CN101427668B - Breeding method for densovirus disease resistant and fluorine resistant cultivated silkworm breed variety - Google Patents
Breeding method for densovirus disease resistant and fluorine resistant cultivated silkworm breed variety Download PDFInfo
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Abstract
The invention discloses a breeding method of anti-densonucleosis and fluorin-resistant solkworn strain. The breeding method uses the Chinese race 'Jiang Song' and the Japanese race 'Hao Yue' as recurrent parents and the polyvoltine race Lan10 is used as the anti-densonucleosis high-grade object character donor parent and the T6 is used as the fluoride-resistant high-grade object character donor parent. The first hybridization generation obtained by hybridizing the recurrent parents with the Lan10 is hybridized with the T6 and then the obtained filial generation is continuously backcrossed with the recurrent parents and each generation is subjected to individual selection during breeding and the selected individual is marked by molecule to select the genotype thereof two anti-densonucleosis and fluorin-resistant silkworn strains 'JsFnsd-Z' and 'HyFnsd-Z' are selected after self-cross homozygosis for six generations. The 'JsFnsd-Z' and 'HyFnsd-Z' have strong physical constitution and dons not infected by the Bombyxmori densovirus and the whole-instars fluoride-resistance rate is above 100mg/kg and the fluoride-resistance performance is higher than the Chinese race 'Jiang Song' and the Japanese race 'Hao Yue' by 50% and the imago fin has no palea scale, in addition the practical character corresponds with the utility breed level.
Description
Technical field
The present invention relates to a kind of selection of cultivated silkworm breed variety, more particularly, relate to a kind of breeding method that adopts molecular marker assisted selection method seed selection densovirus disease resistant and fluorine resistant cultivated silkworm breed variety.
Technical background
The sericulture filature has had the history in more than 5,000 year in China, being one of most characteristic traditional advantage industry of China, also is that China can dominate one of minority product of international market, at present, silk cocoon of China and raw silk output all rank first in the world, and account for about 75% of Gross World Product respectively.
The silk industry not only has important effect to the aspect of increasing farmers' income and expand employment; and the food of silkworm--mulberry tree also has important function and significance at aspects such as environmental protection, conceding the land to forestry; therefore; many areas are in ecological protection and conceding the land to forestry, developing sericulture industry as first-selection and key project.
The new silkworm variety seed selection is one of the basis of silk industry scientific and technological progress and core, after founding of New, the cultivated silkworm breed variety seed selection of China has obtained huge achievement, has successively carried out the update of 4-5 silkworm kind in China, has greatly promoted the development of silk industry.Yet along with China's rapid economy development, labor cost also increases thereupon, environmental pollution especially fluoride pollution serious harm silkworm is already produced, the seed selection economic characters are good, and the cultivated silkworm breed variety of resistance has been extremely urgent, also are one of important behaves of stablizing the silk industry.
Development along with molecular marking technique, silkworm does not infect gene nsd-Z to the strain of desonucleosis Zhenjiang SSR molecular labeling (Muwang Li etc. have successively screened in this seminar, Genome, 2006,49:397-402) and the SSR molecular labeling (Bai Huichai etc. of the silkworm Def of anti-the key-gene, silkworm industry science, 2008,34:191-196).Up to the present, also do not have the type molecular labeling to be used for the silkworm assistant breeding and select, quicken the report of breeding process.
Summary of the invention
Technical problem: the objective of the invention is to be used for the technical problem that the silkworm assistant breeding is selected, a kind of breeding method that adopts molecular marker assisted selection method seed selection densovirus disease resistant and fluorine resistant cultivated silkworm breed variety is provided in order to solve molecular labeling.
Technical scheme: in order to realize above-mentioned target, the selection of a kind of densovirus disease resistant of the present invention and fluorine resistant cultivated silkworm breed variety, its technical scheme comprises the steps:
(1) the cyanines pine of extensively promoting with the whole nation is a recurrent parent, and polyvoltine kind blue 10 is hybridized for densovirus disease resistant high-quality objective trait donor parents, must hybridize for 1 generation; With 1 generation of hybridization and anti-fluorine high-quality objective trait donor parents T6 hybridization, obtain to hybridize for 2 generations again; With recurrent parent and hybridization 2 generation continuous backcross, obtain to backcross for 6 generations afterwards; Each is carried out economical character from generation to generation in the process of raising select, the individual molecular labeling that adopts of reserving seed for planting is selected its genotype, it is identical with the cyanines pine to obtain genetic background, and to the strain of bombyx mori densovirus Zhenjiang do not infect and full age the 100mg/kg of anti-the fluorine target material JsFnsd-Z;
(2) bright moon of extensively promoting with the whole nation is a recurrent parent, and polyvoltine kind blue 10 is hybridized for desonucleosis high-quality objective trait donor parents, must hybridize for 1 generation; With 1 generation of hybridization and anti-fluorine high-quality objective trait donor parents T6 hybridization, obtain to hybridize for 2 generations again, with recurrent parent and hybridization 2 generation continuous backcross, obtain to backcross for 6 generations afterwards; Each is carried out economical character from generation to generation in the process of raising select, the individual molecular labeling that adopts of reserving seed for planting is selected its genotype, it is identical with bright moon to obtain genetic background, and to the strain of bombyx mori densovirus Zhenjiang do not infect and full age the 100mg/kg of anti-the fluorine target material HyFnsd-Z;
(3) hybridize with target material JsFnsd-Z and target material HyFnsd-Z, obtain to hybridize for 1 generation, be densovirus disease resistant and fluorine resistant cultivated silkworm breed variety.
The described JsFnsd-Z of above-mentioned steps (1) is Chinese system;
The described HyFnsd-Z of above-mentioned steps (2) is a Japanese system;
The described densovirus disease resistant of above-mentioned steps (3) is not for to infect the strain of bombyx mori densovirus Zhenjiang; Described anti-fluorine is the 100mg/kg of anti-fluorine the in full age.
Beneficial effect: the selection of a kind of densovirus disease resistant provided by the present invention and fluorine resistant cultivated silkworm breed variety is compared with traditional hybridization back cross breeding technology has following advantage:
Traditional crossbreeding technology exists workload big, select poor reliability, particularly must be for many generations for the resistance seed selection or the throwback method of adding food pathogeny or fluoride guarantee that objective trait do not lose, not only the seed selection cycle is long, and when adding the food pathogeny, might cause environmental pollution, and the present invention can rapidly and efficiently cultivate the silkworm new lines of high yield, high-quality, double densovirus disease resistant and anti-fluorine by the genetic background of molecular marker assisted selection recurrent parent and the objective trait of donor parents in 3-4.
The kind that the inventive method is bred " JsFnsd-Z ";
Principal character is, in be bivoltine four dormancy kinds, larva is plain spot, the silkworm body is sturdy, food Sang Wangsheng grows neatly, agingly gushes together, physique is strong, does not infect the strain of desonucleosis Zhenjiang, the full 100mg/kg of anti-fluorine the in age (mulberry leaf fluorinated volume).Hasten the hatching of silkworms through 10 days, larval phase,, pupa time, process was 14-15 days through 23-24 days, the cocoon shape ellipse, and the cocoon look pure white, and the wrinkle that contracts is medium, cocoon weight 1.8-2.0g, cocoon layer rate about 23%, long about 1300 meters of silk, Xie Shuhao.Sprout wings and concentrate, male moth is active, the mating performance is good, and female moth egg is functional, and single moth ovum is counted the 500-600 grain, begins in 2nd to raise to handing over to need to postpone with HyFnsd-Z.
The kind of breeding " HyFnsd-Z ";
Principal character is, Japanese bivoltine four dormancy kinds, and larva is general spot, and the silkworm body is sturdy, and food Sang Wangsheng grows neatly, and aging neater, physique is strong, does not infect the strain of desonucleosis Zhenjiang, the full 100mg/kg of anti-fluorine the in age (mulberry leaf fluorinated volume).Hasten the hatching of silkworms through 10 days, larval phase,, pupa time, cocoon shape was shallow with a tight waist through 15-16 days through 24-25 days, and the cocoon look pure white, and the wrinkle that contracts is medium, cocoon weight 1.8-2.0g, cocoon layer rate about 23%, long about 1300 meters of silk, Xie Shuhao.Sprout wings and concentrate, male moth is active, the mating performance is good, and female moth egg is functional, and single moth ovum is counted the 500-600 grain, needs to begin in 2nd in advance to raise to handing over JsFnsd-Z.
Embodiment
One, to adopting the explanation of material
Existing result of study shows, silkworm is not infectious by recessive single-gene nsd-Z control (opposing fully) to the strain of desonucleosis Zhenjiang, only under the gene pure situation, could show not the infecting of desonucleosis Zhenjiang strain (opposing fully) (Hu Xuefang, Qian Yuanjun, Wang Honglin. bombyx mori densovirus (DNV) is to the repellence that infects of different silkworm races. silkworm industry science, 1984,10 (2): 87～90); Silkworm is controlled (anti-fluorine threshold value is 200mg/kg) (Lin Changlin to the tolerance of fluoride by dominance major gene resistance Def, Mi Yidian, Yao Qin, Deng. the discovery of the anti-fluorine dominance of silkworm major gene resistance, silkworm industry science, 1997,23 (4): 237-239), as long as this gene exists, and no matter is heterozygous or homozygous, all show tolerance to fluoride.
(1), parent's cultivated silkworm breed variety orchid 10 (public, " fortunatus kind will " agriculture publishing house, in May, 1987, the 193rd page) is not infected in the strain of desonucleosis Zhenjiang;
1, source: the cultivated silkworm breed variety of preserving is collected by Inst. of Silkworm, Chinese Academy of Agricultural Sciences, and externally shares by national economy insect resources platform.
2, principal character characteristic: Chinese system, diapause polyvoltine four dormancy kinds are arranged, the plain spot of larva, the silkworm body is tiny, grows neatly, and physique is strong, agingly gushes Duo Ying upper strata cocoon together.Hasten the hatching of silkworms through 10 days, larval phase,, pupa time was through 12 days through 8 hours on the 17th, cocoon shape is with a tight waist, shallow with a tight waist, the cocoon look pure white, and the wrinkle that contracts is thin, about cocoon weight 0.77g, cocoon layer rate about 11%, long about 343 meters of silk is separated and is relaxed well, sprouts wings and concentrates, several about 300 of single moth egg grain does not infect the strain of desonucleosis Zhenjiang.
(2), the silkworm parental breed of anti-fluorine T6 (public, document sees reference: Lin Changlin, Mi Yidian, Yao Qin, etc. the discovery of the anti-fluorine dominance of silkworm major gene resistance, silkworm industry science, 1997,23 (4): 237-239),
1, source: the cultivated silkworm breed variety of preserving is collected by the Chinese Academy of Agricultural Sciences, and external cooperation is shared.
2, principal character characteristic: Japanese system bivoltine four dormancy kinds, the general spot of larva, the silkworm body is elongated, and the food mulberry is fast, grows neatly, and physique is strong, agingly gushes Duo Ying upper strata cocoon together.Hasten the hatching of silkworms through 10 days, larval phase,, pupa time, process was 15-16 days through about 26 days hours, the shallow bundle of the long tube of cocoon shape, the cocoon look white, and the wrinkle that contracts is medium, cocoon weight 1.6-1.8g, cocoon layer rate about 24%, sprout wings and concentrate several about 500 of single moth egg grain, highly anti-fluorine (threshold value 200mg/kg).。
(3), recurrent parent cyanines pine, bright moon
Cyanines pine * bright moon is the most widely used a pair of silkworm hybrid combination of China, and its output height, silk quality is excellent, physique is strong, but to desonucleosis responsive and anti-fluorine ability.
Two, concrete implementation step:
(1), be recurrent parent with the cyanines pine, blue 10 do not infect genetic donor for desonucleosis, T6 is the major gene resistance of anti-a fluorine donor, after hybridizing with blue 10 with the cyanines pine, gets its F1 and hybridizes with T6, i.e. (cyanines pine * orchid 10) * T6, use cyanines pines continuous backcross afterwards, raise that the economical character to kind comprehensively screens in the process, adopt molecular labeling that objective trait is screened, it is identical with the cyanines pine to obtain genetic background, and the strain of desonucleosis Zhenjiang is not infected and the JsFnsd-Z of highly anti-fluorine.
(2) be recurrent parent with bright moon, blue 10 do not infect genetic donor for desonucleosis, T6 is the major gene resistance of anti-a fluorine donor, after hybridizing with blue 10 with bright moon, gets its F1 and hybridizes with T6, i.e. (bright moon * orchid 10) * T6, use the bright moon continuous backcross afterwards, raise that the economical character to kind comprehensively screens in the process, adopt molecular labeling that objective trait is screened, it is identical with bright moon to obtain genetic background, and the strain of desonucleosis Zhenjiang is not infected and the HyFnsd-Z of highly anti-fluorine.
(3) hybridize with JsFnsd-Z and HyFnsd-Z, obtain to hybridize for 1 generation, be densovirus disease resistant and fluorine resistant cultivated silkworm breed variety.
Three, desonucleosis is not infected the detection of the PCR of gene and the key-gene of anti-the fluorine
Extract the genomic DNA of bombyx mori to be detected, get 20-50ng and make template, increasing with SSR labeled primer F10316 and F10568 with nsd-Z gene linkage, (2 pairs of primers are public, see document MuwangLi etc., Linkage and mapping analyses of the densonucleosis non-susceptible genensd-Z in the silkworm Bombyx mori using SSR markers.Genome, 2006,49:397-402); With SSR labeled primer S1214 and S121201 with Def gene linkage increase (it is public that the 2 pairs of primers are, and sees document Bai Huichai etc., utilizes the SSR mark anti-fluorine-based because of carrying out chain positioning analysis silkworm industry science to silkworm, 2008,34:191-196).
The F10316 primer sequence:
Left end primer sequence: 5`-GCGATAAGACCGCCTATTGAAC-3`
Right-hand member primer sequence: 5-GTGTATTAGGCACGAGAACTGACG-3`
The F10568 primer sequence:
Left end primer sequence: 5`-TCGTCCTACACTTGCGGGTT-3`
Right-hand member primer sequence: 5-TGTTTCGTCAAGTCTGCTCGGT-3`
The S121201 primer sequence:
Left end primer sequence: 5`-AGTTCTTGCTTGTTTCCGTTCT-3`
Right-hand member primer sequence: 5-CGCCGTATGTATTTATTCCTCA-3`
The S1214 primer sequence:
Left end primer sequence: 5`-AATGGATGCGTGCCAGTTTG-3`
Right-hand member primer sequence: 5-CGTTAGTTGCTCTACGCTTTTGG-3`
The PCR response procedures is according to the program of 2 pieces of documents: 95 ℃ of sex change 3min, and 63 ℃ of annealing 40s, 72 ℃ are extended 1min; 16 Touchdown circulations, 94 ℃, 45s, annealing temperature is successively decreased 0.5 ℃ successively up to 56 ℃ of 40s, 72 ℃ of 1min; 94 ℃ of 40s, 56 ℃ of 40s, 72 ℃ of 1min, 24 circulations; 72 ℃ of 10min.The PCR product detects with the full-automatic capillary foranalysis of nucleic acids of the HAD-GT12 system of eGene.
It is 2 bands that the individual PCR product of selecting in the backcross process detects through the full-automatic capillary foranalysis of nucleic acids of HAD-GT12 system, is heterozygous, proves that this individuality carries target gene, can use, and eliminates the individuality with the consistent banding pattern of recurrent parent;
The individuality of selfing after backcrossing, after testing for consistent homozygous (band) of L10 (detect densovirus disease resistant gene) or T6 (detect anti-fluorine-based because of), show that the target gene that this individuality carries isozygotys, can use, eliminate the individuality of banding pattern and heterozygous (2 band) identical with recurrent parent.
Claims (4)
1. the selection of densovirus disease resistant and fluorine resistant cultivated silkworm breed variety is characterized in that, comprises the steps:
(1) the cyanines pine of extensively promoting with the whole nation is a recurrent parent, and polyvoltine kind blue 10 is hybridized for densovirus disease resistant high-quality objective trait donor parents, must hybridize for 1 generation; With 1 generation of hybridization and anti-fluorine high-quality objective trait donor parents T6 hybridization, obtain to hybridize for 2 generations again; With recurrent parent and hybridization 2 generation continuous backcross, obtain to backcross for 6 generations afterwards; Each is carried out economical character from generation to generation in the process of raising select, the individual molecular labeling that adopts of reserving seed for planting is selected its genotype, it is identical with the cyanines pine to obtain genetic background, and to the strain of bombyx mori densovirus Zhenjiang do not infect and full age the 100mg/kg of anti-the fluorine target material JsFnsd-Z;
(2) bright moon of extensively promoting with the whole nation is a recurrent parent, and polyvoltine kind blue 10 is hybridized for desonucleosis high-quality objective trait donor parents, must hybridize for 1 generation; With 1 generation of hybridization and anti-fluorine high-quality objective trait donor parents T6 hybridization, obtain to hybridize for 2 generations again, with recurrent parent and hybridization 2 generation continuous backcross, obtain to backcross for 6 generations afterwards; Each is carried out economical character from generation to generation in the process of raising select, the individual molecular labeling that adopts of reserving seed for planting is selected its genotype, it is identical with bright moon to obtain genetic background, and to the strain of bombyx mori densovirus Zhenjiang do not infect and full age the 100mg/kg of anti-the fluorine target material HyFnsd-Z;
(3) target material JsFnsd-Z and target material HyFnsd-Z are hybridized, obtain and hybridized for 1 generation, be densovirus disease resistant and fluorine resistant cultivated silkworm breed variety.
2. the selection of a kind of densovirus disease resistant according to claim 1 and fluorine resistant cultivated silkworm breed variety is characterized in that: the described JsFnsd-Z of step (1) is Chinese system.
3. the selection of a kind of densovirus disease resistant according to claim 1 and fluorine resistant cultivated silkworm breed variety is characterized in that: the described HyFnsd-Z of step (2) is a Japanese system.
4. the selection of a kind of densovirus disease resistant according to claim 1 and fluorine resistant cultivated silkworm breed variety is characterized in that: the described densovirus disease resistant of step (3) is not for to infect the strain of bombyx mori densovirus Zhenjiang; Described anti-fluorine is the 100mg/kg of anti-fluorine the in full age.
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| CN103070138A (en) * | 2013-02-04 | 2013-05-01 | 江苏科技大学 | Method for breeding hypersilk, bombyx mori densovirus-resistant and fluoride-resistant silkworm variety |
| CN104839099B (en) * | 2015-04-17 | 2017-07-14 | 江苏科技大学 | A kind of hybridization, the method for the anti-nuclear polyhedrosis of backcross improvement silkworm |
| CN112369376B (en) * | 2020-11-27 | 2022-04-26 | 江苏科技大学 | Breeding method of silkworm with densonucleosis resistance |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1418543A (en) * | 2002-09-05 | 2003-05-21 | 浙江省农业科学院 | Method for utilization of biological reaction device specially used for variety development of silkworm |
| CN1419821A (en) * | 2001-11-20 | 2003-05-28 | 成都天友生物科技股份有限公司 | Temp-sensitive lethality male silkworm breeding method |
| CN1515150A (en) * | 2003-08-26 | 2004-07-28 | 苏州大学 | Sex discrimination of mulberry silkworm pod and its silkworm varietly assortive breeding method |
| CN101107925A (en) * | 2007-08-27 | 2008-01-23 | 贵州省蚕业研究所 | Multicomponent cross breeding method of selection and breeding of silkworm |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1419821A (en) * | 2001-11-20 | 2003-05-28 | 成都天友生物科技股份有限公司 | Temp-sensitive lethality male silkworm breeding method |
| CN1418543A (en) * | 2002-09-05 | 2003-05-21 | 浙江省农业科学院 | Method for utilization of biological reaction device specially used for variety development of silkworm |
| CN1515150A (en) * | 2003-08-26 | 2004-07-28 | 苏州大学 | Sex discrimination of mulberry silkworm pod and its silkworm varietly assortive breeding method |
| CN101107925A (en) * | 2007-08-27 | 2008-01-23 | 贵州省蚕业研究所 | Multicomponent cross breeding method of selection and breeding of silkworm |
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