CN101617638B - Construction method of portunus trituberculatus miers disease-resistant variety - Google Patents
Construction method of portunus trituberculatus miers disease-resistant variety Download PDFInfo
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- CN101617638B CN101617638B CN2008101381611A CN200810138161A CN101617638B CN 101617638 B CN101617638 B CN 101617638B CN 2008101381611 A CN2008101381611 A CN 2008101381611A CN 200810138161 A CN200810138161 A CN 200810138161A CN 101617638 B CN101617638 B CN 101617638B
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Abstract
The invention relates to the aquatic product heredity and breeding field, in particular to a construction method of a portunus trituberculatus miers disease-resistant variety. The method comprises the following concrete steps: information of mitochondrial control zone sequence of breeding materials serves as a molecular marker, group with rich heredity mutation serves as base group of group breeding according to the molecular marker, and groups which are genetically independent serve as the supplementary materials of family breeding; first, group breeding is carried out on the base group to obtain group, family breeding is carried out on the obtained group and the supplementary materials of family breeding, then, interfamily hybridization combination is carried out on groups with stable disease-resistant character obtained from family breeding, and the family with stable disease-resistant character is the disease-resistant variety; wherein breeding materials in each step are individuals which survive with half lethal concentration after being soaked and infected by source of infection. The invention shortens the breeding time while making the most use of species genetic resources simultaneously, combines the adaptability of local crabs, and effectively avoids the inbreeding depression of small groups, thus facilitating scientifically cultivation of the portunus trituberculatus miers disease-resistant variety suitable for local aquaculture.
Description
Technical field
The present invention relates to aquatic products genetic breeding field, is the construction method of the disease-resistant strain of a kind of Portunus trituberculatus Miers specifically.
Background technology
The genetic breeding of aquatic livestock adopts the method for colony's seed selection or hybridization to carry out traditionally, and the method for attempting using for reference the family selective breeding that adopts in holding fowl is also arranged at present.Because general aquatic livestock egg laying amount is big, progeny size is many, makes family selective breeding very loaded down with trivial details, operating difficulties.Colony's seed selection length consuming time, it is slow to produce effects.In recent years, Chinese scholars is paid special attention to molecular mark always, but especially yet there are no effect on the genetic breeding of aquatic livestock crab class in actual applications.
The present invention by a large amount of practices and scientific experiment, successfully develops a kind of breeding method that is fit to the disease-resistant strain development of Portunus trituberculatus Miers under the situation that takes into full account various breeding methods, breeding approach characteristics.Through the enforcement in 3 years, prove that the method has significantly reduced the seed selection link, accelerated breeding process, the inbreeding depression phenomenon of effectively having avoided the microcommunity breeding to cause simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of construction method of the disease-resistant strain of Portunus trituberculatus Miers quick, effective and easy and simple to handle.
For achieving the above object, the technical solution used in the present invention is:
The construction method of the disease-resistant strain of Portunus trituberculatus Miers: with the information of the mitochondria control region sequence of seed selection material as molecular labeling, obtain the basic population of the abundant colony of genetic variation according to molecular labeling, obtain the supplementary material as family selective breeding simultaneously with colony separate in the heredity as colony's seed selection; At first the supplementary material with seed selection gained colony of the capable colony of basic population and family selective breeding carries out family selective breeding, carry out tame line cross combination between then that the disease-resistant proterties of family selective breeding gained is the stable colony, the family that the disease-resistant proterties of gained is stable is disease-resistant strain, and wherein each step seed selection material is the individuality of half lethal concentration survival after the source of infection is soaked infection.
The information of the mitochondria control region sequence of described seed selection material is that template adopts primer upstream primer sequence 5 '-AGGAATTAAGAAACAATCAAACTTT-3 ' and downstream primer sequence 5 '-GCCGTTGAGTGAAGAGTGTTCAGATAG-3 ' to carry out the sequence fragment that pcr amplification obtains the mitochondria control district as molecular labeling for the genomic DNA with the seed selection material, and described seed selection material is the colony of each department crab.The PCR total reaction volume is 25uL during described pcr amplification, wherein contains 10mM dNTP 0.5ul, each primer 5uM 1ul, 0.4U Taq enzyme, 10 * PCR Buffer2.5ul, 25mM Mgcl
21.5ul; PCR response procedures: 95 degree 3min; 95 spend 30s then, 55 degree 30s, and 72 degree 1min circulate 29 times; Last 72 degree 7min.The abundant colony of described genetic variation for the mitochondria control region sequence of the colony of each department crab as Dandong colony that filters out in the molecular labeling, Qingdao colony, Yingkou colony, Lianyun Harbour colony or Ningbo colony.In the described heredity separate colony for the mitochondria control region sequence of the colony of each department crab as Laizhou Wan colony that filters out in the molecular labeling and the North Sea, Guangxi colony.Described each step seed selection material is through 72 hours half lethal concentrations of vibrio alginolyticus and soaks metainfective residue individuality.
The advantage that the present invention had:
1. the genetic background of breeding is clear, has avoided the blindness of traditional breeding method.Utilize the mitochondria control region sequence as molecular labeling, distinguished the genetic structure of China wild Portunus trituberculatus Miers colony, genetic affinity and each geographical population genetic diversity between clear and definite each geographical population make the selection of breeding material have stronger purpose.
With genetic variation abundant when land carb be the stock of colony's seed selection, help keeping its adaptive inherent cause, strengthen the adaptive faculty of the disease-resistant strain of selecting, cultivate and be fit to local excellent strain of culturing.
3. with the additional genetic stocks of colony separate in the heredity, when effectively utilizing genetic resources, also effectively alleviated because the inbreeding depression phenomenon that the microcommunity breeding causes as family selective breeding.
4. adopt the parallel method of molecular selection, colony's seed selection and family selective breeding to make up the disease-resistant strain of Portunus trituberculatus Miers, accelerated breeding process, also improved the validity of breeding.
Description of drawings
Fig. 1 is genetic structure and the genetic diversity collection of illustrative plates that utilizes the coastal Portunus trituberculatus Miers colony of mitochondria control district information analysis China.(it utilizes the phylogenetic tree of MEGA3.1 software (Kumar et al.1994) structure based on the Kimura two-parameter model, the node supporting rate is used 1000 bootstrap checks, wherein, DD refers to Dandong colony, and YK refers to Yingkou colony, and LZ refers to Laizhou colony, QD refers to Qingdao colony, LYG refers to Lianyun Harbour colony, and NB refers to Ningbo colony, and BH refers to North Sea colony.The numeral of capitalization back is meant the individuality arrangement number of colony's sampling tense marker.)
Fig. 2 infects the graph of a relation of concentration and lethality for vibrio alginolyticus.
Embodiment
Embodiment 1
As molecular labeling, described seed selection material is the colony of each department crab with the information of the mitochondria control region sequence of seed selection material.Use Auele Specific Primer (upstream primer sequence 5 '-AGGAATTAAGAAACAATCAAACTTT-3 ', downstream primer sequence 5 '-GCCGTTGAGTGAAGAGTGTTCAGATAG-3 '), template is for to carry out the PCR amplification in vitro with seed selection material genomic DNA.The PCR total reaction volume is 25uL, wherein contains dNTP (10mM) 0.5ul, each primer 5uM 1ul, 0.4U Taq enzyme, 10 * PCR Buffer2.5ul, 25mM Mgcl
21.5ul.PCR is reflected on the Bio-rad MyCycler PCR instrument and carries out.PCR response procedures: 95 degree 3min; 95 spend 30s then, 55 degree 30s, and 72 degree 1min circulate 29 times; Last 72 degree 7min.The PCR product reclaims the kit purifying through 1% agarose gel electrophoresis, glue.Use ABI Prism 3100 DNAAnalyzer to carry out unidirectional order-checking, get the information of the mitochondria control region sequence of seed selection material; Utilize the phylogenetic tree of MEGA 3.1 softwares (Kumar et al.1994) structure based on the Kimura two-parameter model, the abundant breeding population of genetic variation will be as the basic population of colony's seed selection, as Dandong colony or Qingdao colony or Yingkou colony or Lianyun Harbour colony or Ningbo colony (referring to Fig. 1); The separate North Sea, Guangxi colony and Shandong Laizhou Wan colony (referring to Fig. 1) are as the supplementary material of family selective breeding in the heredity.At first, the basic population of first generation seed selection for working as land carb with cultivating, and the method for employing is colony's seed selection; Secondly, obtain F1 after generation, replenish North Sea colony and Laizhou colony, the method for employing is a family selective breeding; At last, obtain F2 after generation, carry out hybrid combination between the family that disease-resistant proterties is stable and make up new family, the family that still has stable disease-resistant proterties is formed disease-resistant strain.It is that the source of infection is soaked infection that all seed selection materials are all selected with the vibrio alginolyticus, 72 hours half lethal concentration (1.0-2.0 * 10
10) individuality of survival is as the parent of seed selection.
Be specially and work as the colony of land carb parent and comprise that each 50 of ♀, ♂ carry out random mating, its offspring is cultured to adult be the first filial generation through infect handling survival; Then, handle through same infection the first filial generation, simultaneously, the North Sea, Guangxi colony and Shandong Laizhou Wan colony also handle through same infection, carrying out hybrid combination in such a way and make up family: A1-5, ♀ is big * and ♂ is big, B1-5, ♀ is big * and ♂ is little, C1-5, ♀ is little * and ♂ is big, D1-5, ♀ is big * and ♂ Guangxi is big, E1-5, ♀ is big * and ♂ Guangxi is little, F1-5, greatly * ♂ Shandong is big, G1-5, ♀ is big * and ♂ Shandong is little, and wherein 1-5 refers to every group of 5 repetitions, amount to 35 familys, its offspring is cultured to adult be second filial; At last, select big individuality in the family that disease-resistant proterties is stable from second filial and carry out tame line cross combination, make up new family, the family that wherein still shows stable disease-resistant proterties is the disease-resistant strain of Portunus trituberculatus Miers.
Dandong colony or Qingdao colony or Yingkou colony or Lianyun Harbour colony or Ningbo colony all are the abundant geographical population of genetic variation as can be seen from Figure 1, and the North Sea, Guangxi colony (BH) has formed hereditary two the separate colonies of with Shandong Laizhou Wan colony (LZ).
Embodiment 2
Place: Ninghai County, Zhejiang Province.
In August, 2005, Ninghai County, Ningbo City, Zhejiang Province double plate be coated with the aquatic product sprout field advance sea area, the Xiangshan Bay East Sea, Ningbo gonadal maturation female and male swimming crab each 50, with 1.0 * 10
10The vibrio alginolyticus bacterium liquid of concentration soaks the swimming crab parent, and a survival body and function clear water is cleaned after 72 hours, puts back to storage pond, holding pond and supports temporarily.The offspring that random mating produces cultures to adult.
In October, 2006, getting water aquatic product sprout field will go up the disease-resistant candidate colony of culturing in a year to adult and handle through same infection in Ninghai County, Ningbo City, Zhejiang Province, simultaneously, will through two separate on Shandong Laizhou Wan colony that embodiment filters out and the North Sea, Guangxi population genetic colonies through same pathogeny infect handle after, hybridize, a same embodiment of hybrid combination amounts to 35 groups.Wherein, refer to Ningbo colony when land carb.Each hybrid combination is supported separately temporarily, and hatching successively, through conventional artificial breeding, goes out ten thousand/kg of seed C1-C2 phase 2.4-3.12 in June, 2007 altogether, amounts to 4.45kg, puts in a suitable place to breed in 1 pond, separates with enclosure between each family and cultures.
Get water aquatic product sprout field in October, 2007 in Ninghai County, Ningbo City, Zhejiang Province, culturing the survival rate height in the family of selecting to culture the previous year is that the strong family of premunition is as the parent, with 2.0 * 10
10The vibrio alginolyticus of concentration is that the source of infection is carried out the processing same with last one year, and the big individuality of survival carries out the hybrid combination between each family, makes up new family.Then, new family through with last one year of same supporting temporarily, mating, lay eggs, through conventional artificial breeding, go out ten thousand/kg of seed C1-C2 phase 2.4-3.12 in June, 2008 altogether, amount to 10.26kg, put in a suitable place to breed in 1 pond, separate with enclosure between each family and culture.One of them family is disease-resistant strain, compares with the seed of wild population, has shown tangible premunition as shown in Figure 2.Its Smalt is represented wild population, and pink colour is represented disease-resistant strain, and the vibrio alginolyticus source of infection of variable concentrations is 3.2 * 10
4, 1.8 * 10
5, 1.0 * 10
6With 5.6 * 10
6Cfuml
-1.
The premunition experiment of disease-resistant as can be seen from Figure 2 strain and wild population, the result proves that under the vibrio alginolyticus of variable concentrations infected, disease-resistant strain improved more than 20% than the survival rate of wild population.
Claims (5)
1. the construction method of the disease-resistant strain of Portunus trituberculatus Miers, it is characterized in that: with the information of the mitochondria control region sequence of seed selection material as molecular labeling, obtain the basic population of the abundant colony of genetic variation according to molecular labeling, obtain the supplementary material as family selective breeding simultaneously with colony separate in the heredity as colony's seed selection; At first the supplementary material with seed selection gained colony of the capable colony of basic population and family selective breeding carries out family selective breeding, carry out tame line cross combination between then that the disease-resistant proterties of family selective breeding gained is the stable colony, the family that the disease-resistant proterties of gained is stable is disease-resistant strain, and wherein each step seed selection material is the individuality of half lethal concentration survival after the source of infection is soaked infection;
The information of the mitochondria control region sequence of described seed selection material is that template adopts primer upstream primer sequence 5 '-AGGAATTAAGAAACAATCAAACTTT-3 ' and downstream primer sequence 5 '-GCCGTTGAGTGAAGAGTGTTCAGATAG-3 ' to carry out the sequence fragment that pcr amplification obtains the mitochondria control district as molecular labeling for the genomic DNA with the seed selection material, and described seed selection material is the colony of each department crab.
2. by the construction method of the disease-resistant strain of claim 1 Portunus trituberculatus Miers, it is characterized in that: the PCR total reaction volume is 25ul during described pcr amplification, wherein contains 10mM dNTP 0.5ul, each primer 5uM1ul, 0.4U the Taq enzyme, 10 * PCR Buffer2.5ul, 25mM Mgcl
21.5ul; PCR response procedures: 95 degree 3min; 95 spend 30s then, 55 degree 30s, and 72 degree 1min circulate 29 times; Last 72 degree 7min.
3. by the construction method of the disease-resistant strain of claim 1 Portunus trituberculatus Miers, it is characterized in that: the abundant colony of described genetic variation for the mitochondria control region sequence of each department crab as Dandong colony that filters out in the molecular labeling, Qingdao colony, Yingkou colony, Lianyun Harbour colony or Ningbo colony.
4. by the construction method of the disease-resistant strain of claim 1 Portunus trituberculatus Miers, it is characterized in that: in the described heredity separate colony for the mitochondria control region sequence of each department crab as Laizhou Wan colony that filters out in the molecular labeling and the North Sea, Guangxi colony.
5. by the construction method of the disease-resistant strain of claim 1 Portunus trituberculatus Miers, it is characterized in that: described each step seed selection material is through 72 hours half lethal concentrations of vibrio alginolyticus and soaks metainfective residue individuality.
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| KR20130050406A (en) | 2011-11-07 | 2013-05-16 | 오수미 | Method for generating prediction block in inter prediction mode |
| CN104412914A (en) * | 2013-08-23 | 2015-03-18 | 盘锦光合蟹业有限公司 | Breeding method of large-size river crab |
| CN107535398A (en) * | 2017-09-12 | 2018-01-05 | 钦州学院 | By controlling concentration of formaldehyde to improve Eriocheir hepuensis breeding survival rate and the method for taking the photograph bait amount |
| CN108624670B (en) * | 2018-07-23 | 2019-09-03 | 中国水产科学研究院黄海水产研究所 | A kind of the molecular labeling Marker Sex 1 and identification method of Rapid identification Portunus trituberculatus Miers genetic sex |
| CN110432200A (en) * | 2019-09-05 | 2019-11-12 | 中国水产科学研究院淡水渔业研究中心 | A kind of freshwater shrimp pyramiding breeding method |
| CN110724749B (en) * | 2019-12-02 | 2021-06-04 | 中国水产科学研究院黄海水产研究所 | Molecular marker C104 of portunus trituberculatus resistant vibrio parahaemolyticus and application thereof |
| CN115053842B (en) * | 2022-08-03 | 2023-05-16 | 中国水产科学研究院黄海水产研究所 | Breeding method of new strain of portunus trituberculatus for resisting vibrio parahaemolyticus |
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| WO2005005613A2 (en) * | 2003-07-02 | 2005-01-20 | Medical University Of South Carolina | Dsrna induced specific and non-specific immunity in crustaceans and other invertebrates and biodelivery vehicles for use therein |
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| WO2005005613A2 (en) * | 2003-07-02 | 2005-01-20 | Medical University Of South Carolina | Dsrna induced specific and non-specific immunity in crustaceans and other invertebrates and biodelivery vehicles for use therein |
| CN100998320A (en) * | 2006-12-21 | 2007-07-18 | 张玉满 | Outdoor earth pond ecological culturing of portunid juvenile crabs |
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