CN103478025B - Cultivating method of hyriopsis cumingii with golden yellow shell - Google Patents
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Abstract
The invention relates to a cultivating method of hyriopsis cumingii with a golden yellow shell. The method is characterized by comprising the following steps of (1), selecting 4-6 parent hyriopsis cumingii; (2) taking the margin tissues of the pallium of the parent hyriopsis cumingii, extracting the total RNA (ribonucleic acid) and synthetic single stranded c RNA, performing SRAP (sequence-related amplified polymorphism) amplification by adopting primer combination, and selecting male and female individuals with specific strip sequences with the golden yellow shells after the amplification products are subjected to electrophoresis; (3) using the selected male and female parent hyriopsis cumingii to build a family and cultivating in a water flowing pool; (4) periodically examining the pregnant condition of the female hyriopsis cumingii, and if mature glochidiums are found, selecting the mature female hyriopsis cumingii to reproduce to obtain infant hyriopsis cumingii; cultivating the infant hyriopsis cumingii in the water flowing pool until the shell reaches 1-2cm long, removing into to a net cage, and suspending the net cage into a pond to perform second stage cultivating on the infant hyriopsis cumingii until the shell length reaches 5-8cm; (5) removing the family parent hyriopsis cumingii with the non-golden yellow shells, and using the rest parent hyriopsis cumingii to build a core cultivating population with the golden yellow shells. According to the cultivation method of the hyriopsis cumingii with the golden yellow shell, the ratio of the hyriopsis cumingii with the golden yellow shell can reach more than 99.0%, the shell color is stable in heredity and the postoperative survival rate is high.
Description
Technical field
The breeding method that the present invention relates to a kind of golden yellow shell color triangle sail freshwater mussel, belongs to fishery breeding technical field.
Background technology
Hydriopsis cumingii (
hyriopsis cumingii(Lea)) be under the jurisdiction of Unionidae (Unionidae) pearl oyster subfamily (Unioninae) sail freshwater mussel belong to (
hyriopsis), be China endemic species.Lake, tributary that is mainly distributed in the Yangtze river basin, Basin of Huaihe River etc. has in the large and medium-sized water body of miniflow water, particularly abundant in Poyang Lake, Dongting Lake, Taihu Lake, Chaohu and Wu great fresh water lake, Hongchehu Lake resource.20 century 70s, China's hydriopsis cumingii artificial breeding is obtained successfully.Subsequently several years, hydriopsis cumingii with the good performance of growing cultured pearls substitute cristaria plicata (
cristaria plicata), anodonta woodiana pacifica (
anodonta woodiana woodiana) etc., for fresh water pearl, cultivate, become the main fresh water high-quality pearl culturing clam kind of China, greatly promoted the development of China's water pearl culture industry.At present, more than 70% fresh water pearl in the world is cultivated and is formed by the hydriopsis cumingii of China.Hydriopsis cumingii is that China becomes world's water pearl culture and solid foundation has been established by outlet the first big country in the large-scale application of growing cultured pearls on producing.The fast development that China's water pearl culture is spent more than 40 year already, also expose gradually a series of problem: due in hydriopsis cumingii seed breeding process, the general seed selection of not focusing on close freshwater mussel, long-term many for inbreeding, cause seed germplasm degenerate and mix, the problems such as in addition highdensity cultivation, environmental pollution take place frequently extensive popular disease, and pearl quality declines, direct and the indirect economic loss causing is huge, has seriously hindered the sustainable health development of China's water pearl culture industry.At present, China fresh water pearl produces above and can use without hydriopsis cumingii breeding.Cultivate hydriopsis cumingii new varieties (being), improving breeding coverage rate and culture benefit is industry key technology in the urgent need to address in China's water pearl culture industry.
Domestic scholars is utilized different kinds of molecules biological method, comprises that RAPD, IST-1, cytochrome enzyme, micro-satellite, 16S rRNA sequence etc. analyze the genetic background of hydriopsis cumingii, for carrying out the genetic breeding of hydriopsis cumingii, has established solid foundation.The method of application hybridization in recent years, colony's seed selection has obtained some useful results, but because the sexual maturation cycle of hydriopsis cumingii is long, selection cross is made slow progress.
The shell look of shellfish is to be formed by mantle tissue secretion.Because most of seashells have colourful shell look, from twentieth century sixties, the polymorphic research of shell look is subject to Marine Biologist's common concern.The correlative study of nearly 50 years shows: the shell look of shellfish is as a heritable qualitative character, not only relevant with their ecology and behavior, also relevant with its growth, survival isophenous proterties.The selfing of different shell look family, hybridization result of study show: the shell color table type of shellfish is mainly subject to individual gene or minority gene is controlled, and can give of future generation by genetic stability.Therefore, the shell look of take has been subject to numerous breeding experts' favor as the shellfish breeding of new variety of assist-breeding means or final goal, and has obtained great successes.Wada, to the seed selection of Japanese pearl freshwater mussel shell prismatic layer color, has increased substantially the ratio that white breeding line produces white pearl.Domestic, by the seed selection of shell look has successfully been selected to a plurality of shellfish new varieties (strain), as grow fast, premunition is strong " agate Bao ", " middle section is red " bay scallop, " Chinese red " haliotis discus hannai Ino strain and " southern section pearl is red " Ma Shi pearl oyster etc. of high yield.The seed selection of shell look has become the most effectively one of technological means of China's seashells breeding of new variety.
By contrast, the shell color table type of Non-marine Bivalves is simple, often out in the cold, and correlative study is at present still almost blank.By the research of shell look directive breeding, can accelerate hydriopsis cumingii fine-variety breeding process, promote the sustainable health development of China's water pearl culture industry.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, the breeding method of a kind of golden yellow shell color triangle sail freshwater mussel is provided, it is good to make the shell look inheritance stability of this strain, postoperative survival rate is high and cultivates pearly luster.
According to technical scheme provided by the invention, the breeding method of a kind of golden yellow shell color triangle sail freshwater mussel, feature is to comprise the following steps:
(1) select the hydriopsis cumingii parent freshwater mussel in 4～6 age that water spray is strong, abdomen border is soft, shell is complete, and differentiate male and female;
(2) get the outer embrane edge tissues 50mg of male and female parent freshwater mussel, adopt pillar total RNA from animal tissues extracting and purifying kit to extract total RNA; Adopt the synthesizing single-stranded cDNA of AMV the first chain cDNA synthetic agent box; Adopt specific primer combination to carry out SRAP amplification, amplified production is observed after agarose electrophoresis under uviol lamp, selects the male and female individuality with golden yellow shell look specific band sequence, as golden yellow shell color triangle sail freshwater mussel line breeding parent freshwater mussel; The gene order of described specific primer is: forward primer is gactgcgtacgaattcga, and reverse primer is tgagtccaaaccggtcc;
(3) the male and female parents freshwater mussel of selecting through step (2) is set up to 1 female 1 male or 1 male how female family, cultivate in the flowing water pond that every group of family is placed in respectively mutual isolation, and flowing water pool area is 1～2 ㎡, and water level is 15～25 cm, flow rate of water flow 2～3 m/min;
(4) make regular check on bosom children's situation of the maternal outer gill filament in each family, find that there is ripe hook Jie larva, immediately ripe female parent is selected, adopt conventional method to collect seedling, obtain young freshwater mussel; Cultivate in the flowing water pond that the young freshwater mussel of each family is placed in respectively to mutual isolation, flow rate of water flow 1～4 m/min, children freshwater mussel is cultivated to the long 1～2cm of shell and moves in the net cage that mesh diameter is 2.5～3cm, breeding density is 200～300/net cage, layer of plastic film is laid in net cage bottom, in net cage bottom, places the thick yellow mud of 2～3cm, and net cage keeps hanging on and in pond, carries out second stage children freshwater mussel and cultivate, cultivate to the long 5～8cm of shell, observe each family F
1for young clam shell color table type, 1000 F of casual inspection
1for young clam shell look, if golden yellow shell color ratio example is greater than 99%, show that close clam shell look genotype selection is correct;
(5) reject the family parent freshwater mussel of the non-golden yellow shell of offspring, by setting up golden yellow shell look core by the close freshwater mussel of step (4) checking, breed colony, the close freshwater mussel breeding as golden yellow shell color triangle sail freshwater mussel, adopts the mode of step (4) to breed, and obtains golden yellow shell color triangle sail freshwater mussel.
In described step (2), the system of SRAP amplification comprises: the synthetic strand cDNA 3 μ L that obtain, deoxyribonucleoside triphosphate (dNTP) 1.5 μ L, 10 * Taq DNA polymerase buffer liquid, 2.5 μ L, the concentration that concentration is 2.5 mmol/L are the MgCl of 25 mmol/L
22.5 μ L, forward primer and reverse primer each 0.5 μ L, Taq archaeal dna polymerase 0.2 μ L, aseptic redistilled water 14.3 μ L; Amplification procedure is: by system at 94 ℃ of sex change 5 min; Then 94 ℃ of sex change 45 s, 38 ℃ of annealing 45 s, 72 ℃ of extension l min, carry out 5 circulations; 94 ℃ of sex change 1 min, 50 ℃ of annealing 1 min, 72 ℃ of extension l min, carry out 30 circulations again; Last 72 ℃ are extended 7 min.
The running gel process for preparation of agarose electrophoresis in described step (2): every 100ml Ago-Gel adds the ethidium bromide solution that 3 μ l concentration are 10mg/ml; Deposition condition: 120～150V, the time is 15～25min.
In described step (4), when young clam shell length is less than 2mm, flow rate of water flow is 1～2 m/min; During children's clam shell length >=2mm, flow rate of water flow is 2～4 m/min.
Described golden yellow shell look specific band sequence is: TGAGTCCAAACCGGAGCACGTCGTAGTTTGGCATACAACATCGTAGTGTAGGCATA CTACTTCTTAGTACGTATACCACATCTTAGTGTAGACATAGCACATTGTAGTGAAA TTCGAGCATACGGCAGTATAATCATAGTACTTCGTAGTGTAGGCATAGCACACCGT AGTGTAGGCATATTAATTCGTACGCAG.
Compared with the prior art the present invention has the following advantages:
(1) the present invention by close freshwater mussel basic population select, shell form and aspect correlation gene type is selected, family pairing is bred and the golden yellow shell color triangle of the technology quickly breeding sail freshwater mussel new lines such as F1 generation children clam shell look check; By secondary parent freshwater mussel, select, in offspring, the ratio of golden yellow shell look can reach more than 99.0%; This new lines has shell look inheritance stability, postoperative survival rate is high and cultivate the advantages such as pearly luster is good;
(2) the present invention utilizes specific primer combination, by golden yellow shell color characteristic gene band, close freshwater mussel is screened, and has shortened jib clam shell look seed selection process and has improved seed selection accuracy;
(3) the present invention cultivates family F1 generation to long 5～8cm stage of shell and carries out shell look Phenotypic Observation, further verifies that whether correct genotype select, and the golden yellow shell related gene of close freshwater mussel is confirmed and purifying;
(4) the golden yellow shell hydriopsis cumingii new lines that the present invention cultivates has shell look inheritance stability, postoperative survival rate is high and cultivates the features such as pearly luster is good.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
The present invention by close freshwater mussel basic population select, shell form and aspect correlation gene type is selected, family pairing is bred and the golden yellow shell color triangle of the technology quickly breeding sail freshwater mussel new lines such as F1 generation children clam shell look check; Golden yellow shell look specific band sequence involved in the present invention is: TGAGTCCAAACCGGAGCACGTCGTAGTTTGGCATACAACATCGTAGTGTAGGCATA CTACTTCTTAGTACGTATACCACATCTTAGTGTAGACATAGCACATTGTAGTGAAA TTCGAGCATACGGCAGTATAATCATAGTACTTCGTAGTGTAGGCATAGCACACCGT AGTGTAGGCATATTAATTCGTACGCAG.
Embodiment mono-: the breeding method of a kind of golden yellow shell color triangle sail freshwater mussel, comprises the following steps:
On March 10th, (1) 2010,581 of the hydriopsis cumingii parent freshwater mussels in 4～6 ages that selection water spray is strong from Poyang Lake wild population, abdomen border is soft, shell is complete, by observing gill filament spacer region, differentiate male and female, select 323 of female freshwater mussels, 258 of male freshwater mussels, at the shell surface mark of the male and female parent freshwater mussel of selecting;
(2) get the outer embrane edge tissues 50mg of male and female parent freshwater mussel, adopt pillar total RNA from animal tissues extracting and purifying kit (Shanghai Sheng Gong biotechnology Co., Ltd) to extract total RNA; Adopt the synthesizing single-stranded cDNA of AMV the first chain cDNA synthetic agent box; Adopt specific primer combination to carry out SRAP amplification, amplified production is observed after agarose electrophoresis under uviol lamp, selects and has 30 female freshwater mussels of golden yellow shell look specific band sequence, 23 male freshwater mussels, as golden yellow shell color triangle sail freshwater mussel line breeding parent freshwater mussel; The gene order of described specific primer is: forward primer is gactgcgtacgaattcga, and reverse primer is tgagtccaaaccggtcc;
The system of described SRAP amplification comprises: the synthetic strand cDNA 3 μ L that obtain, deoxyribonucleoside triphosphate (dNTP) 1.5 μ L, 10 * Taq DNA polymerase buffer liquid, 2.5 μ L, the concentration that concentration is 2.5 mmol/L are the MgCl of 25 mmol/L
22.5 μ L, forward primer and reverse primer each 0.5 μ L, Taq archaeal dna polymerase 0.2 μ L, aseptic redistilled water 14.3 μ L; Amplification procedure is: by system at 94 ℃ of sex change 5 min; Then 94 ℃ of sex change 45 s, 38 ℃ of annealing 45 s, 72 ℃ of extension l min, carry out 5 circulations; 94 ℃ of sex change 1 min, 50 ℃ of annealing 1 min, 72 ℃ of extension l min, carry out 30 circulations again; Last 72 ℃ are extended 7 min;
The running gel process for preparation of described agarose electrophoresis: every 100ml Ago-Gel adds the ethidium bromide solution that 3 μ l concentration are 10mg/ml; Deposition condition: 120V, the time is 25min;
On April 1st, (3) 2010, the male and female parents freshwater mussel of selecting through step (2) is set up to 1 female 1 male or 1 male how female family, cultivate in the flowing water pond that every group of family is placed in respectively mutual isolation, and flowing water pool area is 1 ㎡, and water level is 15 cm, flow rate of water flow 2 m/min;
(4) make regular check on bosom children's situation of the maternal outer gill filament in each family, finding that there is 20 female freshwater mussels of family on May 1 has ripe hook Jie larva, immediately ripe female parent is selected, and adopts yellow cartfish to carry out artificial seeding as host fish, obtains young freshwater mussel; The young freshwater mussel of each family is cultivated respectively at the flowing water pond of mutual isolation, and when young clam shell length is less than 2mm, flow rate of water flow is 1 m/min, and during clam shell length>=2mm, flow rate of water flow is 2m/min; Children freshwater mussel is cultivated to the long 1～2cm of shell, on July 10th, 2010, move in 50cm * 40cm * 10cm net cage and cultivate, the mesh diameter of net cage is 2.5cm, breeding density is 200/net cage, layer of plastic film is laid in net cage bottom, in net cage bottom, places the thick yellow mud of 2cm, and net cage keeps hanging on and in pond, carries out second stage children freshwater mussel and cultivate, cultivate to the long 5～8cm of shell, observe each family F
1for young clam shell color table type, 1000 F of casual inspection
1for young clam shell look, golden yellow shell color ratio example is greater than 99%, shows that close clam shell look genotype selection is correct;
(5) reject the family parent freshwater mussel of the non-golden yellow shell of offspring, cultivation to 2011 year May, obtain altogether 20 groups of family full-sibs F1 generation children freshwater mussels, remaining close freshwater mussel is set up to golden yellow shell look core breeding population, the close freshwater mussel breeding as golden yellow shell color triangle sail freshwater mussel, adopt the mode of step (3), step (4) to cultivate, obtain golden yellow shell color triangle sail freshwater mussel.
Embodiment bis-: the breeding method of a kind of golden yellow shell color triangle sail freshwater mussel, comprises the following steps:
On March 20th, (1) 2011,483 of the hydriopsis cumingii parent freshwater mussels in 4～6 ages that selection water spray is strong from Poyang Lake wild population, abdomen border is soft, shell is complete, by observing gill filament spacer region, differentiate male and female, select 272 of female freshwater mussels, 211 of male freshwater mussels, at the shell surface mark of the male and female parent freshwater mussel of selecting;
(2) get the outer embrane edge tissues 50mg of male and female parent freshwater mussel, adopt pillar total RNA from animal tissues extracting and purifying kit (Shanghai Sheng Gong biotechnology Co., Ltd) to extract total RNA; Adopt the synthesizing single-stranded cDNA of AMV the first chain cDNA synthetic agent box; Adopt specific primer combination to carry out SRAP amplification, amplified production is observed after agarose electrophoresis under uviol lamp, selects and has 25 female freshwater mussels of golden yellow shell look specific band sequence, 20 male freshwater mussels, as golden yellow shell color triangle sail freshwater mussel line breeding parent freshwater mussel; The gene order of described specific primer is: forward primer is gactgcgtacgaattcga, and reverse primer is tgagtccaaaccggtcc;
The system of described SRAP amplification comprises: the synthetic strand cDNA 3 μ L that obtain, deoxyribonucleoside triphosphate (dNTP) 1.5 μ L, 10 * Taq DNA polymerase buffer liquid, 2.5 μ L, the concentration that concentration is 2.5 mmol/L are the MgCl of 25 mmol/L
22.5 μ L, forward primer and reverse primer each 0.5 μ L, Taq archaeal dna polymerase 0.2 μ L, aseptic redistilled water 14.3 μ L; Amplification procedure is: by system at 94 ℃ of sex change 5 min; Then 94 ℃ of sex change 45 s, 38 ℃ of annealing 45 s, 72 ℃ of extension l min, carry out 5 circulations; 94 ℃ of sex change 1 min, 50 ℃ of annealing 1 min, 72 ℃ of extension l min, carry out 30 circulations again; Last 72 ℃ are extended 7 min;
The running gel process for preparation of described agarose electrophoresis: every 100ml Ago-Gel adds the ethidium bromide solution that 3 μ l concentration are 10mg/ml; Deposition condition: 150V, the time is 15min;
On March 28th, (3) 2011, the male and female parents freshwater mussel of selecting through step (2) is set up to 1 female 1 male or 1 male how female family, and cultivate in the flowing water pond that every group of family is placed in respectively mutual isolation, and flowing water pool area is 2 ㎡, water level is 25 cm, flow rate of water flow 3 m/min;
(4) make regular check on bosom children's situation of the maternal outer gill filament in each family, finding that there is 18 female freshwater mussels of family on April 28 has ripe hook Jie larva, immediately ripe female parent is selected, and adopts yellow cartfish to carry out artificial seeding as host fish, obtains young freshwater mussel; The young freshwater mussel of each family is cultivated respectively at the flowing water pond of mutual isolation, and when young clam shell length is less than 2mm, flow rate of water flow is 2 m/min, and during clam shell length>=2mm, flow rate of water flow is 4 m/min; Children freshwater mussel is cultivated to the long 1～2cm of shell, on June 30th, 2011, move in 50cm * 40cm * 10cm net cage and cultivate, the mesh diameter of net cage is 3cm, breeding density is 300/net cage, layer of plastic film is laid in net cage bottom, in net cage bottom, places the thick yellow mud of 3cm, and net cage keeps hanging on and in pond, carries out second stage children freshwater mussel and cultivate, cultivate to the long 5～8cm of shell, observe each family F
1for young clam shell color table type, 1000 F of casual inspection
1for young clam shell look, golden yellow shell color ratio example is greater than 99%, shows that close clam shell look genotype selection is correct;
(5) reject the family parent freshwater mussel of the non-golden yellow shell of offspring, cultivation to 2012 year May, obtain altogether 18 groups of family full-sibs F1 generation children freshwater mussels, remaining close freshwater mussel is set up to golden yellow shell look core breeding population, the close freshwater mussel breeding as golden yellow shell color triangle sail freshwater mussel, adopt the mode of step (3), step (4) to cultivate, obtain golden yellow shell color triangle sail freshwater mussel.
Embodiment tri-: the breeding method of a kind of golden yellow shell color triangle sail freshwater mussel, comprises the following steps:
On March 20th, (1) 2012,1710 of the hydriopsis cumingii parent freshwater mussels in 4～6 ages that selection water spray is strong from Poyang Lake wild population, abdomen border is soft, shell is complete, by observing gill filament spacer region, differentiate male and female, select 1124 of female freshwater mussels, 586 of male freshwater mussels, at the shell surface mark of the male and female parent freshwater mussel of selecting;
(2) get the outer embrane edge tissues 50mg of male and female parent freshwater mussel, adopt pillar total RNA from animal tissues extracting and purifying kit (Shanghai Sheng Gong biotechnology Co., Ltd) to extract total RNA; Adopt the synthesizing single-stranded cDNA of AMV the first chain cDNA synthetic agent box; Adopt specific primer combination to carry out SRAP amplification, amplified production is observed after agarose electrophoresis under uviol lamp, selects and has 98 female freshwater mussels of golden yellow shell look specific band sequence, 53 male freshwater mussels, as golden yellow shell color triangle sail freshwater mussel line breeding parent freshwater mussel; The gene order of described specific primer is: forward primer is gactgcgtacgaattcga, and reverse primer is tgagtccaaaccggtcc;
The system of described SRAP amplification comprises: the synthetic strand cDNA 3 μ L that obtain, deoxyribonucleoside triphosphate (dNTP) 1.5 μ L, 10 * Taq DNA polymerase buffer liquid, 2.5 μ L, the concentration that concentration is 2.5 mmol/L are the MgCl of 25 mmol/L
22.5 μ L, forward primer and reverse primer each 0.5 μ L, Taq archaeal dna polymerase 0.2 μ L, aseptic redistilled water 14.3 μ L; Amplification procedure is: by system at 94 ℃ of sex change 5 min; Then 94 ℃ of sex change 45 s, 38 ℃ of annealing 45 s, 72 ℃ of extension l min, carry out 5 circulations; 94 ℃ of sex change 1 min, 50 ℃ of annealing 1 min, 72 ℃ of extension l min, carry out 30 circulations again; Last 72 ℃ are extended 7 min;
The running gel process for preparation of described agarose electrophoresis: every 100ml Ago-Gel adds the ethidium bromide solution that 3 μ l concentration are 10mg/ml; Deposition condition: 125V, the time is 20min;
On March 30th, (3) 2012, the male and female parents freshwater mussel of selecting through step (2) is set up to 1 female 1 male or 1 male how female family, and cultivate in the flowing water pond that every group of family is placed in respectively mutual isolation, and flowing water pool area is 1.5 ㎡, water level is 20cm, flow rate of water flow 2.5 m/min;
(4) make regular check on bosom children's situation of the maternal outer gill filament in each family, finding that there is 68 female freshwater mussels of family on April 30 has ripe hook Jie larva, immediately ripe female parent is selected, and adopts yellow cartfish to carry out artificial seeding as host fish, obtains young freshwater mussel; The young freshwater mussel of each family is cultivated respectively at the flowing water pond of mutual isolation, and when young clam shell length is less than 2mm, flow rate of water flow is 1.5 m/min, and during clam shell length>=2mm, flow rate of water flow is 3m/min; Children freshwater mussel is cultivated to the long 1～2cm of shell, on July 3rd, 2012, move in 50cm * 40cm * 10cm net cage and cultivate, the mesh diameter of net cage is 2.6cm, breeding density is 250/net cage, layer of plastic film is laid in net cage bottom, in net cage bottom, places the thick yellow mud of 2.5cm, and net cage keeps hanging on and in pond, carries out second stage children freshwater mussel and cultivate, cultivate to the long 5～8cm of shell, observe each family F
1for young clam shell color table type, 1000 F of casual inspection
1for young clam shell look, golden yellow shell color ratio example is greater than 99%, shows that close clam shell look genotype selection is correct;
(5) reject the family parent freshwater mussel of the non-golden yellow shell of offspring, cultivation to 2013 year June, obtain altogether 65 groups of family full-sibs F1 generation children freshwater mussels, remaining close freshwater mussel is set up to golden yellow shell look core breeding population, the close freshwater mussel breeding as golden yellow shell color triangle sail freshwater mussel, adopt the mode of step (3), step (4) to cultivate, obtain golden yellow shell color triangle sail freshwater mussel.
<160> 3
<210> SEQ ID NO: 1
<211> 195
<212> DNA
<213>
<400> 1
TGAGTCCAAA CCGGAGCACG TCGTAGTTTG GCATACAACA TCGTAGTGTA GGCATACTAC TTCTTAGTAC GTATACCACA TCTTAGTGTA GACATAGCAC ATTGTAGTGA AATTCGAGCA TACGGCAGTA TAATCATAGT ACTTCGTAGT GTAGGCATAG CACACCGTAG TGTAGGCATA TTAATTCGTA CGCAG；
<210> SEQ ID NO: 2
<211> 18
<212> forward primer
<213>
<400> 2
gactgcgtac gaattcga；
<210> SEQ ID NO: 3
<211> 17
<212> reverse primer
<213>
<400> 3
tgagtccaaa ccggtcc；
Claims (5)
1. a breeding method for golden yellow shell color triangle sail freshwater mussel, is characterized in that, comprises the following steps:
(1) select the hydriopsis cumingii parent freshwater mussel in 4～6 age that water spray is strong, abdomen border is soft, shell is complete, and differentiate male and female;
(2) get the outer embrane edge tissues 50mg of male and female parent freshwater mussel, adopt pillar total RNA from animal tissues extracting and purifying kit to extract total RNA; Adopt the synthesizing single-stranded cDNA of AMV the first chain cDNA synthetic agent box; Adopt specific primer combination to carry out SRAP amplification, amplified production is observed after agarose electrophoresis under uviol lamp, selects the male and female individuality with golden yellow shell look specific band sequence, as golden yellow shell color triangle sail freshwater mussel line breeding parent freshwater mussel; The gene order of described specific primer is: forward primer is gactgcgtacgaattcga, and reverse primer is tgagtccaaaccggtcc;
(3) the male and female parents freshwater mussel of selecting through step (2) is set up to 1 female 1 male or 1 male how female family, cultivate in the flowing water pond that every group of family is placed in respectively mutual isolation, and flowing water pool area is 1～2 ㎡, and water level is 15～25 cm, flow rate of water flow 2～3 m/min;
(4) make regular check on bosom children's situation of the maternal outer gill filament in each family, find that there is ripe hook Jie larva, immediately ripe female parent is selected, adopt conventional method to collect seedling, obtain young freshwater mussel; Cultivate in the flowing water pond that the young freshwater mussel of each family is placed in respectively to mutual isolation, flow rate of water flow 1～4 m/min, children freshwater mussel is cultivated to the long 1～2cm of shell and moves in the net cage that mesh diameter is 2.5～3cm, breeding density is 200～300/net cage, layer of plastic film is laid in net cage bottom, in net cage bottom, places the thick yellow mud of 2～3cm, and net cage keeps hanging on and in pond, carries out second stage children freshwater mussel and cultivate, cultivate to the long 5～8cm of shell, observe each family F
1for young clam shell color table type, 1000 F1 generations children clam shell looks of casual inspection, if golden yellow shell color ratio example is greater than 99%, show that close clam shell look genotype selection is correct;
(5) reject the family parent freshwater mussel of the non-golden yellow shell of offspring, by setting up golden yellow shell look core by the close freshwater mussel of step (4) checking, breed colony, the close freshwater mussel breeding as golden yellow shell color triangle sail freshwater mussel, adopts the mode of step (4) to breed, and obtains golden yellow shell color triangle sail freshwater mussel.
2. the breeding method of golden yellow shell color triangle sail freshwater mussel as claimed in claim 1, it is characterized in that: in described step (2), the system of SRAP amplification comprises: the synthetic strand cDNA 3 μ L that obtain, deoxyribonucleoside triphosphate (dNTP) 1.5 μ L, 10 * Taq DNA polymerase buffer liquid, 2.5 μ L, the concentration that concentration is 2.5 mmol/L are the MgCl of 25 mmol/L
22.5 μ L, forward primer and reverse primer each 0.5 μ L, Taq archaeal dna polymerase 0.2 μ L, aseptic redistilled water 14.3 μ L; Amplification procedure is: by system at 94 ℃ of sex change 5 min; Then 94 ℃ of sex change 45 s, 38 ℃ of annealing 45 s, 72 ℃ of extension l min, carry out 5 circulations; 94 ℃ of sex change 1 min, 50 ℃ of annealing 1 min, 72 ℃ of extension l min, carry out 30 circulations again; Last 72 ℃ are extended 7 min.
3. the breeding method of golden yellow shell color triangle sail freshwater mussel as claimed in claim 1, is characterized in that: the running gel process for preparation of agarose electrophoresis in described step (2): every 100mL Ago-Gel adds the ethidium bromide solution that 3 μ L concentration are 10mg/mL; Deposition condition: 120～150V, the time is 15～25min.
4. the breeding method of golden yellow shell color triangle sail freshwater mussel as claimed in claim 1, is characterized in that: in described step (4), when young clam shell length is less than 2mm, flow rate of water flow is 1～2 m/min; During children's clam shell length >=2mm, flow rate of water flow is 2～4 m/min.
5. the breeding method of golden yellow shell color triangle sail freshwater mussel as claimed in claim 1, is characterized in that: described golden yellow shell look specific band sequence is: TGAGTCCAAACCGGAGCACGTCGTAGTTTGGCATACAACATCGTAGTGTAGGCATA CTACTTCTTAGTACGTATACCACATCTTAGTGTAGACATAGCACATTGTAGTGAAA TTCGAGCATACGGCAGTATAATCATAGTACTTCGTAGTGTAGGCATAGCACACCGT AGTGTAGGCATATTAATTCGTACGCAG.
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