Patent Application: US-22662707-A

Abstract:
the present invention relates to a sex - specific marker for shrimps and prawns . more specifically , it relates to a sex - specific pcr - based molecular marker , derived from penaeus monodon , that can be used to determine the sex in shrimps and prawns and can be used for any and all requirements for the determination of genetic sex in shrimp and prawn including , but not limited to , sex determination of very young animals , determination of genetic sex on any animals and setting up monosex cultures .

Description:
four half - sib mapping populations were generated by crossing two female animals ( ic100 and ic67 ) with two male animals ( al91 and al99 ) in all four possible combinations ( see table 1 ). all four parents were recently caught in the wild . all crosses were performed at moana technologies , inc . the mapping populations were harvested at post - larval stage ( pl ) 120 , when the sex of each individual could be scored with an acceptable degree of certainty . in addition , a set of 52 unrelated wild - caught animals was available at moana technologies , inc . these individuals were used to assess the tightness of the physical linkage between the sex - locus and the potential sex - markers identified in the mapping populations . dna was prepared from snap - frozen pleopod tissue using a ctab method optimized for shrimp tissue . aflp analysis was performed as described by vos et al . ( vos et al . 1995 ). genomic dna of the parents and progeny was restricted using ecori and msei . double - stranded adaptors were ligated to the ends of the restriction fragments . the digestion product was diluted and pre - amplified using adaptor - specific primers with a single selective nucleotide on each primer . two pools , or bulks , of five individuals of population a were made at the pre - amplification level . within each pool , or bulk , the individuals were identical for sex but arbitrary for all other loci . selected subsets of restriction fragments were amplified using aflp - primers containing two additional selective nucleotides . amplification reactions were separated on aflp sequencing gels and visualized using li - cor ir 2 technology . aflp markers identified as sex - specific in the bulk segregant analysis ( bsa ) on population a ( i . e ., that were present in the two female bulks but not in the two male bulks ) were considered candidate sex - specific markers . subsequently , these candidate sex - specific markers were tested on the bulked samples from the three remaining mapping populations . markers that confirmed their association to sex in the three additional bsa analyses were then tested for linkage to the sex - locus in each of the four mapping populations . to finally assess the strength of the identified marker - trait - associations by linkage , we genotyped the set of 52 unrelated wild - caught ( broodstock ) animals ( the four parents from the mapping populations and 48 additional samples ) with the aflp markers found to be in linkage with sex . to obtain sequence information from aflp markers , the ecori - specific primer was radioactively labeled using 33 p - atp and amplification products were separated on a 5 % denaturing sequencing gel . gels were dried and visualized by autoradiography . after visualization , the bands were excised from the gel , and eluted fragments were amplified and sequenced . most pcr reactions were performed in 1 × pcr buffer supplemented with 1 . 5 ng / μl of each primer , 0 . 2 mm of each dntp , 2 . 5 mm of mgcl2 , 0 . 025 u of taq polymerase and 100 ng of template dna . pcr using primers attgca - 1 and attgca - 2 were performed in a total volume of 20 μl using 100 ng of genomic dna as a template . the reaction mixture was heated to 95 ° c . for four minutes , followed by 35 cycles of 30 seconds of denaturation at 95 ° c ., 30 seconds of annealing at 52 ° c . and 30 seconds of elongation at 72 ° c . finally , an additional elongation step of two minutes at 72 ° c . was performed . pcrs using primer attgca - 1 in combination with primers msei + gca or msei + aaa and pcr using primer attgca - 2 in combination with primer ecori + att were performed in a total volume of 50 μl using 5 μl of a 1 / 20 diluted pre - amplification reaction . pcr reactions were as follows : 35 cycles of 30 seconds of denaturation at 95 ° c ., 30 seconds at the appropriate annealing temperature ( 55 ° c . for attgca - 1 and 53 ° c . for attgca - 2 ) and 30 seconds of elongation at 72 ° c . followed by an additional elongation step of two minutes at 72 ° c . pcr for the indel - marker were performed using either radioactively or fluorescently labeled primer indel - 4 . the reaction was performed in 1 × pcr buffer supplemented with 0 . 3 ng / μl of each primer , 0 . 2 mm of each dntp , 2 . 5 mm of mgcl2 , 0 . 025 u of taq polymerase and 50 ng of genomic dna . the reaction mixture was heated to 95 ° c . for four minutes , followed by 35 cycles of 30 seconds of denaturation at 95 ° c ., 30 seconds of annealing at 55 ° c ., and 30 seconds of elongation at 72 ° c . finally , an additional elongation step of two minutes at 72 ° c . was performed . for the fluorescent analysis , the reaction was essentially the same as for the radioactive analysis except that 0 . 03 mm of ird700 - labeled indel - 4 primer was added and 0 . 3 mm of indel - 5 primer . primer sequences are given in table 2 . genomic dna was prepared from a small sample of tissue ( e . g ., 5 mm of the tip of a walking leg ) by heating for 30 minutes at 95 ° c . in 75 μl of alkaline lysis buffer ( 25 mm naoh , 0 . 2 mm edta ph = 12 ). the samples were cooled on ice and 75 μl of neutralization buffer ( 40 mm tris - hcl ph = 5 ) was added . pcr was performed on 5 μl of the 1 / 20 diluted dna . pcr was performed using the “ platinum sybr green qpcr supermix - udg ” kit ( invitrogen ) using primers femaleforward — 2 , maleforward — 2 and reverse — 2 . an initial denaturation step ( five minutes at 95 ° c .) was followed by 40 cycles of 20 seconds at 95 ° c ., 30 seconds at 55 ° c . and 20 seconds at 72 ° c . reactions were subsequently denatured ( one minute at 95 ° c .) and renatured ( one minute at 40 ° c .). melting curve analysis was performed on an icycler system ( bio - rad ). femaleforward — 2 : gcgggcgttagctgatattcataattcatgctc ( seq id no : 9 ) maleforward — 2 : gcgggcagggcggcgttagctgatattcataatccatgcaa ( seq id no : 10 ) reverse — 2 : aaggggtcgcgaatgtaaaata ( seq id no : 11 ) in a first screening , we analyzed approximately 1 , 408 aflp primer combinations on the bulked samples from mapping population a . the aflp ecori + 3 / msei + 3 primer combinations generate 50 to 80 aflp fragments . hence , the bulks were fingerprinted with more than 70 , 400 aflp fragments . of these fragments , 13 were identified by the bsa analysis as sex - specific . to confirm this , bulked samples from the other three mapping populations ( b , c and d ) were fingerprinted for these 13 sex - specific markers . in nine cases , the marker - sex association could be confirmed . all of these markers were present in the female bulks but absent from the male bulks . to determine how tightly these nine markers were linked to the sex - locus , markers were scored in all offspring from the four mapping populations . the recombination frequency ( i . e ., the number of females that did not show a band and the number of males that did show a band , expressed as a proportion of the total number of individuals analyzed ) is expressed in table 3 . to assess the degree of ld of the aflp marker haplotypes with the sexual dimorphism in a population of genetically unrelated individuals , the four parental animals and 48 additional broodstock animals recently caught in the wild at several locations throughout the pacific ocean were genotyped at the nine sex - linked aflp marker loci . at two loci , aflp marker alleles ( ecori + aag / msei + cgc - 72 . 8 and ecori + att / msei + gca - 347 . 0 ) were in complete ld with the sex of p . monodon . the corresponding ecori - specific primers were radioactively labeled . the aflp amplification products were separated on a denaturing polyacrylamide sequencing gel and visualized using autoradiography . the female ecori + aag / msei + cgc - 72 . 8 and ecori + att / msei + gca - 347 . 0 marker alleles were cut from the aflp gel , eluted , amplified and sequenced . because we obtained multiple possible sequences , we increased the selectivity of the aflp reaction by two additional selective nucleotides (+ 3 /+ 3 aflp reaction →+ 4 /+ 4 aflp reaction ). the female - specific fragments were now obtained as ecori + aagt / msei + cgct - 72 . 8 and ecori + atta / msei + gcat - 347 . 0 . isolation and subsequent sequencing of the ecori + atta / msei + gcat - 347 . 0 marker band resulted in a unique sequence . a non - specific band hampered the isolation of the ecori + aagt / msei + cgct - 72 . 8 fragment and its subsequent generation of a unique sequence . furthermore , the sequence was short , making it even more difficult to design a specific pcr for this fragment . therefore , marker ecori + atta / msei + gcat - 347 . 0 was chosen to further develop into a co - dominant pcr - marker . for marker ecori + atta / msei + gcat - 347 . 0 , we designed pcr primers to amplify the marker in both female and male individuals . using primers attgca - 1 and attgca - 2 , we were able to amplify a fragment of approximately 285 base pairs ( bp ) on the genomic dna of both female and male individuals . these pcr fragments were isolated from the gel and sequenced . this resulted in a female - and male - specific sequence for this marker . careful examination of these sequences revealed nine sex - specific polymorphisms in these sequences : six single nucleotide polymorphisms ( snps ) and three insertion / deletion ( indel ) polymorphisms . one of the indels caused the presence of an additional msei restriction site in the male . this is the polymorphism most probably responsible for the absence of the ecori + atta / msei + gcat - 347 . 0 aflp fragment in the males . to obtain sequence information of the part of flanking regions of the aflp fragments , we performed pcr using primers attgca - 1 and the corresponding msei - specific aflp primer ( m + gca for the female and m + aaa for the male ) and using primers attgca - 2 and the corresponding ecori - specific aflp primer ( ecori + att for both female and male ) ( see fig2 ). the resulting pcr fragments were sequenced and the previously obtained sequences were updated using this additional sequence information ( seq id nos : 3 and 4 ). as a result , an additional sequence polymorphism ( indel ) between the male and female sequence was detected , which was previously not identified because it is located in the sequence targeted by primer attgca - 1 . to convert the e + atta / m + gcat - 347 . 0 aflp marker into a simple single locus marker , we designed primers to specifically amplify the genomic locus harboring the indel polymorphism identified to be sex - specific . using primers indel - 4 and indel - 5 , an allelic fragment of 76 bp was amplified in five males and five females coming from population a , and an allelic fragment of 82 bp was amplified in the five females only . this proved that females are the heterogametic sex in penaeus monodon . a similar result was obtained for the set of 52 unrelated broodstock animals , showing that the indel polymorphism is in complete ld with the sex ( see fig1 ). another set of 33 unrelated broodstock animals was genotyped with this marker and again the indel polymorphism was found to be in complete ld with the sex . the pcr - amplified marker alleles described here are in complete ld with the sex dimorphism in penaeus monodon . this marker allows the determination of the genetic sex of any p . monodon individual regardless of its developmental stage . development of an rt - pcr - based snp genotyping assay for sex in p . monodon to facilitate the screening of large numbers of shrimp , we developed an rt - pcr - based snp genotyping assay for the sex marker . specific forward primers were designed for the male and the female allele and used in a pcr in combination with a common reverse primer . after amplification , melting curve analysis was performed . in males , only one peak was observed , while in the female , two peaks corresponding to the two alleles were observed ( fig3 ). hansford s . w . and d . r . hewitt ( 1994 ). growth and nutrient digestibility by male and female penaus monodon : evidence of sexual dimorphism . aquaculture 125 , 147 - 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