |
| CN112813171A (en) * | 2020-12-17 | 2021-05-18 | 水利部中国科学院水工程生态研究所 | MHC gene primer for cupreous rotundifolia fish and application thereof |
| CN113265470A (en) * | 2021-05-09 | 2021-08-17 | 湖州师范学院 | SNP (single nucleotide polymorphism) locus related to adverse resistance of Chinese lateolabrax japonicus and application thereof |
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Application publication date: 20101124 |