CN101418347B - Yellow catfish sex chromosome specific molecular markers and genetic sex identification method - Google Patents
Yellow catfish sex chromosome specific molecular markers and genetic sex identification method Download PDFInfo
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Abstract
The invention discloses specific molecular markers of sex chromosomes of yellow catfish and a method for identifying the genetic sex of the yellow catfish. The method comprises the following steps of screening and cloning of specific AFLP markers of XY sex chromosomes of the yellow catfish, design of specific SCAR markers of the XY sex chromosomes of the yellow catfish, and establishment of a method for identifying genotype (XX/XY/YY) PCR of the sex chromosomes of the yellow catfish. The invention establishes the method for screening the specific AFLP markers of the sex chromosomes of the yellow catfish, screens 3 primer combinations from 256 AFLP primer combinations to generate 4 specific AFLP segments of an X sex chromosome or a Y sex chromosome, and establishes a method for identifying the genotype PCR of the sex chromosomes of the yellow catfish. The invention initially screens specific molecular markers of X and Y chromosomes of the yellow catfish to establish a method for identifying genetic sex of the yellow catfish, and the method is applied to identification of the genetic sex during the process of cultivation of holandric yellow catfish. The method has the characteristics of high efficiency, accuracy and stability, and has important application value in control of the sex of the yellow catfish and continuous mass production of holandric yellow catfish seedlings.
Description
Technical field
The invention belongs to fish genetic sex identification and sex control technology in the aquatic living things technical field, more specifically relate to a kind of Yellow catfish sex chromosome specific molecular markers and genetic sex identification method.The present invention has important use and is worth in the cultivation of the sex control of Yellow catfish and complete male seed, also have application prospect simultaneously in other fish sex control and the production of unisexuality seed.
Background technology
Yellow catfish [Pelteobagrus fulvidraco (Richardson)] is the Silurformes Chang section fish that dwell the end with higher economic worth, is distributed widely in waters such as rivers, lake, reservoir.Its delicious meat, nutritious, thorn is few between flesh, thereby favored by the human consumer.Price is higher in recent years, and market demand increases day by day.The Yellow catfish milter is faster than the raun growth.According to investigations, under identical cultivating condition, 1 year male Yellow catfish is than the fast growth about 30% of raun born of the same parents.At 1 year that cultures, its milter grew to the 150-200 gram, and raun has only the 50-75 gram, and the male and female growth differences is near 3 times.Therefore, if can be in breeding the sex of Artificial Control Yellow catfish, cultivate complete male Yellow catfish, will improve output greatly, reduce the cost of breeding fish, increase economic efficiency.
Production unisexuality milter has several method available: as hand picking, the direct inducibility reverse of male hormone, androgenesis, species hybridization, artificial induction triploid and genetic method etc.At present, using genetic method to produce the full milter of XY used at the bolti monosex cultivation.This GMT technology that is called is applicable to the fish of male gamete abnormal shape, and method is reliable, stable, and environmentally friendly, no bad influence.
Nearest Liu Han duty etc. has successfully produced YY supermale fish from XY physiology raun gynogenesis, and by having obtained complete male filial generation with XX raun test cross, verified the existence of YY supermale fish, the feasibility of this technological line has been described, also laid a solid foundation for the production of complete male Yellow catfish.But in the time of in being applied to scale operation, there are some technical bottlenecks in this technology, hindered the production of full milter, such as: find in the production practice, when producing YY supermale fish by Yellow catfish XY raun gynogenesis, also produced the normal milter of XY, its quantity is equally matched with YY supermale fish, has hindered the application of YY supermale fish in full milter seedling is cultivated.Therefore how identify accurately and quickly and open YY and the XY milter becomes a key point that realizes in the large-scale production of full realgar forehead fry.Traditional method has only by after the test cross experiment, and according to the sex of children ratio, just can judge its male parent is hereditary YY or XY milter; And in the test cross experiment, tester is cultured could dissect more than 3 months according to histology for needs and is judged its sex, so experimental period is long, and the aquaculture cost height is unfavorable for scale operation; Moreover, multiply test does not for many years find that also YY supermale fishing gear has the natural propagation ability the same with normal X Y milter; And the Yellow catfish spermary is microphyll type, almost can't extrude seminal fluid when being expert at artificial propagation, so during the test cross experiment, will kill milter usually and obtain seminal fluid from spermary and carry out artificial insemination.Therefore can't be used for continuing to produce full milter seedling by the YY supermale fish that the test cross experiment directly obtains to live.
In sum, realize the large-scale production of full realgar forehead fry, the YY supermale fish authentication method of being badly in need of a kind of economy, fast, accurately and must not murdering Yellow catfish.
Along with the development and the maturation of dna molecular marker technology, utilize dna marker to identify that sex becomes possibility, for we provide a kind of economy simple and direct and genetic sex identification of means accurately, but prerequisite is to screen sex specific DNA mark earlier.At present, in some economic fishs, by various dna molecular marker technology (RAPDs, RFLPs, SSRs, AFLPs etc.) the existing many reports of screening sex specific mark, as: Kovacs etc. utilize RAPD to scan female, the male gene pool of African catfish (Clarias gariepinus), find the relevant RAPD mark of two male sexs; Sakamoto etc. find that two SSR marks (OmyFGT19 TUF and OmyRGT28 TUF) are linked with the sex determination site of male rainbow trout (Oncorhynchusmykiss).Simultaneously, the sex specific DNA mark of fish has species or strain specificity usually, the sex specific mark of a kind of fish can not be striden species usually and be used, and therefore will utilize molecule marker to realize the genetic sex evaluation of Yellow catfish, at first will screen the sex specific mark of Yellow catfish.
The AFLP molecular marking technique is a kind of multidigit point molecular marking technique of PCR-based technology, has the ability that demonstrates very strong polymorphism.Every pair of AFLP selective amplification primer can obtain 50-100 band, can obtain a large amount of amplified fragments by limited combination of primers.Therefore the AFLP molecule marker is particularly suitable for the species that still do not have the molecular genetic background of information are studied.The AFLP molecular marking technique has obtained than widespread use in screening sex marker research over nearly 5 years, and some successful reports are arranged, as usefulness AFLP technology to analyze bolti (Oreochromis niloticus L.) genomes such as Ezaz, find the AFLP mark of 3 Y linkages (OniY425.OniY382.OniY227) and an X-linkage (OniX420); Application AFLP such as Brunelli have also found a new special mark of king salmon Y chromosome.
Summary of the invention
The objective of the invention is to be to provide a kind of Yellow catfish sex chromosome specific molecular markers and genetic sex identification method; method is simple; easy to operate; cultivate route in conjunction with traditional YY supermale fish; can realize the large-scale production of complete male advanced fry of yellow catfish, and then, finally reach the output that improves Yellow catfish for the Yellow catfish aquaculture provides full milter seedling; reduce the cost of breeding fish, improved the purpose of economic benefit.
To achieve these goals, the present invention adopts following technical measures:
As mentioned above, realize the scale operation of complete male Yellow catfish, its key point is the YY supermale fish authentication method being badly in need of a kind of economy, fast, accurately and must not murdering Yellow catfish.Therefore, the present invention obtains Yellow catfish sex chromosome specific DNA mark by screening, and utilizes this mark to realize fast identifying the genetic sex of Yellow catfish exactly, is tested and appraised and obtains the production that big YY supermale fish is used for full realgar forehead fry.
Basic design of the present invention is: at first, adopt the AFLP molecular marking technique, screening obtains Yellow catfish X and Y chromosome specific AFLP mark, change into the SCAR mark of simple and direct use after, set up Yellow catfish sex chromosome genotype (XX/XY/YY) PCR authentication method; Secondly, present method is applied to the genetic sex of complete male Yellow catfish in cultivating identifies, for the production of full realgar forehead fry provides reliable YY supermale fish.
Method of the present invention is: adopt AFLP molecular marking technique screening Yellow catfish X and Y sex chromosome specific AFLP mark, clone these X or Y sex chromosome specific AFLP mark and sequential analysis, clone the flanking DNA sequence of these specific mark dna fragmentations by chromosome walking, compare by sequential analysis, select to be fit to the sex chromosome specific site of design primer, the design special primer, these marks are converted into the convenient SCAR mark that uses, in conjunction with X and the special SCAR mark of Y chromosome, set up Yellow catfish sex chromosome genotype (XX/XY/YY) PCR authentication method, the genetic sex that present method is applied to various breedings combinations in the complete male Yellow catfish cultivation is identified, finally identifies the production that YY supermale fish is used for complete male Yellow catfish.
Therefore, the present invention includes four big steps: one) screening and clone Yellow catfish XY sex chromosome specific AFLP mark; Two) be converted into the special SACR mark of XY sex chromosome; Three) set up Yellow catfish sex chromosome genotype PCR authentication method; Four) carrying out genetic sex in complete male Yellow catfish is cultivated identifies.
Above-mentioned one) screening and clone Yellow catfish XY sex chromosome specific AFLP mark comprise:
1.XY the AFLP label screening that sex chromosome is special; 2. the clone of sex chromosome specific AFLP mark and sequential analysis;
The screening of the AFLP mark that wherein above-mentioned 1.XY sex chromosome is special:
Extract genomic dna according to ordinary method from Yellow catfish XX raun, XY milter and YY supermale fish tail fin sample, by agarose electrophoresis and divide tube photometer to detect the concentration of DNA, the DNA concentration dilution becomes 30ng/ μ l the most at last.
AFLP screening may further comprise the steps: 1) use the MseI of commercial acquisition and EcoRI restriction enzyme (NEB company) that dna profiling is carried out enzyme and cut, 2) the T4DNA ligase enzyme (NEB company) of the commercial acquisition of use, special joint is connected to the endonuclease bamhi two ends, 3) increase in advance, 4) carry out selective amplification, 5) electrophoretic analysis.
Use the Taq archaeal dna polymerase of commercial acquisition to carry out selective amplification, (16 MseI aligning primers make up with 16 EcoRI aligning primers respectively to adopt the Shanghai synthetic AFLP of bio-engineering corporation primer, totally 256 combination of primers), to 8 XX rauns of Yellow catfish, the genome of 8 XY milters and 8 YY supermale fish individualities carries out the AFLP amplification and analyzes.Wherein 3 combination of primers amplify 2 X chromosome specific AFLP fragments and 2 Y chromosome specific AFLP fragments.Be specially: number 62 combination of primers (sequence is that upstream primer E6 and the sequence of SEQ ID NO.1 is the downstream primer M2 of the SEQ ID NO.2) specific fragment that all to amplify a size in all XY and YY individuality be 233bp, number 33 combination of primers (sequence is that upstream primer E3 and the sequence of SEQ ID NO.3 is the downstream primer M3 of the SEQ ID NO.4) specific fragment that all to amplify a size in all XY and YY individuality be 226bp simultaneously, and these two specific fragments all do not occur in the amplified production of all XX individualities, therefore this class is only occurred in all XY and YY individuality, and in the XX individuality absent variable fragment as the Y chromosome specific AFLP fragment; In addition, number the 62 combination of primers specific fragment that all to amplify a size in all XX and XY individuality be 239bp, the specific fragment that while No. 63 combination of primers (sequence is that upstream primer E6 and the sequence of SEQ ID NO.1 is the downstream primer M2 of SEQ ID NO.4) all amplify a size in all XX and XY individuality be 226bp, and these two fragments all do not occur in all YY individualities, therefore this class is only occurred in all XX and XY individuality, and in the YY individuality absent variable fragment as the X chromosome specific AFLP fragment.
The clone and the sequential analysis of 2. wherein above-mentioned sex chromosome specific AFLP marks may further comprise the steps: the 1) recovery of Yellow catfish XY sex chromosome specific AFLP fragment; 2) clone of sex chromosome specific AFLP fragment; 3) sequential analysis of sex chromosome specific AFLP fragment;
Above-mentioned 1) recovery of Yellow catfish XY sex chromosome specific AFLP fragment:
The AFLP amplified production downcuts XY sex chromosome specific fragment from polyacrylamide gel after electrophoretic separation, behind the single steaming of 50 μ l water rinse, add the single water that steams of 50 μ l, and gel smashed to pieces, in 95 ℃, heat 15min, the target dna fragment is discharged from gel.Of short duration centrifugal after, supernatant solution carries out AFLP selective amplification once more as the template of selective amplification respectively with pairing selective amplification primer of each specific fragment and same PCR condition.
Amplified production downcuts specific fragment through 1% agarose gel electrophoresis separation detection from gel under the ultra violet lamp, it is standby to reclaim test kit (Omega company) recovery purifying specific fragment by gel.
Above-mentioned 2) clone of sex chromosome specific AFLP fragment:
After 4 specific fragments that reclaim are connected to the pMD18-T carrier (Takara company) of commercial acquisition, adopt standard method transformed competence colibacillus bacillus coli DH 5 alpha (available from DSMZ of Wuhan University), adopt conventional PCR method to sift out the positive colony that contains target fragment.
Above-mentioned 3) sequential analysis of sex chromosome specific AFLP fragment:
Choose positive colony, deliver to the order-checking of Shanghai associating genome company.Sequencing result is by DNAMAN4.0 software analysis (Lynnon company).Above-mentioned 2 Yellow catfish Y chromosome specific AFLP fragments and 2 X chromosome specific AFLP fragments have been cloned, difference called after Pf-Y62, Pf-Y33, Pf-X62 and Pf-X63, its sequence is respectively the nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQID NO.7 and the SEQ ID NO.8.Sequential analysis shows: sex chromosome specific AFLP fragment Pf-X62 and Pf-Y62 are a pair of allelic sequence on Yellow catfish X and the Y chromosome, and their sequence difference is the insertion of 6 bases or the replacement of disappearance and 1 base; Sex chromosome specific AFLP fragment Pf-X33 and Pf-Y63 also are a pair of allelic sequences on Yellow catfish X and the Y chromosome, and their sequence difference is the replacement of 1 base.
Above-mentioned two) being converted into the special SACR mark of XY sex chromosome comprises:
1. the flanking sequence of the acquired karyomit(e) specific fragment of chromosome walking; 2. sequential analysis comparison, selectively staining body specific site design primer; 3. be converted into the SCAR mark;
The flanking sequence of the above-mentioned acquired karyomit(e) specific fragment of 1. chromosome walkings:
Use chromosome walking test kit (the Genome Walking Kit of commercial acquisition, Takara company), by the three-wheel amplification, the flanking sequence of 2 pairs of allelic sequences that clone Yellow catfish XY sex chromosome is special, according to the requirement of test kit specification sheets, its process comprises:
1) the SP1 primer respectively with this test kit in AP1, AP2, AP3, AP4 combination of primers, be template with Yellow catfish XX and YY genes of individuals group DNA, carry out first round amplification;
2) the SP2 primer respectively with this test kit in AP1, AP2, AP3, AP4 combination of primers, be template with first round amplified production, carry out second and take turns amplification;
3) the SP3 primer respectively with this test kit in AP1, AP2, AP3, AP4 combination of primers, taking turns amplified production with second is template, carries out third round amplification;
4) the third round amplified production separates by 1% agarose gel electrophoresis, downcut clear band, reclaim test kit (Gel Extraction Kit by sepharose, Omiga company) reclaims, and after being connected to the pMD18-T carrier (Takara company) of commercial acquisition, adopt standard method transformed competence colibacillus bacillus coli DH 5 alpha (available from DSMZ of Wuhan University), adopt conventional PCR method to sift out the positive colony that contains target fragment.
Above-mentioned 2. sequential analyses comparison, selectively staining body specific site design primer:
Choose positive colony, deliver to the order-checking of Shanghai associating genome company.Sequencing result finally obtains 2 couples of each about 1.5Kb of allelic sequence that sex chromosome is special by DNAMAN4.0 software (Lynnon company) and former specific fragment splicing and analysis.Selecting X and Y chromosome difference site from the flanking sequence that chromosome walking obtains is that the fragment of 311bp-624bp is used for sequential analysis than the size of horn of plenty, its sequence is respectively the nucleotide sequence shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ IDNO.12 and the SEQ ID NO.13, and wherein SEQ ID NO.9 and SEQ IDNO.10 are the copy of 2 kinds of sizes existing on X chromosome of same gene; By the sequence difference that the special allelic sequence of analyses and comparison sex chromosome copies on Yellow catfish X and Y chromosome respectively, find a collection of stable X and Y chromosome sequence difference site, can be used for distinguishing X or Y chromosome; Therefrom select part X and Y sex chromosome to have the site of sequence difference and suitable design primer, designed 4 pairs of special primers, be respectively:
1) Y chromosome specific mark Pf-Y62 primer:
By sequence is the upstream primer Pf-XY62L of SEQ ID NO.14 and downstream primer Pf-Y62R that sequence is SEQ IDNO.15 combination;
2) X chromosome specific mark Pf-X62 primer:
By sequence is the upstream primer Pf-XY62L of SEQ ID NO.14 and downstream primer Pf-X62R that sequence is SEQ IDNO.16 combination;
3) Y chromosome specific mark Pf-Y33 primer:
By sequence is the upstream primer Pf-Y33L of SEQ ID NO.17 and downstream primer Pf-Y33R that sequence is SEQ ID NO.18 combination;
4) X chromosome specific mark Pf-X63 primer:
By sequence is the upstream primer Pf-X63L of SEQ ID NO.19 and downstream primer Pf-X63R that sequence is SEQ ID NO.20 combination;
Above-mentioned 3. are converted into the SCAR mark:
Adopt 4 pairs of sex chromosome specific mark primers of previous step design, at Yellow catfish XX, increase among XY and the YY genes of individuals group DNA, the PCR reaction system is 25 μ l:2.5 μ l, 10 * PCRbuffer, 2.0 μ l 25mM Mgcl
2, 1U TaqDNA polysaccharase, 0.5 μ l 10mM dNTP, 0.5uM upstream and downstream primer, 20ng DNA adds water to 25 μ l; Amplification condition is respectively:
Pf-Y62 or Pf-X62:94 ℃ 30s, 58 ℃ of 30s, 72 ℃ of 30s, cycle number is 36;
Pf-Y33 or Pf-X63:94 ℃ 30s, 65 ℃/-0.5 ℃ 30s, 72 ℃ of 30s, cycle number is 10; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 30s, cycle number is 25;
Amplification shows: the specific fragment that the Pf-Y62 combination of primers can amplify a clip size from Yellow catfish XY milter and YY supermale fish genome be 568bp, the specific DNA fragment that the Pf-Y33 combination of primers can amplify a clip size from Yellow catfish XY milter and YY supermale fish genome be 462bp, these two combination of primers then all can not amplify this specific fragment from XX raun genes of individuals group.The specific DNA fragment that the Pf-X62 combination of primers can amplify a clip size from Yellow catfish XX raun and XY milter genome be 598bp or 285bp, the specific DNA fragment that the Pf-X63 combination of primers can amplify a clip size from Yellow catfish XX raun and XY milter genome be 462bp, these two combination of primers then all can not amplify this specific fragment from YY supermale fish genes of individuals group.
Above-mentioned three) setting up Yellow catfish sex chromosome genotype PCR authentication method comprises:
1. take a sample and DNA extraction:
Gather Yellow catfish tail fin sample, be stored in the dehydrated alcohol, extract genomic dna according to ordinary method.
2.PCR reaction:
Select for use the special SCAR mark of any one X chromosome in 4 designed SCAR marks and the special SCAR mark of Y chromosome as combination, in Yellow catfish sample gene group DNA to be identified, increase respectively.PCR reaction system and amplification condition are all as step 2) be converted into 3. in the special SACR mark of XY sex chromosome and be converted into PCR reaction system and the amplification condition described in the SCAR mark.
3. the result judges:
Judge its sex chromosome genotype according to the amplification of each sample: be merely able to amplify the special SCAR specific mark of X chromosome fragment, and the individuality of the special SCAR labeled fragment of the Y chromosome that can not increase is hereditary XX individuality; Can amplify the special SCAR labeled fragment of X chromosome, the individuality that can amplify Y chromosome SCAR labeled fragment again is hereditary XY individuality; Can only amplify the special SCAR labeled fragment of Y chromosome, and the individuality of the special SCAR labeled fragment of the X chromosome that can not increase is hereditary YY individuality.
Above-mentioned four) carrying out genetic sex in complete male Yellow catfish is cultivated identifies and comprise: 1.XX raun * XY milter mating combination filial generation genetic sex is identified; 2.XY raun gynogenesis combination filial generation genetic sex is identified; 3.YY physiology raun * XY milter mating combination filial generation genetic sex is identified; 4.YY physiology raun * YY supermale fish mating combination filial generation genetic sex is identified; 5.XX raun * YY supermale fish mating combination filial generation genetic sex is identified.
Above-mentioned steps four) 1.XX raun * XY milter mating combination filial generation genetic sex is identified:
Treat that filial generation grows to sexual maturity, judge Yellow catfish sex to be identified from outward appearance after, female, the realgar forehead fish tail fin sample of clip extracts genomic dna according to a conventional method respectively.Adopt above-mentioned steps three) described Yellow catfish sex chromosome genotype PCR authentication method, select each sample DNA of the special SCAR mark of Y chromosome Pf-Y62 primer amplification for use, genetic sex according to the amplification judgement sample: the individuality that can to amplify a size be the 568bp specific fragment is hereditary XY fish, and can not amplify this special pulsating individuality is hereditary XX fish.The result shows: it is the 568bp specific fragment that all individualities that are judged as milter from outward appearance all can amplify a size, and all can not amplify this specific fragment from the individuality that outward appearance is judged as raun.Therefore, the genetic sex and its sex phenotype (Fig. 2) in full accord that go out of molecular markers for identification.
Above-mentioned steps four) 2.XY raun gynogenesis combination filial generation genetic sex is identified:
Gather Yellow catfish XY raun gynogenesis filial generation XX raun, XY milter and each 12 tail of YY supermale fish (by test cross experimental identification idiogenetics sex), clip tail fin sample extracts genomic dna according to ordinary method.Adopt above-mentioned steps three) described Yellow catfish sex chromosome genotype PCR authentication method: select 4 designed pairing combination of primers of SCAR mark for use, in above 36 Yellow catfish sample gene group DNA, increase respectively.As step 3) described method, judge the genetic sex of each sample according to amplification, the result shows: all 12 XX raun samples all can amplify two special SCAR labeled fragments of X chromosome, can not amplify the special SCAR labeled fragment of Y chromosome; All 12 XY milter samples all can amplify two special SCAR labeled fragments of X chromosome, can amplify two Y chromosome SCAR labeled fragments again; All 12 YY supermale fish samples all can amplify two special SCAR labeled fragments of Y chromosome, the special SCAR labeled fragment of the X chromosome that can not increase.Therefore, the result (Fig. 3) in full accord that goes out of each the sample genetic sex that identifies of the special SCAR mark of do as one likes karyomit(e) and test cross experimental identification.
Above-mentioned steps four) 3.YY physiology raun * XY milter mating combination filial generation genetic sex is identified:
From YY raun * XY milter mating combination filial generation, extract 114 tail fishes, clip small pieces tail fin, behind ordinary method extraction genomic dna, adopt above-mentioned steps three) described Yellow catfish sex chromosome genotype PCR authentication method: select for use Pf-Y62 and Pf-X63 mark to carry out genetic sex and identify.The result shows the Pf-Y62 combination of primers specific fragment that all to amplify a clip size in all 114 samples be 568bp; The Pf-X63 primer specific fragment that then only can to amplify a clip size in 57 samples be 462bp meanwhile, all the other 57 samples do not have amplified production, illustrate 114 odd amount in addition to the round numbers in 57 tails (50%) YY supermale fish and 57 tail XY milters (Fig. 4) are arranged.
Above-mentioned steps four) 4.YY physiology raun * YY supermale fish mating combination filial generation genetic sex is identified:
Extracted for 42 odd amount in addition to the round number generations from the combination of YY physiology raun * YY supermale fish mating, clip small pieces tail fin extracts genomic dna with ordinary method.Adopt above-mentioned steps three) described Yellow catfish sex chromosome genotype PCR authentication method: select for use two marks of Pf-Y62 and Pf-X62 to carry out genetic sex and identify.Two mark amplifications show, the Y chromosome specific fragment that the Pf-Y62 combination of primers all can amplify a clip size in 42 samples be 568bp, and the Pf-X62 combination of primers does not all have amplified production in 42 samples, shows that all 42 odd amount in addition to the round numbers are for being YY supermale fish (Fig. 5).
Above-mentioned steps four) 5.XX raun * YY supermale fish mating combination filial generation genetic sex is identified:
From the YY supermale fish that above-mentioned steps 3 and 4 evaluations obtain, respectively choose 5 tail YY supermale fishes, carry out mating with the XX raun respectively after the sexual maturity.When treating that tester generation is grown to 4 months, randomly draw 400 tail prelarvas, dissect the back and judge that according to histology sexual gland is the sex that spermary or ovary are judged fish.Anatomical results shows that all 400 tail fishes are milter; Therefrom randomly draw simultaneously 20 tail fish clip tail fins, extract genomic dna, adopt above-mentioned steps three according to ordinary method) described Yellow catfish sex chromosome genotype PCR authentication method, select for use Pf-Y62 to be marked in the genomic dna of 20 samples and increase.The result shows, the specific fragment that it is 568bp that all individualities all can amplify a clip size illustrates that they are milter in the heredity, has illustrated that YY supermale fish that above-mentioned steps 3 and 4 is identified is the YY fish (Fig. 6) in the heredity simultaneously once more.
Characteristics of the present invention are:
(1) at first screens Yellow catfish X and Y chromosome specific AFLP mark, and they are changed into the convenient SCAR mark that uses, thereby set up Yellow catfish sex chromosome genotype identification PCR method;
(2) at first identify YY supermale fish, thereby avoided the man power and material's that brought by test cross experimental identification YY supermale fish consumption by aforesaid method, when also having avoided carrying out the test cross experiment to the injury of YY supermale fish;
(3) the auxiliary complete male Yellow catfish method of cultivation of molecule marker described in the invention does not all have report at present at home and abroad; present method has efficiently, accurate, stable and economic characteristics; to help to realize the large-scale production of full realgar forehead fry after the popularization, significantly improve the production efficiency of Yellow catfish aquaculture.Present method also extends in the sex control breeding of other economic fish simultaneously, has important economic value and social value.
Description of drawings
Figure 1A. (Pf62XS is respectively the sequence that Pf-X62 is marked at two kinds of different size copies on the X chromosome with Pf62XL to Yellow catfish sex chromosome specific mark fragments sequence compare of analysis; Pf62Y is that Pf-Y62 is marked at the sequence on the Y chromosome); The base of black background is three sequence same locis, and the base of all the other pearl opal backgrounds is the difference site.Sex chromosome special primer site for designing in the square frame.
The dna sequence analysis of B.Pf-Y33 and Pf-X33 mark (33X is the special copy of X chromosome, and 33Y is the special copy of Y chromosome).The base of black background is three sequence same locis, and the base of all the other pearl opal backgrounds is the difference site.In the square frame is sex chromosome special primer site.
Fig. 2 uses the genetic sex that the special SCAR mark of Y chromosome Pf-Y62 identifies XX raun * XY milter mating combination filial generation.
Fig. 3 utilizes X and the special SCAR mark of Y sex chromosome to identify the genetic sex of XY physiology raun gynogenesis combination filial generation.
Fig. 4 usability karyomit(e) special SCAR mark Pf-Y62 and Pf-X63 identify the genetic sex of YY physiology raun and the normal milter mating combination of XY filial generation.
Fig. 5 usability karyomit(e) special SCAR mark Pf-Y62 and Pf-X62 identify the genetic sex of YY physiology raun and YY supermale fish mating combination filial generation.
The special SCAR mark of Fig. 6 usability karyomit(e) Pf-Y62 identifies the genetic sex of YY supermale fish and normal X X raun mating combination filial generation.
Embodiment
Embodiment 1
A kind of Yellow catfish sex chromosome specific molecular markers and genetic sex identification method the steps include:
The present invention includes four big steps: one) screening and clone Yellow catfish XY sex chromosome specific AFLP mark; Two) be converted into the special SACR mark of XY sex chromosome; Three) set up Yellow catfish sex chromosome genotype PCR authentication method; Four) carrying out genetic sex in complete male Yellow catfish is cultivated identifies.
Above-mentioned one) screening and clone Yellow catfish XY sex chromosome specific AFLP mark comprise:
1.XY the AFLP label screening that sex chromosome is special; 2. the clone of sex chromosome specific AFLP mark and sequential analysis;
The AFLP label screening that wherein above-mentioned 1.XY sex chromosome is special:
Extract genomic dna according to ordinary method from Yellow catfish XX raun, XY milter and YY supermale fish tail fin sample, by agarose electrophoresis and divide tube photometer to detect the concentration of DNA, the DNA concentration dilution becomes 30ng/ μ l the most at last.
The AFLP screening may further comprise the steps:
1) genomic dna template enzyme is cut:
Use MseI and each 5U of EcoRI restriction enzyme (NEB company) of commercial acquisition, simultaneously the 300ng genomic dna is carried out double digestion, the enzyme tangent condition is 37 ℃, reacts after 3 hours 70 ℃ to restriction enzyme enzyme-deactivating 15min.
2) special joint is connected to the endonuclease bamhi two ends:
Commercial synthetic special joint,
EcoRI joint: 5-CTC GTA GAC TGC GTA CC-3
CAT?CTG?ACG?CAT?GG?TTAA-5
MseI joint: 5-GACG ATG AGT CCT GAG-3
TAC?TCA?GGA?CTC?AT-5
Cut adding respectively in the product to the previous step enzyme: each 1 μ l of MseI joint (50 μ M) and EcoRI joint (5 μ M), the T4DNA ligase enzyme of commercial acquisition (NEB company) 0.2 μ l, single water, the 4.0 μ l buffer of steaming of 13.8 μ l.4 ℃ of reactions are spent the night.
3) increase in advance:
Use the Taq archaeal dna polymerase (Fermanta company) of commercial acquisition to increase in advance; With 10 times of above-mentioned connection product dilutions, as the template of pre-amplification; Pre-amplification primer is that commercial synthetic primer (E+1:5 ' GACTGCGTACCAATTCA3 ', M+1:5 ' GATGAGTCCTGAGTAAC3 ') increases.
4) carry out selective amplification:
Use the Taq archaeal dna polymerase of commercial acquisition to carry out selective amplification; With 20 times of above-mentioned pre-expansion volume increase thing dilutions, as the template of selective amplification; Primer adds 2 bases at random for pre-amplification primer 3 ' end, by each 16 of commercial synthetic MseI and EcoRI selective amplification primers, is combined into 256 combination of primers altogether and carries out selective amplification.
16 EcoRI selective amplification primers are:
E+3：5’-GACTGCGTACCAATTCANN-3’
E-AAC E-AAG E-ACA E-ACT E-AAA E-AAT E-ACC E-AGAE-AGT
E-ACC E-ACG E-AGC E-AGG E-ATA E-ATC E-ATG E-ATT
16 Mse I selective amplification primers are:
M+3： 5’-GATGAGTCCTGAGTAACNN-3’
M-CAA M-CAC M-CAG M-CAT M-CCA M-CCC M-CCG M-CCT
M-CTA M-CTC M-CTGM-CTT M-CGA M-CGCM-CGG M-CGT
5) electrophoretic analysis:
Past above-mentioned selective amplification product adds 6X Loading buffer (Takara company) sample solution of 1/5 volume, and 94 ℃ of sex change placed stand-by rapidly on ice after 5 minutes.With 6% polyacrylamide gel prerunning, the prerunning condition is: permanent power 100W, 1 hour; Then that sex change is good selective amplification product moves on to electrophoresis on the gel, and deposition condition is: firm power 90W, 50 ℃ of temperature, 2.5 hours.
Electrophoresis finishes the back and adopts argentation, polyacrylamide gel is carried out silver dye: with 6% polyacrylamide gel of separator well fixing 30min in 2 liter of 1% acetate; Behind single steaming water rinse, place silver-colored dye liquor (the single water+3ml formaldehyde+2g Silver Nitrate that steams of 2000ml) silver to dye 30min; Single steam water rinse after, the developing solution (the single water+3ml formaldehyde+10g NaOH of steaming of 2000ml) of putting into precooling develop the color to electrophoretic band clear after, gel is taken out with single water rinse that steams, stop colour developing.To carry out the strip analysis screening by naked eyes behind the gel natural air drying.
Use the Taq archaeal dna polymerase of commercial acquisition to carry out selective amplification, (16 MseI aligning primers make up with 16 EcoRI aligning primers respectively to adopt the Shanghai synthetic AFLP of bio-engineering corporation primer, totally 256 combination of primers), to 8 XX rauns of Yellow catfish, the genome of 8 XY milters and 8 YY supermale fish individualities carries out the AFLP amplification and analyzes.Wherein 3 combination of primers amplify 2 X chromosome specific AFLP fragments and 2 Y chromosome specific AFLP fragments.Be specially: number 62 combination of primers (sequence is that upstream primer E6 and the sequence of SEQ ID NO.1 is the downstream primer M2 of the SEQ ID NO.2) specific fragment that all to amplify a size in all XY and YY individuality be 233bp, number 33 combination of primers (sequence is that upstream primer E3 and the sequence of SEQ ID NO.3 is the downstream primer M3 of the SEQ ID NO.4) specific fragment that all to amplify a size in all XY and YY individuality be 226bp simultaneously, and these two specific fragments all do not occur in the amplified production of all XX individualities, therefore this class is only occurred in all XY and YY individuality, and in the XX individuality absent variable fragment as the Y chromosome specific AFLP fragment; In addition, number the 62 combination of primers specific fragment that all to amplify a size in all XX and XY individuality be 239bp, the specific fragment that while No. 63 combination of primers (sequence is that upstream primer E6 and the sequence of SEQ ID NO.1 is the downstream primer M2 of SEQ ID NO.4) all amplify a size in all XX and XY individuality be 226bp, and these two fragments all do not occur in all YY individualities, therefore this class is only occurred in all XX and XY individuality, and in the YY individuality absent variable fragment as the X chromosome specific AFLP fragment.
The clone and the sequential analysis of 2. wherein above-mentioned sex chromosome specific AFLP marks may further comprise the steps: the 1) recovery of Yellow catfish XY sex chromosome specific AFLP fragment; 2) clone of sex chromosome specific AFLP fragment; 3) sequential analysis of sex chromosome specific AFLP fragment;
Above-mentioned 1) recovery of Yellow catfish XY sex chromosome specific AFLP fragment:
The AFLP amplified production downcuts XY sex chromosome specific fragment from polyacrylamide gel after electrophoretic separation, behind the single steaming of 50 μ l water rinse, add the single water that steams of 50 μ l, and gel smashed to pieces, in 95 ℃, heat 15min, the target dna fragment is discharged from gel.Of short duration centrifugal after, supernatant solution carries out AFLP selective amplification once more as the template of selective amplification respectively with pairing selective amplification primer of each specific fragment and same PCR condition.
Amplified production downcuts specific fragment through 1% agarose gel electrophoresis separation detection from gel under the ultra violet lamp, it is standby to reclaim test kit (Gel Extraction Kit, Omega company) recovery purifying specific fragment by gel.
Above-mentioned 2) clone of sex chromosome specific AFLP fragment:
After 4 specific fragments that reclaim are connected to the pMD18-T carrier (available from Takara company) of commercial acquisition, adopt standard method transformed competence colibacillus bacillus coli DH 5 alpha (available from DSMZ of Wuhan University), adopt conventional PCR method to sift out the positive colony that contains target fragment.
Above-mentioned 3) sequential analysis of sex chromosome specific AFLP fragment:
Choose positive colony, deliver to the order-checking of Shanghai associating genome company.Sequencing result is by DNAMAN4.0 software analysis (Lynnon company).Above-mentioned 2 Yellow catfish Y chromosome specific AFLP fragments and 2 X chromosome specific AFLP fragments have been cloned, difference called after Pf-Y62, Pf-Y33, Pf-X62 and Pf-X63, its sequence is respectively the nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQID NO.7 and the SEQ ID NO.8.Sequential analysis shows: sex chromosome specific AFLP fragment Pf-X62 and Pf-Y62 are a pair of allelic sequence on Yellow catfish X and the Y chromosome, and their sequence difference is the insertion of 6 bases or the replacement of disappearance and 1 base; Sex chromosome specific AFLP fragment Pf-X33 and Pf-Y63 also are a pair of allelic sequences on Yellow catfish X and the Y chromosome, and their sequence difference is the replacement of 1 base.
Above-mentioned two) being converted into the special SACR mark of XY sex chromosome comprises:
1. the flanking sequence of the acquired karyomit(e) specific fragment of chromosome walking; 2. sequential analysis comparison, selectively staining body specific site design primer; 3. be converted into the SCAR mark;
The flanking sequence of the above-mentioned acquired karyomit(e) specific fragment of 1. chromosome walkings:
Use chromosome walking test kit (the Genome Walking Kit of commercial acquisition, Takara company), by the three-wheel amplification, the flanking sequence of 2 pairs of allelic sequences that clone Yellow catfish XY sex chromosome is special, according to the requirement of test kit specification sheets, its process comprises:
1) the SP1 primer respectively with this test kit in AP1, AP2, AP3, AP4 combination of primers, be template with Yellow catfish XX and YY genes of individuals group DNA, carry out first round amplification;
2) the SP2 primer respectively with this test kit in AP1, AP2, AP3, AP4 combination of primers, be template with first round amplified production, carry out second and take turns amplification;
3) the SP3 primer respectively with this test kit in AP1, AP2, AP3, AP4 combination of primers, taking turns amplified production with second is template, carries out third round amplification;
4) the third round amplified production separates by 1% agarose gel electrophoresis, downcut clear band, reclaim test kit (Gel Extraction Kit by gel, Omega company) reclaims, and after being connected to the pMD18-T carrier (available from Takara company) of commercial acquisition, adopt standard method transformed competence colibacillus bacillus coli DH 5 alpha (available from DSMZ of Wuhan University), adopt conventional PCR method to sift out the positive colony that contains target fragment.
Above-mentioned 2. sequential analyses comparison, selectively staining body specific site design primer:
Choose positive colony, deliver to the order-checking of Shanghai associating genome company.Sequencing result finally obtains 2 couples of each about 1.5Kb of allelic sequence that sex chromosome is special by DNAMAN4.0 software (Lynnon company) and former specific fragment splicing and analysis.Selecting X and Y chromosome difference site from the flanking sequence that chromosome walking obtains is that the fragment of 311bp-624bp is used for sequential analysis than the size of horn of plenty, its sequence is respectively the nucleotide sequence shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ IDNO.12 and the SEQ ID NO.13, and wherein SEQ ID NO.9 and SEQ IDNO.10 are the copy of 2 kinds of sizes existing on X chromosome of same gene; By the sequence difference that the special allelic sequence of analyses and comparison sex chromosome copies on Yellow catfish X and Y chromosome respectively, find a collection of stable X and Y chromosome sequence difference site, be used for distinguishing X or Y chromosome; Therefrom select part X and Y sex chromosome to have the site of sequence difference and suitable design primer, designed 4 pairs of special primers, be respectively:
1) Y chromosome specific mark Pf-Y62 primer:
By sequence is the upstream primer Pf-XY62L of SEQ ID NO.14 and downstream primer Pf-Y62R that sequence is SEQ IDNO.15 combination;
2) X chromosome specific mark Pf-X62 primer:
By sequence is the upstream primer Pf-XY62L of SEQ ID NO.14 and downstream primer Pf-X62R that sequence is SEQ IDNO.16 combination;
3) Y chromosome specific mark Pf-Y33 primer:
By sequence is the upstream primer Pf-Y33L of SEQ ID NO.17 and downstream primer Pf-Y33R that sequence is SEQ ID NO.18 combination;
4) X chromosome specific mark Pf-X63 primer:
By sequence is the upstream primer Pf-X63L of SEQ ID NO.19 and downstream primer Pf-X63R that sequence is SEQ ID NO.20 combination;
Above-mentioned 3. are converted into the SCAR mark:
Adopt 4 pairs of sex chromosome specific mark primers of previous step design, at Yellow catfish XX, increase among XY and the YY genes of individuals group DNA, the PCR reaction system is 25 μ l:2.5 μ l, 10 * PCRbuffer, 2.0 μ l 25mM Mgcl
2, 1U TaqDNA polysaccharase, 0.5 μ l 10mM dNTP, 0.5uM upstream and downstream primer, 20ng DNA adds water to 25 μ l; Amplification condition is respectively:
Pf-Y62 or Pf-X62:94 ℃ 30s, 58 ℃ of 30s, 72 ℃ of 30s, cycle number is 36;
Pf-Y33 or Pf-X63:94 ℃ 30s, 65 ℃/-0.5 ℃ 30s, 72 ℃ of 30s, cycle number is 10; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 30s, cycle number is 25;
Amplification shows: the specific fragment that the Pf-Y62 combination of primers can amplify a clip size from Yellow catfish XY milter and YY supermale fish genome be 568bp, the specific DNA fragment that the Pf-Y33 combination of primers can amplify a clip size from Yellow catfish XY milter and YY supermale fish genome be 462bp, these two combination of primers then all can not amplify this specific fragment from XX raun genes of individuals group.The specific DNA fragment that the Pf-X62 combination of primers can amplify a clip size from Yellow catfish XX raun and XY milter genome be 598bp or 285bp, the specific DNA fragment that the Pf-X63 combination of primers can amplify a clip size from Yellow catfish XX raun and XY milter genome be 462bp, these two combination of primers then all can not amplify this specific fragment from YY supermale fish genes of individuals group.
Above-mentioned three) setting up Yellow catfish sex chromosome genotype PCR authentication method comprises:
1. take a sample and DNA extraction:
Gather Yellow catfish tail fin sample, be stored in the dehydrated alcohol, extract genomic dna according to ordinary method.
2.PCR reaction:
Select for use the special SCAR mark of any one X chromosome in 4 designed SCAR marks and the special SCAR mark of Y chromosome as combination, in Yellow catfish sample gene group DNA to be identified, increase respectively.PCR reaction system and amplification condition are all as step 2) be converted into 3. in the special SACR mark of XY sex chromosome and be converted into PCR reaction system and the amplification condition described in the SCAR mark.
3. the result judges:
Judge its sex chromosome genotype according to the amplification of each sample: be merely able to amplify the special SCAR specific mark of X chromosome fragment, and the individuality of the special SCAR labeled fragment of the Y chromosome that can not increase is hereditary XX individuality; Can amplify the special SCAR labeled fragment of X chromosome, the individuality that can amplify Y chromosome SCAR labeled fragment again is hereditary XY individuality; Can only amplify the special SCAR labeled fragment of Y chromosome, and the individuality of the special SCAR labeled fragment of the X chromosome that can not increase is hereditary YY individuality.
Above-mentioned four) carrying out genetic sex in complete male Yellow catfish is cultivated identifies and comprise: 1.XX raun * XY milter mating combination filial generation genetic sex is identified; 2.XY raun gynogenesis combination filial generation genetic sex is identified; 3.YY physiology raun * XY milter mating combination filial generation genetic sex is identified; 4.YY physiology raun * YY supermale fish mating combination filial generation genetic sex is identified; 5.XX raun * YY supermale fish mating combination filial generation genetic sex is identified.
Above-mentioned steps four) 1.XX raun * XY milter mating combination filial generation genetic sex is identified:
Treat that filial generation grows to sexual maturity, judge Yellow catfish sex to be identified from outward appearance after, female, the realgar forehead fish tail fin sample of clip extracts genomic dna according to a conventional method respectively.Adopt above-mentioned steps three) described Yellow catfish sex chromosome genotype PCR authentication method, select each sample DNA of the special SCAR mark of Y chromosome Pf-Y62 primer amplification for use, genetic sex according to the amplification judgement sample: the individuality that can to amplify a size be the 568bp specific fragment is hereditary XY fish, and can not amplify this special pulsating individuality is hereditary XX fish.The result shows: it is the 568bp specific fragment that all individualities that are judged as milter from outward appearance all can amplify a size, and is judged as from outward appearance, and the individuality of raun all can not amplify this specific fragment.Therefore, the genetic sex and its sex phenotype (Fig. 2) in full accord that go out of molecular markers for identification.
Above-mentioned steps four) 2.XY raun gynogenesis combination filial generation genetic sex is identified:
Gather Yellow catfish XY raun gynogenesis filial generation XX raun, XY milter and each 12 tail of YY supermale fish (by test cross experimental identification idiogenetics sex), clip tail fin sample extracts genomic dna according to ordinary method.Adopt above-mentioned steps three) described Yellow catfish sex chromosome genotype PCR authentication method: select 4 designed pairing combination of primers of SCAR mark for use, in above 36 Yellow catfish sample gene group DNA, increase respectively.As step 3) described method, judge the genetic sex of each sample according to amplification, the result shows: all 12 XX raun samples all can amplify two special SCAR labeled fragments of X chromosome, can not amplify the special SCAR labeled fragment of Y chromosome; All 12 XY milter samples all can amplify two special SCAR labeled fragments of X chromosome, can amplify two Y chromosome SCAR labeled fragments again; All 12 YY supermale fish samples all can amplify two special SCAR labeled fragments of Y chromosome, the special SCAR labeled fragment of the X chromosome that can not increase.Therefore, the result (Fig. 3) in full accord that goes out of each the sample genetic sex that identifies of the special SCAR mark of do as one likes karyomit(e) and test cross experimental identification.
Above-mentioned steps four) 3.YY physiology raun * XY milter mating combination filial generation genetic sex is identified:
From YY raun * XY milter mating combination filial generation, extract 114 tail fishes, clip small pieces tail fin, behind ordinary method extraction genomic dna, adopt above-mentioned steps three) described Yellow catfish sex chromosome genotype PCR authentication method: select for use Pf-Y62 and Pf-X63 mark to carry out genetic sex and identify.The result shows the Pf-Y62 combination of primers specific fragment that all to amplify a clip size in all 114 samples be 568bp; The Pf-X63 primer specific fragment that then only can to amplify a clip size in 57 samples be 462bp meanwhile, all the other 57 samples do not have amplified production, illustrate 114 odd amount in addition to the round numbers in 57 tails (50%) YY supermale fish and 57 tail XY milters (Fig. 4) are arranged.
Above-mentioned steps four) 4.YY physiology raun * YY supermale fish mating combination filial generation genetic sex is identified:
Extracted for 42 odd amount in addition to the round number generations from the combination of YY physiology raun * YY supermale fish mating, clip small pieces tail fin extracts genomic dna with ordinary method.Adopt above-mentioned steps three) described Yellow catfish sex chromosome genotype PCR authentication method: select for use two marks of Pf-Y62 and Pf-X62 to carry out genetic sex and identify.Two mark amplifications show, the Y chromosome specific fragment that the Pf-Y62 combination of primers all can amplify a clip size in 42 samples be 568bp, and the Pf-X62 combination of primers does not all have amplified production in 42 samples, shows that all 42 odd amount in addition to the round numbers are for being YY supermale fish (Fig. 5).
Above-mentioned steps four) 5.XX raun * YY supermale fish mating combination filial generation genetic sex is identified:
From the YY supermale fish that above-mentioned steps 3 and 4 evaluations obtain, respectively choose 5 tail YY supermale fishes, carry out mating with the XX raun respectively after the sexual maturity.When treating that tester generation is grown to 4 months, randomly draw 400 tail prelarvas, dissect the back and judge that according to histology sexual gland is the sex that spermary or ovary are judged fish.Anatomical results shows that all 400 tail fishes are milter; Therefrom randomly draw simultaneously 20 tail fish clip tail fins, extract genomic dna, adopt above-mentioned steps three according to ordinary method) described Yellow catfish sex chromosome genotype PCR authentication method, select for use Pf-Y62 to be marked in the genomic dna of 20 samples and increase.The result shows, the specific fragment that it is 568bp that all individualities all can amplify a clip size illustrates that they are milter in the heredity, has illustrated that YY supermale fish that above-mentioned steps 3 and 4 is identified is the YY fish (Fig. 6) in the heredity simultaneously once more.
SEQUENCE?LISTING
＜110〉Inst. of Hydrobiology, Chinese Academy of Sciences
＜120〉Yellow catfish sex chromosome specific molecular markers and genetic sex identification method
＜130〉Yellow catfish sex chromosome specific molecular markers and genetic sex identification method
<160>20
<170>PatentIn?version?3.5
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Claims (4)
1. a specific mark combination of primers that is used to detect the Yellow catfish sex is made up of the specific mark primer of X chromosome and the specific mark primer of Y chromosome, it is characterized in that:
The upstream primer sequence of the specific mark primer of described X chromosome is shown in SEQ ID NO.14, and the downstream primer sequence is shown in SEQ ID NO.16;
The upstream primer sequence of the specific mark primer of described Y chromosome is shown in SEQ ID NO.14, and the downstream primer sequence is shown in SEQ ID NO.15.
2. a specific mark combination of primers that is used to detect the Yellow catfish sex is made up of the specific mark primer of X chromosome and the specific mark primer of Y chromosome, it is characterized in that:
The upstream primer sequence of the specific mark primer of described X chromosome is shown in SEQ ID NO.14, and the downstream primer sequence is shown in SEQ ID NO.16;
The upstream primer sequence of the specific mark primer of described Y chromosome is shown in SEQ ID NO.17, and the downstream primer sequence is shown in SEQ ID NO.18.
3. a specific mark combination of primers that is used to detect the Yellow catfish sex is made up of the specific mark primer of X chromosome and the specific mark primer of Y chromosome, it is characterized in that:
The upstream primer sequence of the specific mark primer of described X chromosome is shown in SEQ ID NO.19, and the downstream primer sequence is shown in SEQ ID NO.20;
The upstream primer sequence of the specific mark primer of described Y chromosome is shown in SEQ ID NO.14, and the downstream primer sequence is shown in SEQ ID NO.15.
4. a specific mark combination of primers that is used to detect the Yellow catfish sex is made up of the specific mark primer of X chromosome and the specific mark primer of Y chromosome, it is characterized in that:
The upstream primer sequence of the specific mark primer of described X chromosome is shown in SEQ ID NO.19, and the downstream primer sequence is shown in SEQ ID NO.20;
The upstream primer sequence of the specific mark primer of described Y chromosome is shown in SEQ ID NO.17, and the downstream primer sequence is shown in SEQ ID NO.18.
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| CN106011300A (en) * | 2016-08-04 | 2016-10-12 | 淮阴师范学院 | PCR primer pair used for identifying genetic sex of Pseudobagrus ussuriensis, and rapid identification method thereof |
| CN109880893B (en) * | 2019-04-12 | 2020-02-04 | 中国科学院水生生物研究所 | Specific DNA fragment for sex identification of mystus guttatus and application |
| CN110551808B (en) * | 2019-09-25 | 2021-07-06 | 西南大学 | Southern catfish sex chromosome specific molecular marker, genetic sex identification method based on molecular marker and parthenocarpic fish production method |
| CN111944887A (en) * | 2020-08-26 | 2020-11-17 | 扬州大学 | Method for screening and verifying female sex-determining gene of chicken |
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| CN115807058B (en) * | 2022-12-02 | 2023-06-30 | 中国科学院水生生物研究所 | Low-bias single sperm whole genome amplification method |
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