CN104920255A - F3-generation rapid-growth flounder pure line construction method - Google Patents
F3-generation rapid-growth flounder pure line construction method Download PDFInfo
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- A—HUMAN NECESSITIES
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
A breeding technique method for selecting, inducing and cultivating excellent flounder lines belongs to the technical field of marine organisms. The method mainly comprises screening and construction of F1-generation and F2-generation rapid-growth flounder excellent families, induced establishment of mitotic and meiotic F3-generation gynogenetic pure lines, and genetic monitoring of flounder pure lines. According to the method, a rapid-growth F1-generation family F0750 and a rapid-growth F2-generation excellent family F09119 are screened and constructed; a mitotic F3-generation gynogenetic line F1346 and a meiotic F3-generation gynogenetic line F1324 are constructed through heterogenous frozen semen induction, hydrostatic pressure induction and cold shock induction and are compared on growth with an inbred line F1313, wherein the growth speed of the F1346 is obviously higher than those of the F1324 and the F1313; the genetic monitoring is performed through microsatellite markers scaffold288_1492; the genetypes of the F1346 and the F1324 are pure AA and BB respectively, while that of the F1313 is hybrid. Therefore, excellent pure lines of rapid-growth new flounder species can be constructed.
Description
One, technical field: this technology belongs to field of marine biotechnology is the breeding method of a kind of selection, induction and the excellent pure lines of cultivation lefteye flounder.
Two, background technology:
Lefteye flounder (Paralichthys olivaceus) is the Main Commercial Fishes being distributed in China coast, be naturally to fish for, batch production, pond and cage culture main object, annual production reaches 26477.5t, account for 30% (Shanghai Ocean University of flounder flounder class output, Yang Zhengyong, 2009), in left-eyed flounder output, larger proportion is occupied.But along with the rapid expansion of lefteye flounder aquaculture industry, there are problems in lefteye flounder kind matter, as: slow, the various disease of growth rate frequently occurs (as: balantidiasis, Lymphocystis disease, Vibrio anguillarum disease, albinism etc.), the culture-cycle long, cultivation efficiency is low, has had a strong impact on the development of the popularization of lefteye flounder seed cultivating large area and industrialization.Therefore the breeding core population of lefteye flounder Different population and artificial screening is utilized to carry out lefteye flounder selection and use, build the excellent pure lines of lefteye flounder with good development and degeneration-resistant proterties, and then cultivate lefteye flounder improved seeds, develop significant for the recovery of lefteye flounder kind matter, raising cultured product quality and economic benefit, promotion China left-eyed flounder aquaculture industryization.In the research of lefteye flounder cultivation new varieties, the screening such as Kanako Fuji cultivates one can cultured population (the Faculty of Marine Science of disease-resistant Lymphocystis disease, Tokyo University of Marine Science and Technology, Kanako Fuji et al, Aquaculture, 2006).This seminar collects and establishes a large amount of lefteye flounder breeding core populations in recent years, establish a large amount of lefteye flounder familys, and filter out the lefteye flounder family (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science with merits such as growth are fast, survival rate is high, field Yongsheng etc., aquatic product journal, 2009; Ocean journal, 2011), within 2010, cultivate China's lefteye flounder hybrid new breed " excellent No. 1 of flounder " (national Fishery technical Center for Popularization, 2011).The excellent pure lines of lefteye flounder build has important effect for continuing to cultivate the lefteye flounder new varieties with different merits such as growth are fast, resistance against disadvantage is strong, but the excellent pure lines of lefteye flounder to build be a comprehensive and complicated technology, at home and abroad have not been reported at present.
Three, summary of the invention:
Mainly comprise following several aspect: 1, lefteye flounder F1 generation grows foundation and the screening of fast family, 2, in lefteye flounder F2 generation, grows foundation and the screening of fast superior families, 3, the induction that lefteye flounder F3 is sheerly for mitosis is set up, 4, the induction that lefteye flounder F3 is sheerly for subtrahend gynogenesis is set up, and 5, the Genetic Detection of lefteye flounder pure lines.
1, lefteye flounder F1 generation grows foundation and the screening of fast family
Utilize the sick colony (RS) of lefteye flounder anti-vibrio anguillarum, Japanese lefteye flounder colony (JS) and Huanghai Sea lefteye flounder colony (YS) to carry out artificial orientation's mating in mating season, mating pattern is:
set up lefteye flounder family, family is growing into average age in days to 90, about 524d,, body weight long to the body of each family is measured, utilize one-way analysis of variance and Student-Newman-Keuls (SNK) method to carry out Multiple range test to different family growth traits, analyze the growth differences between family.Calculate absolute gain rate (AGR simultaneously
w, g.d
-1)=(W
2-W
1)/(t
2-t
1), compared by rate of body weight gain and filter out the fast family F0750 of growth.
2, in lefteye flounder F2 generation, grows foundation and the screening of fast superior families
F0750 screening obtained, lefteye flounder colony of Korea S (KS), Japanese lefteye flounder colony (JS), the sick colony (RS) of lefteye flounder anti-vibrio anguillarum cultivate sexual maturity, and carry out directed artificial propagation mating in mating season, modes of reproduction is
establish lefteye flounder family, family grow into 164 days and 340 days time measure its body weight, utilize one-way analysis of variance and Student-Newman-Keuls (SNK) method to carry out Multiple range test to different family body weight, analyze the growth differences between family.Utilize absolute gain rate (AGR
w, g.d
-1)=(W
2-W
1)/(t
2-t
1) formula, each family growth absolute gain rate is calculated, sorts according to the growth rate of absolute gain rate to all familys, filter out the fastest family F09119 of growth
3, the induction that lefteye flounder F3 is sheerly for mitosis is set up
Gather ripe perch sperm in advance, utilize MPRS+20%DMOS solution with the dilution proportion perch sperm of volume ratio 1: 1, adopt 2.0ml cryovial freezen protective in liquid nitrogen, set up perch sperm freezing storehouse, each constituent concentration of MPRS solution is: NaCl 60.35mM, NaH
2pO
41.8mM; NaHCO
33mM, KCl 5.23mM, CaCl
2.2H
2o 1.13mM, MgCl
2.6H
2o 1.13mM, D-Glucose 55.55mM.DMSO is commercially available dimethyl sulfoxide (DMSO).
F09119 is cultivated to sexual maturity, utilizes the mode of manual compression belly to obtain the unfertilized egg of its maturation.Utilize 37 DEG C of water-bath solution frozen sea-bass sperms thereupon, utilize MPRS dilution by sperm with 1: 10 dilution proportion, at diameter be add in the culture dish of 10cm 1ml dilute seminal fluid pave, at 40000 μ J/cm
2irradiate under intensity ultraviolet, make its genetic inactivation, add in lefteye flounder ovum and carry out dry method insemination, fertilized egg is 100: 0.4 with seminal fluid ratio.At water temperature 17 DEG C, after fertilization 58min, utilizes hydrostatic press by pressure adjusting to 590kg/cm
2, the hydrostatic pressing processing time is 6min, and induction fertilized egg carries out mitosis gynogenesis, obtains pure lines F1346.
4, lefteye flounder F3 sets up for the induction of subtrahend gynogenosis system
Superior families F09119 is cultivated to sexual maturity, utilizes manual compression belly method to gather unfertilized egg.Utilize 37 DEG C of water-bath solution frozen sea-bass sperms thereupon, utilize MPRS dilution by sperm with 1: 10 dilution proportion, at diameter be add in the culture dish of 10cm 1ml dilute seminal fluid pave, at 40000 μ J/cm
2irradiate under intensity ultraviolet, make its genetic inactivation, add in lefteye flounder ovum and carry out dry method insemination, fertilized egg is 100: 0.4 with seminal fluid ratio, cold shock treatment 45min in 2-4 DEG C of seawater is immersed after insemination 3min, make its chromosome doubling, then ovum is put into 17 DEG C of seawater hatchings, obtain lefteye flounder subtrahend gynogenosis system F1324.So far complete the structure of the excellent pure lines of lefteye flounder, the gene of superior families is further purified, reaches the object setting up excellent pure lines.
5, the Genetic Detection of lefteye flounder pure lines
Lefteye flounder is sheerly F1346, F1324 and cultivates 10-15cm, random selecting 30 tail individual clip tail fin bar respectively, phenol chloroform method is utilized to extract DNA, microsatellite marker scaffold288_1492 in selection lefteye flounder genetic linkage maps No. 21 linkage groups is as primer, primer sequence F:AGTGGATTAGCGTGGTGGATAC, R:AGATCAGTGTGGTCAAGGTGG; By PCR to its DNA cloning, 8% native polyacrylamide gel electrophoresis detects the genotype of lefteye flounder pure lines.
Four, accompanying drawing explanation
Fig. 1 and Fig. 2 is respectively subtrahend thelykaryon family (F1324) and spilting of an egg thelykaryon family (F1346) the offspring genotype electrophoretogram that lefteye flounder grows fast family F09119, utilizing microsatellite marker (scaffold288_1492) to detect offspring individuals is all homozygote, has 16 AA genotype and 14 BB genotype in offspring respectively.Fig. 3 is F09119 inbreeding family (F1313) offspring genotype electrophoretogram, and Parent genotype is AC and BC, 2 AB types in 30 filial generations, 10 AC types, 12 BC types, and 5 is CC type.
Five, embodiment
1, lefteye flounder F1 generation grows the foundation of fast family and screening
Utilize the sick colony (RS) of lefteye flounder anti-vibrio anguillarum, Japanese lefteye flounder colony (JS) and Huanghai Sea lefteye flounder colony (YS) to carry out artificial orientation's mating in mating season, mating pattern is:
establish family 59.
Set up lefteye flounder family and grow into average age in days to 90, about 524d, the body weight of each family is measured, utilize one-way analysis of variance and Student-Newman-Keuls (SNK) method to carry out Multiple range test to different family growth traits, analyze the growth differences between family.Utilize absolute gain rate (AGR
w, g.d
-1)=(W
2-W
1)/(t
2-t
1), each family growth 90d to 524d absolute gain rate is calculated, average age in days (t
2-t
1) calculate (table 1) by 286d, sort according to the growth rate of absolute gain to all familys, filter out the growth of F0750 family the fastest.
Table 1. lefteye flounder F1 generation family mean body weight and absolute gain rate compare
Note: * has significant difference (P < 0.05) in growth
2, in lefteye flounder F2 generation, grows foundation and the screening of fast superior families
F0750 screening obtained, lefteye flounder colony of Korea S (KS), Japanese lefteye flounder colony (JS), the sick colony (RS) of lefteye flounder anti-vibrio anguillarum cultivate sexual maturity, directed artificial propagation mating is carried out in mating season, mode is as table 2, establish 22 lefteye flounder familys, family grow into 164 days and 340 days time measure its body weight, utilize one-way analysis of variance and Student-Newman-Keuls (SNK) method to carry out Multiple range test to different family body weight, analyze the growth differences between family.Utilize absolute gain rate (AGR
w, g.d
-1)=(W
2-W
1)/(t
2-t
1) formula, each family growth 164d to 340d absolute gain rate is calculated, average age in days (t
2-t
1) calculate (table 2) by 176d, sort according to the growth rate of absolute gain to all familys, filter out the fastest family F09119 of growth.
Table 2. lefteye flounder F2 compares for family mean body weight and absolute gain rate
Note: * has significant difference (P < 0.05) in growth
3, the induction that lefteye flounder F3 is sheerly for mitosis is set up
Gather ripe perch sperm in advance, utilize MPRS+20%DMOS solution with the dilution proportion perch sperm of volume ratio 1: 1, adopt 2.0ml cryovial freezen protective in liquid nitrogen, set up perch sperm freezing storehouse, freezen protective perch sperm 200ml.Each constituent concentration of MPRS solution is: NaCl 60.35mM, NaH
2pO
41.8mM; NaHCO
33mM, KCl 5.23mM, CaCl
2.2H2O 1.13mM, MgCl
2.6H2O 1.13mM, D-Glucose 55.55mM.DMSO is commercially available dimethyl sulfoxide (DMSO).
F09119 family is cultivated to sexual maturity, utilizes manual compression belly method to gather unfertilized egg.Utilized by freezen protective perch sperm 37 DEG C of water-baths to thaw sperm simultaneously, utilize MPRS dilution by sperm with 1: 10 dilution proportion, at 40000 μ J/cm
2irradiate under intensity ultraviolet, make its genetic inactivation.Add in the ovum of above collection by the perch sperm of inactivation, carry out dry method insemination, ovum essence is than being 100: 0.4.At water temperature 17 DEG C, after fertilization 58min, utilizes hydrostatic press by pressure adjusting to 590kg/cm
2, the hydrostatic pressing processing time is 6min, and induction fertilized egg carries out mitosis gynogenesis, hatches under afterwards fertilized egg being placed on 17 DEG C of water temperatures, obtains lefteye flounder pure lines F1346.
4, lefteye flounder F3 sets up for the induction of subtrahend gynogenosis system
F09119 family is cultivated to sexual maturity, utilizes manual compression belly method to gather unfertilized egg.Utilized by freezen protective perch sperm 37 DEG C of water-baths to thaw sperm simultaneously, utilize MPRS dilution by sperm with 1: 10 dilution proportion, at 40000 μ J/cm
2irradiate under intensity ultraviolet, make its genetic inactivation.The perch sperm of inactivation is added in the ovum of above collection, carry out dry method insemination, ovum essence is than being 100: 0.4,2-4 DEG C of seawater cold shock treatment 45min is put into after insemination 3min, then put into 17 DEG C of seawater hatchings and fry rearing, establish subtrahend gynogenosis system F1324 (table 3) of F09119.
Gather the ovum of F09119 family raun and the sperm of milter simultaneously, carry out inbreeding fertilization, fertilized egg put into 17 DEG C of seawater hatchings and cultivate fry, establishing the inbred line F1313 of F09119 family.
Cultivate setting up F1346, F1324 and F1313 tri-familys above, cultivation to when 80d, 105d, 125d, 388d respectively long to the body of family, body is wide and body weight measures and compare, result shows, and the body of F1346 cleavage gynogenesis system is long, body is wide and body weight 3 proterties are all significant higher than subtrahend gynogenosis system F1324 and inbred line F1313 (P < 0.05) (table 3).The growth rate of cleavage gynogenesis system F1346 is apparently higher than subtrahend gynogenosis system F1324 and inbred line F1313 (P < 0.05).
The mitosis of table 3 lefteye flounder, reduction division gynogenesis and inbred line growth fraction are comparatively
Note: 1313 is inbred line, 1324 is subtrahend gynogenosis system, and 1346 is cleavage gynogenesis system
5, lefteye flounder cleavage gynogenesis system, subtrahend gynogenosis system and inbred line Genetic Detection
Lefteye flounder is sheerly F1346, F1324 and inbred line 1313 to cultivate and grow into 10-15cm, random selecting 30 tail individual clip tail fin bar respectively, phenol chloroform method is utilized to extract DNA, the microsatellite marker scaffold288_1492 in lefteye flounder genetic linkage maps No. 21 linkage groups is selected to design primer, by PCR to its DNA cloning, 8% native polyacrylamide gel electrophoresis detects the genotype of lefteye flounder pure lines.
1) DNA extracts and Primer selection synthesis
Clip fin ray, by lysate (50mmol/LNaCl, 30mmol/LTris-HCl, 200mmol/LEDTA pH8.0,1%SDS, 200mg/L Proteinase K) add in the tail fin shredded, 55 DEG C of digestion are to clarification, isopyknic saturated phenol: chloroform: isoamyl alcohol (25: 24: 1) extracting twice, equal-volume deionized water is diluted, the absolute ethyl alcohol precipitation of 2.5 times of volume precoolings, finally arrive out alcohol to dry, add ddH2O, concentration is adjusted to 50-100ng/ μ L ,-20 DEG C of preservations.
Choose scaffold288_14927 as microsatellite marker from lefteye flounder genetic linkage maps No. 21 linkage groups, from GenBank, search flag sequence, by Hua Da genome company synthetic primer.
2) PCR reaction and electrophoresis
PCR reaction system is 16 μ L, comprises that 10 × buffer, dNTP, upstream and downstream primer are each, template DNA 1 μ L, rTaqDNA polymerase 1U, adds appropriate ddH
2o.PCR response procedures comprises: 94 DEG C of denaturation 10min; 94 DEG C of sex change 30s, annealing 30s, 72 DEG C extend 30s, 35 circulations; Last 72 DEG C extend 10min.Pcr amplification product detects through 8% native polyacrylamide gel electrophoresis, 3% cma staining 5min after electrophoresis, and develop the color nitrite ion (1% formaldehyde, 2% sodium hydroxide) 5min.
Result shows, and lefteye flounder subtrahend thelykaryon family F1324 and spilting of an egg thelykaryon family F1346 is homozygote (Fig. 1, Fig. 2), has 16 AA genotype and 14 BB genotype in offspring respectively.Inbreeding family F1313 Parent genotype is AC and BC, 2 AB types in 30 filial generations, 10 AC types, 12 BC types, and 5 is CC type (Fig. 3).Thus prove that spilting of an egg thelykaryon family F1346 and subtrahend thelykaryon family F1324 is lefteye flounder pure lines.In Fig. 1, Fig. 2 and Fig. 3, P is parent, and M is Marker..
Claims (1)
1. select, induce and cultivate a breeding technique method for the excellent pure lines of lefteye flounder, technical characteristics comprises:
1) lefteye flounder F1 generation grows foundation and the screening of fast family: utilize the sick colony (RS) of lefteye flounder anti-vibrio anguillarum, Japanese lefteye flounder colony (JS) and Huanghai Sea lefteye flounder colony (YS) to carry out artificial orientation's mating, set up family, by comparing growth traits measurement, variance analysis, absolute gain rate, filter out the fast family F0750 of growth;
2) in F2 generation, grows foundation and the screening of fast superior families: utilize F0750, lefteye flounder colony of Korea S (KS), Japanese lefteye flounder colony (JS), the sick colony (RS) of lefteye flounder anti-vibrio anguillarum to carry out artificial orientation's mating, set up family, by comparing growth traits measurement, variance analysis, absolute gain rate, filter out the fast family F09119 of growth;
3) induction that F3 is sheerly for mitosis is set up: the perch sperm utilizing freezen protective, at 40000 μ J/cm
2irradiate under intensity ultraviolet, make its genetic inactivation, inseminate with the ovum of F09119, at water temperature 17 DEG C, after fertilization 58min, utilizes 590kg/cm
2hydrostatic pressing process 6min, induction fertilized egg carries out mitosis gynogenesis, obtains pure lines F1346;
4) induction that F3 is sheerly for subtrahend gynogenesis is set up: with the perch sperm of freezen protective, at 40000 μ J/cm
2irradiate under intensity ultraviolet, make its genetic inactivation, inseminate with the ovum of F09119, cold shock treatment 45min in 2-4 DEG C of seawater is immersed after insemination 3min, make its chromosome doubling, then ovum is put into 17 DEG C of seawater hatchings, obtain lefteye flounder subtrahend gynogenosis system F1324;
5) Genetic Detection of lefteye flounder pure lines: utilize lefteye flounder microsatellite marker scaffold288_1492, Genetic Detection is carried out to F1346, F1324 and inbred line F1313, F1346 and F1324 genotype is homozygous AA and BB, F1313 is heterozygous, thus establishes the fast excellent pure lines of growth.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105660490A (en) * | 2016-03-24 | 2016-06-15 | 中国水产科学研究院北戴河中心实验站 | Method for improving induction efficiency of mitotic gynogenetic dihaploid of bastard halibut |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101120660A (en) * | 2007-08-28 | 2008-02-13 | 中国水产科学研究院黄海水产研究所 | Method for inducing female nucleus growth with heterologous frozen sperm |
| CN101699998A (en) * | 2009-11-03 | 2010-05-05 | 中国水产科学研究院黄海水产研究所 | Selection and culture method of excellent breed large-tooth flounder featuring fast growth and high survival rate |
-
2015
- 2015-06-01 CN CN201510306390.XA patent/CN104920255A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101120660A (en) * | 2007-08-28 | 2008-02-13 | 中国水产科学研究院黄海水产研究所 | Method for inducing female nucleus growth with heterologous frozen sperm |
| CN101699998A (en) * | 2009-11-03 | 2010-05-05 | 中国水产科学研究院黄海水产研究所 | Selection and culture method of excellent breed large-tooth flounder featuring fast growth and high survival rate |
Non-Patent Citations (3)
| Title |
|---|
| 杨景峰等: "异源精子诱导犬齿牙鲆的雌核发育", 《水产学报》 * |
| 田永胜等: "牙鲆选育Ｆ３代家系的建立及遗传效应分析", 《海洋学报》 * |
| 齐文山: "牙鲆卵裂雌核发育家系和F3 代家系建立及遗传分析", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105660490A (en) * | 2016-03-24 | 2016-06-15 | 中国水产科学研究院北戴河中心实验站 | Method for improving induction efficiency of mitotic gynogenetic dihaploid of bastard halibut |
| CN105660490B (en) * | 2016-03-24 | 2018-04-17 | 中国水产科学研究院北戴河中心实验站 | A kind of method for improving lefteye flounder mitosis gynogenesis dihaploid induction efficiency |
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