WO1997021835A2

WO1997021835A2 - Dna markers for shrimp selection

Info

Publication number: WO1997021835A2
Application number: PCT/US1996/019568
Authority: WO
Inventors: Joseph C. Bagshaw; Michael A. Buckholt
Original assignee: Worcester Polytechnic Institute
Priority date: 1995-12-12
Filing date: 1996-12-12
Publication date: 1997-06-19
Also published as: WO1997021835A3; AU1684197A

Abstract

Isolated DNA from Penaeus vannamei was digested with restriction enzymes, restriction fragments of genomic DNA inserted into a plasmid vector and screened for recombinant plasmids containing repeated sequences. Ten of the resulting isolates contained representatives of the same repeated element, a satellite sequence present in one or more blocks of tandemly repeated units. The cloned repeat units range in size from about 100 to 200 base pairs, more typically between about 139 to 188 base pairs. Embedded within each cloned repeat unit are 6 to 15 copies of a tandemly repeated pentanucleotide microsatellite. The genome of P. vannamei contains approximately one million copies of this satellite/microsatellite unit. The sequences are useful as markers for the selection of shrimp having a genetically-transmitted favorable growth characteristic, such as increased reproduction, enhanced growth rate, increased size, disease-resistance, and the ability to grow in colder waters, for improved aquacultured shrimp production. Hybridization of the marker to an isolated Peaneus shrimp mucleic acid molecule can be used to identify species, strains or individual shrimp having the desired characteristics. Once identified, these shrimps can be bred to shrimp having the same or an additional desired characteristic to produce a high quality, genetically superior seedstock or larvae useful for the economic production of farmed shrimp.

Description

DNA MARKERS FOR SHRIMP SELECTION

Background of the Invention

The United States government has rights in this invention by virtue of U.S. Department of Agriculture CSRS Grant Nos CSRS88-38808- 3320 to the Gulf Coast Research Laboratory Consortium. This relates generally to the field ot shrimp farming and more particularly relates to the identification ot nucleic acid sequences in shrimp useful as genetic markers.

Shrimp is a very popular seafood, highly favored for human consumption worldwide, especially in Japan and the United States According to consumer surveys, shrimp dominates consumer prelerences in all regions of the United States The preterred species, Penaeus vannamei, lives only in warm water and can, therefore, be harvested in only limited areas of the world, including the southernmost region of the United States Shrimp farming, or aquiculture, is the production of marine shrimp in impoundments, ponds and tanks. The requirement tor a warm water environment has limited shrimp tarming in the United States to a few locations in Florida, South Carolina, Texas and Hawaii with the result that only one hundredth of one percent of the annual U.S shrimp consumption is supplied by these farms Shrimp farms can be established on previously unused land, normally maintain a high yield, and avoid the environmental problems associated with the unintended capture and injury of marine wildlife, such as dolphins and turtles, in the shrimp trolling nets On a typical shrimp tarm, juvenile shrimp are cultured at high densities in shrimp nurseries or small ponds, and, upon maturation, are transferred to a growout operation until they attain sufficient market-size for consumption. Juvenile shrimp are supphed from a shrimp hatchery or are captured from the wild. In a shrimp hatchery, egg-laden females are spawned, and young shrimp are raised through several larval and post-larval stages. Hatcheries normally provide two types of juveniles tor transfer to a nursery nauplu (tiny, newly-hatched larvae) and post-larvae (juveniles that have passed through three larval stages, namely, naup us, zoea, and mysis) The problems associated with shrimp hatcheries include disease and poor water quality A disease-infested or polluted hatchery can cause illness or death of all or most of the larvae, resulting in the abrupt termination ot juvenile shrimp production

Disease represents the biggest obstacle to the future of shrimp farming Farms and hatcheries have tew defenses against protozoa, fungi and bacteπa, and viral infections can be devastating Viral infections have already contributed to shrimp crop failures throughout the world No pharmaceutical products are currently available to treat shrimp viruses When faced with a rampant viral infection, shrimp farmers are often forced to discard the entire crop, drain the ponds or tanks, disinfect the premises, and resupply the tanks with uninfected larvae oi shrimp obtained from the wild or another hatchery

Over the past few years, farmers have developed strains of poultry and livestock that are resistant to disease and exhibit enhanced growth rates or size These strains are obtained by screening and breeding animals for the desired characteristics, thus optimizing production of genetically superior progeny DNA markers have been developed to aid in identification of animals having advantageous characteristics Mapping of factors using Restriction Fragment Length Polymorphisms (RrLPs) IS descπbed by Lander and Bostem, Genetics 121 , 185-199 (1989), DNA fingerprint (DFP) bands in marker-assisted selection programs for chickens is descπbed by Dunnmgton, et al , Poultry Sci 71 , 1251 -1258 (1992) and Plotsky, et al Animal Genetics 24, 105-110 (1993) Shrimp farmers have not yet established strains of shrimp having such improved characteristics, nor developed the DNA markers to identify these shrimp, although Chow and Sandifer have reported that differences in growth, morphometπc traits, and sexual maturity among shrimp can be identified in different commercial hatcheries (Aquaculture 92, 165-178 ( 1991 ) 3

Mitochondrial DNA from several species of Penaeid shrimp have been sequenced by Palumbi and Benzie, Mol Marine Biol 1 27-34 (1991) A comparison of these sequences between morphologically similar species reveals a high degree of genetic divergence Nuclear DNA from Penaeid shrimp has not yet been isolated or subjected to restπction fragment length polymorphism analysis

The development of shrimp exhibiting superior characteristics such as disease resistance would enable shrimp farmers to hatch, grow and market increased quantities of high quality shrimp at a lower cost It is therefore an object of the present invention to provide a method and reagents for selecting shrimp having genetically-transmitted favorable characteristics such as disease resistance, enhanced growth rate, increased size, or the ability to grow in colder waters

Summary of the Invention Isolated DNA from Penaeus vannamei was digested with restriction enzymes, restriction fragments of genomic DNA inserted into a plasmid vector and screened for recombinant plasmids containing repeated sequences Ten ot the resulting isolates contained representatives of the same repeated element (SEQ ID NO 1), a satellite sequence present in one or more blocks ot tandemly repeated units The cloned repeat units range in size from 139 to 188 base pairs Embedded within each cloned repeat unit are 6 to 15 copies of a tandemly repeated pentanucleotide microsatellite (SEQ ID NO 2) The genome of P vannamei contains approximately one million copies of this satel te/microsatellite unit Additional studies demonstrate that the repeated element was not present in other species of shrimp Others studies demonstrate that ribosomal RNA genes and histone genes can be isolated from shrimp and used to demonstrate polymorphisms between individuals

The sequences are useful as markers for the selection ot shrimp having a genetically-transmitted favorable growth characteristic, such as increased reproduction, enhanced growth rate, increased size, disease- resistance, and the ability to grow in colder waters, for improved aquacultured shrimp production Hybridization of the marker to an isolated Penaeus shrimp nucleic acid molecule can be used to identify species, strains or individual shrimp having the desired characteristics Once identified, these shrimp can be bred to shrimp having the same or an additional desired characteristic to produce a high quality, genetically superior seedstock or larvae useful for the economic production of farmed shrimp

Brief Description of the Drawings Figure 1 is an alignment of the sequences of seven different isolates ot the P vannamei satellite The names of the isolates are given on the left, pPV451 and pPV452 are the two copies of the satellite found in plasmid pPV45

Detailed Description of the Invention Repeated or Satellite DNA

Repeated DNA sequences are a common feature of the genomes of all eukaryotes (Brutlag, Annu Rev Genet 14 (1980) 124-144, Singer, Int Rev Cytol 76 (1982) 67-112) Tandemly repeated structures of a few hundred base pairs or less are frequently referred to as satellite DNA, although this term originally derives from the physical separation of such DNA from the rest of the genome by lsopycmc centrifugation (Skinner. Proc Natl Acad Sci USA 58, 103-1 10 (1967) The utility of satellite DNAs as tools for identification ot closely related strains or species is well documented Another category of repeated sequence elements, commonly called microsatelhtes or simply sequence repeats, consist of repeats of one or a few nucleotides (Tautz, Nucleic Acids Res 17 (1989) 6463-6471) Such sequences are valuable for DNA typing because of the occurrence of variable numbers of tandem repeats (VNTRs) (Bentzen et al , "Cloning of hypervanable minisatelhte and simple sequence microsatellite repeats for DNA fingerprinting of important aquacultural species of salmonids and tilapia" In Burke, T . Dolf, G , Jeffreys, A J and Wolff, R (Eds ), DNA Fingerprinting Approaches and Applications Birkhausen Verlag, Basel, Switzerland, 1991 , pp 243- 262), Edwards et ai , Am J Human Genet 49 (1991) 746-756, Goft et al , Genomics 14 (1992) 200-202, Hubert et al , Am J Human Genet 51 (1992) 985-991)

A number of repeated DNA sequences have now been isolated and cloned from the commercially important marine shrimp Penaeus vannamei Five different repeated DNA elements have been sequenced and DNA sequencing has revealed a five-bp sequence (SEQ ID NO 1), variable numbers of which are embedded within or interspersed with a larger repeat, a sequence of 139-188 bp that is repeated in tandem (a) Identification of repeated DNAs in Penaeus vannamei Restriction enzyme digestion of Penaeus vannamei DNA Genomic DNA from post-larvae was digested with various restriction enzymes and the resulting fragments were separated by agarose gel electrophoresis in TAE buffer DNA digested with Aluϊ, Ddel, Ilaelll, Hhal, Hin l, HinPl, Hpaϊl, Mspl, and Sau3 Al was electrophoresed on a 1 % agarose gel at 25 volts for 16 hours DNA digested with Sau Al, Rsal and Hind was electrophoresed on a 1 5 % agarose gel at 25 volts for 16 hours

DNA was isolated from post-larvae of Penaeus vannamei as described by Palumbi and Benzie (1991) with the following modification, as described in PCT/US93/06577 by Worcester Polytech Institute After dialysis against TE buffer (10 mM Tris, pH 8 0, 1 mM EDTA), the DNA solution was adjusted to 0 7 M NaCl, 1 % cetyl trimethylammonium bromide (CTAB) and extracted twice with chloroform The DNA was ethanol precipitated, collected by centrifugation. air dried and dissolved in sterile TE butter The results demonstrated that geonomic DNA isolated from post- larvae of Penaeus vannamei, when digested with a number of restriction enzymes, produced discrete fragments visible in stained agarose gels, indicating the presence of repeated DNA sequences in the genome Of particular interest was a band of approximately 150 base pairs in DNA digested with Sau3Al, Rsal or Hinϊl The latter observation, together with the occasional appearance of a ladder of bands in SauAl digests, suggested that this sequence might be tandemly repeated (b) Cloning and sequencing of the SauAl repeat

In order to clone and isolate this repeated DNA sequence, a plasmid mini-library was constructed by inserting size-selected SauAl fragments of genomic DNA into the plasmid vector pBluescript A plasmid mini-library was then constructed by inserting size-selected SauAl fragments ot genomic DNA into the plasmid vector pBluescript and screened with a probe made by random priming from genomic DNA Because sequences repeated in the genome are abundant in the probe, this screening strategy selects for sequences repeated in the genome, and the signal strength is roughly proportional to the copy number of the hybridizing sequence Fifty of these clones were screened by Southern blot hybridization of plasmid DNA with a genomic DNA probe, which selects tor sequences repeated in the genome Among 37 probe-positive clones, several displayed a stronger signal than the others, and one, pPV45, produced a particularly strong signal, suggesting that it represented a highly repeated sequence The insert size of pPV45. approximately 300 bp, indicated that it was large enough to contain two units of the Sau3Al repeat sequence

A fragment of about 150 base pairs was obtained by digesting genomic DNA from P vannanei with Sau3Al, Rsal or Hinϊl This insert from pPV45 was used as a probe to screen the original 50 clones containing genomic DNA inserts, obtaining nine additional plasmids that cross-hybridized with the insert of pPV45 Restπction digests ol these plasmids suggested some heterogeneity in the length ot the inserts Five of these plasmids, as well as pPV45 , were sequenced and the sequences aligned as shown in Figure 1 The two plasmids shown in Figure 1 , pPV451 and pPV452, are the two copies ot the satellite found in plasmid pPV45 Plasmid DNA was sequenced using the Cyclist DNA Sequencing Kit (Strategene) Alignment and determination of the consensus sequence were performed with the ALIGN Plus program version 2 (Scientific and Educational Software) The gaps or dashes in Figure 1 were introduced to maximize alignment The dots in Figure 1 represent identity with the consensus sequence, SEQ ID NO 1 The length heterogeneity was found to be the result of a variable number ot tandem repeats ot the pentanucleotide sequence CCTAA (SEQ ID NO 1), each embedded within a larger repeated sequence Among the clones isolated and sequenced thus far, the number of internal repetitions of the CCTAA sequence (SEQ ID NO 1) range from six to fifteen and the cloned repeat units range in size from 139 to 188 base pairs The sequences flanking the internal tandem repeats are nearly identical in all clones thus far sequenced, differing from the consensus sequence by only 1 to 5 % A sixth clone contains a truncated version ot the repeat unit (not shown), and may represent a border between a block of repeats and other DNA

This combined satelhte/microsatellite structure, including both the repeated pentanucleotide and the larger repeated sequence, is referred to as PVS1, for Penaeus vannamei satellite number 1 The consensus sequence is SEQ ID NO 1 The description of this structure as a repeated pentanucleotide embedded within a larger repeat is an operational definition It stems from the fact that this strucmre was first observed and subsequently cloned as a Sau3A I fragment Thus the plasmid inserts actually sequenced begin and end at the conserved SauSA I sites, and the pentanucleotide repeats he between these sues The overall structure could equally well be described as a tandemly repeated sequence of 162- 168 bp interspersed with variable numbers of a repeated pentanucleotide (c) Organization of PVS1 in the P. vannamei genome

Southern blots of genomic DNA were probed with plasmid pPV45 DNA was digested with Sau3Al, Rsal. Hinϊl, and Apol DNA was digested with Ddel, Seal and Alul DNA fragments were electrophoresed on 1 5 % agarose geis at 25 volts for 16 hours, blotted onto charged nylon and probed with radiolabelled plasmid pPV45 Signals were detected by exposure to X-ray film

When pPV45 was used as a probe against Southern blots of genomic DNA digested with various enzymes, three different kinds of results were obtained DNA digested with Seal, EcoRI, BamHI, and other enzymes having six base recognition sequences produced only large probe-positive fragments indistinguishable from undigested DNA, as expected of a sequence tandemly repeated in large blocks and lacking sites for these restriction enzymes The enzymes Sau3Al, Rsal, HinϊΛ and Apol each produced a ladder ot probe-positive fragments with a spacing of approximately 150 bp, as expected of a tandemly repeated sequence having these restriction sites in most but not all units Indeed, SauAl, Rsal and Hmϊl have one site each in the repeat unit, and Apol has two closely spaced sites Multimers in the Apol ladder were less abundant than in digests with other enzymes Digestion with Alul and Ddel produced an intermediate pattern with a visible ladder beginning at about 800 bp and extending into a smear above about 2 kb This suggested that sites for these two enzymes were present within PVS1, but less frequently than the Sau3Al site The repeated pentanucleotide sequence, CCTAA (SEQ ID NO 2), differs from the restriction sites of both Alul and Ddel by a smgle base each Thus occasional variants in the embedded VNTR sequence would produce the ladder structure seen in digests with these two enzymes From these results it is clear that the sequence element that has been isolated includes a tandemly repeated satellite sequence, interspersed with a tandemly repeated microsatellite sequence

The copy number of the repeated satellite sequence was estimated as described by He et al , Mol Marine Biol Biotech ] (1992) 125-135 Known amounts of genomic DNA and a clone containing a single repeat unit, pPV16, were slot blotted onto a charged nylon membrane and hybridized for 72 hours with a genomic DNA probe The amount of hybridization was quantitated by liquid scintillation counting The plasmid data were used to construct a standard curve of copy number versus radioactivity, from which the copy number per slot and then the percentage of the genome represented by the repeat were determined. This analysis showed that PVS1 represents 7% of the P vannamei genome, or approximately one million copies per haploid genome (d) Comparison with other known sequences

Microsatellites of more than one base are often found in eukaryotic genomes, but a microsatellite interspersed with a larger tandemly repeated sequence is very rare, and the structure of PVS1 is unique in size and sequence. A sequence complementary to the CCTAA microsatellite in PVS1 has been identified in telomeres of Bombxv mon and other insects by Okazaki et al. , Mol. Cell Biol. 13 (1993) 1424-1432. They found variable numbers ot the pentanucleotide repeat in clones of probable sub- telomeπc regions, but these structures were not themselves tandemly repeated. The crustacean genus A emia has two different satellites that occupy 1 % and 2% of the genome respectively (Cruces et al. , Gene 44 ( 1986) 341-345; Badarro et al. , J. Mol. Evol 32 (1991) 31-36) Compaπson of PVS1 with the sequences of these two Anemia satellites revealed less than 50% sequence identity, thus it is unlikely that they represent a crustacean-specific satellite sequence. (e) Cloning and Sequencing of Additional Repeats.

Genomic DNA isolated from post-larvae of the marine shrimp Penaeus vannamei was digested with the restπction enzyme Sau 3AI Restriction fragments were ligated into the plasmid vector pBluescript, and used to transform competent bacterial cells. Recombinant colonies were screened by hybridization with a genomic DNA probe to identify clones containing repeated DNAs. Plasmid DNAs were then puπfied and sequenced by the chain termination method

The partial sequence of the insert in plasmid ρPV12 is shown in SEQ. ID NO. 3. The complete sequence of the insert in plasmid vPV13 is shown in

SEQ. ID NO. 4 The partial sequence of the insert in plasmid pPV19 is shown in SEQ ID NO 5

The partial sequence ot the insert in plasmid pPV9 is shown in SEQ ID NO 6 The complete sequence of the insert in plasmid pPV49 is shown in

SEQ ID NO 7

In summary, approximately 7% of the genome of Penaeus vannamei consists of tandem repetitions of a sequence element containing one copy of a 162-168 bp sequence and variable numbers of a pentanucleotide sequence The number of separate blocks of this repeated element is unknown, but the total number ot repeat units is approximately one million per hapioid genome This repeated element resembles other satellite and microsatellite sequences in some respects, but is unique in its size and structure (g) Polymorphisms and Detection of Individual Differences.

The following studies were then conducted to demonstrate that one can detect molecular genetic differences between individual shrimp using either cloned genes or sequence data derived from the shrimp The first two studies were analyzed by Southern blots probed with genes cloned from P vannamei, the third is the result of a primer extension assay using DNA sequence data described above

Polymorphism of a repeated DNA sequence in P vannamei DNA was extracted from individual shrimp and digested with the restπction enzyme Sau 3AI to determine the polymorphism of ribosomal RNA genes in Penaeus vannamei Restriction fragments were separated by agarose gel electrophoresis and transterred to a charged nylon membrane (Southern blot) The blot was hybridized with a probe made by random radiolabelling of plasmid pPVr 9 8 After the blot was washed, hybridization was detected by autoradiography Plasmid pPVr 9 8 contains ribosomal RNA eenes cloned lrom P vannamei Polymoφhism of histone genes in P vannamei

DNA was extracted from individual shrimp and digested with the restriction enzyme Sau 3AI to determine the degree of polymorphism of histone genes in Penaeus vannamei Restriction fragments were separated by agarose gel electrophoresis and transferred to a charged nylon membrane (Southern blot) The blot was hybridized with a probe made by random radiolabelling of plasmid pPVh 7 2 After the blot was washed, hybridization was detected by autoradiography Plasmid pPVh 7 2 contains histone genes cloned from P vannamei DNA was extracted from individual shrimp and digested with the restriction enzyme Sau 3AI to determine the degree ot polymoφhism of a repeated DNA sequence in Penaeu vannamei Restriction fragments were denatured by boiling and annealed with a synthetic oligonucleotide The sequence of this oligonucleotide was based on the repeated sequence structure descπbed above The synthetic oligonucleotides were extended by DNA polymerase to the ends of the template strands in the presence of radiolabelled dATP The resulting fragments were separated by acrylamide gel electrophoresis and detected by autoradiography

Comparison of lanes containing DNA from a different individual shrimp shows that different individual shrimp present different DNA "fingeφπnts" Similarly, in Figure 6, comparison of adjacent lanes shows that individual shrimp present different band patterns

These examples demonstrate that a number of individual specific markers can be obtained as described herein, and that these markers can then be used to identity individual shrimps within a single species having desirable phenotypes (h) Detection of Individual Differences.

Selection ot Markers

The results presented above demonstrate that it is possible to determine differences between individual shrimp The sequences are therefore useful for the selection ot shrimp having one or more favorable growth characteπstics In the preferred embodiment, the sequences descπbed above are shown to be associated with shrimp having desirable characteristics Alternatively, where the markers are shown to be physically in juxtaposition with a gene identified with a desirable characteristic, DNA can be extracted from Penaeus shrimp, digested with one or more restriction enzymes for identification and subsequent hybridization with a Penaeus shrimp nucleic acid sequence associated with or comprising a gene or genes conferring the favorable growth characteristic The marker is hybridized to an isolated shrimp nucleic acid molecule, such as DNA or RNA, for detection of the sequence of interest using well known hybridization techniques as described by Sambrook. Fπsch & Maniatis, Molecular Cloning A Laboratory Manual, 2nd Ed. , (Cold Spring Harbor Laboratory, NY 1989) Shrimp species, strains or individual shrimp having a nucleic acid molecule that hybridizes to the marker will have the favorable growth characteristics and can be bred to shrimp having the same or an additional favorable characteristic to produce genetically superior seedstock or larvae useful for the economic production of aquacultured shrimp

Favorable growth characteristics include, but are not limited to, increased reproduction, enhanced growth rate, increased size, disease- resistance, and the ability to grow in colder waters Genes confeirmg favorable growth characteristics include, but are not limited to, homeotic genes, genes encoding transcription factors, genes encoding peptide hormones and their receptors, genes encoding digestive and other metabolic enzymes, genes encoding major strucmral proteins such as collagen, myosin, and actin, and genes encoding components of exosk on and its construction such as chitin synthase

The marker can be either a nucleic acid probe, labelled with a detectable label for detection of the sequence of interest, or a nucleic acid primer specific tor amplification of a nucleic acid sequence or sequences conferring the favorable characteristics Preferably, amplification is achieved by utilizing the poiymerase chain reaction, or variations thereof, in combination with two pπmers that hybridize to nucleic acid sequences flanking the sequence to be amplified tor subsequent detection

The term "shrimp" is defined herein as shrimp eggs, shrimp larvae, shrimp post-larvae and adult shrimp The shrimp are preterably Penaeus shrimp and include the species Penaeus vannamei, Penaeus chinensis, Penaeus monodon, Penaeus sty lirostris, Penaeus japonicus, Penaeus penitillatus, Penaeus merguiensis, Penaeus indicus, Penaeus subtilis, Penaeus paulensis, Penaeus setiferus, Penaeus brasiliensis, Penaeus duorarum, Penaeus occidentalis, Penaeus schmitu, Penaeus cahforniensis, Penaeus semisulcatus, Penaeus latisulcatus Metapenaeus monoceros. Metapenaeus dobsoni, Metapenaeus affinis, and Metapenaeus brivicornis

The term "marker" is defined herein as a nucleic acid sequence (DNA or RNA) that hybridizes to a genetically similar nucleic acid sequence (DNA or RNA) under standard hybridization conditions and includes probes and primers Standard hybridization conditions aie defined herein as hybridization at a temperamre approximately 20-40°C or more below the melting temperamre ot a perfectly base-paired double stranded DNA molecule The melting temperature of a double stranded DNA molecule can be determined by methods well known to those skilled

The nucleic acid sequence marker is prepared from the genomic or cDNA library or by digestion of isolated high molecular weight shrimp DNA with one or more restriction enzymes into fragments approximately 15,000 bases or less as described above The marker is preferably 20 bases in length or longer and can be a nucleic acid probe specific for a particular gene, specific tor a restriction fragment length polymoφhism, specific tor variable number tandem repeats, specific for dispersed repeated DNA sequences, or the probe can be a specific for a gene or gene sequence flanking a gene In the preferred embodiment, the markers are SEQ ID Nos 1 or 2, or sequences sharing substantial sequence identity thereto, as shown in Figure 1 The sequence ot an isolated DNA fragment can be determined by the dideoxy chain termination method of Sanger et al , Proc Natl Acad Sci USA 74 5463-5467 (1977), using a Sequenase™ kit (U S Biochemical Coφ Cleveland, OH) according to the manufacturer s instructions or by other methods known to those skilled in the art

Specified polynucleotide markers can be synthesized either in a biological system or in a chemical reaction in vitro in accordance with methods well known to those skilled in the art Biological systems include both prokaryotic organisms such as bacteria and eukaryotic organisms such as yeast, isolated cells in culture, germ line cells in multicellular organisms, somatic tissue cells in multicellular organisms, or plant cells

Isolated fragments or synthetic nucleic acid sequences capable ol hybridization to isolated shrimp nucleic acid sequences associated with favorable growth characteristics can be labelled and used as a probe to select shrimp having the desired characteristics

Preferred fragments are derived from regions ot the Penaeus genome that contain the genes that confer desirable growth characteristic. on the species, strain or individual shrimp Fragments derived from regions exhibiting restriction fragment length polymoφhisms. variable number tandem repeats, and dispersed repeats, as described above should exhibit enhanced specificity for the desired characteristics and be useful as probes

Preparation of Labelled Probes tor Markers

The probes may be labelled with an atom or inorganic radical, most preferably using radionucleotides, such as ³²P, 'H, ^l4C, ^3SS ^12<iI, ^πιI, or heavy metals A ³²P label can be incoφorated into the sequence of the probe by nick-translation, end-labelling or incoφoration of a labelled nucleotide A ³H, ¹⁴C or ³⁵S label can be incoφorated into the sequence of the probe by incoφoration of a labelled precursor or by chemical modification An ^I2SI or ¹³¹I label can be incoφorated into the sequence of the probe by chemical modification Detection ot a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography

The label can also be a Mass or Nuclear Magnetic Resonance (NMR) label such as, for example, ⁿC, ^!<iN, or ^I90 Detection ot such a label can be by Mass Spectrometry or NMR

Preferably the label is attached to the probe by chemical conjugation Any label may be used that provides an adequate signal and has a sufficiently long half-life Other preferred labels include dyes, ligands, fluorescers, chemilummescers, enzymes, antibodies and similar compounds For example, biotin can be bound to the probe and detected by binding an avidin-conjugated enzyme or streptavidin conjugated enzyme to the biotin followed by washing to remove non-specifically bound enzyme Upon addition of an appropriate substrate for the enzyme, the substrate is converted to a colored or chemiluminescent product that can be detected Examples of such enzymes include alkaline phosphatase and horseradish peroxidase as descπbed by Renz et al , Nuc Acids Res 12 3435-3444 (1984) Examples of dyes include ethidium bromide, actidines, propidium and other intercalating dyes, and 4' ,6'-dιamιdιno-2-phenylιndole (DAPI)(Sιgma Chemical Company, St Louis, MO) or other proprietary nucleic acid stains Examples ot fluorogens include fluorescein and derivatives, phycoerythπn, allo-phycocyanin, phycocyanin, rhodamine, Texas Red or other proprietary fluorogens The fluorogens are generally attached by chemical modification The dye labels can be detected by a spectrophotometer and the fluorogens can be detected by a fluorescence detector

Recognition sites for enzymes, such as restriction enzyme sites, can also be incoφorated into the probes to provide a detectable label A label can also be made by incoφorating any modified base or precursor containing any label, incoφoration ot a modified base containing a chemical group recognizable by specific antibodies, or by detecting any bound antibody complex by various means including immunofluorescence or lmmuno-enzymatic reactions Such labels can be detected using enzyme-linked immunoassays (ELISA) or by detecting a color change with the aid of a spectrophotometer

The labelled probe can be hybridized to DNA or mRNA in cells in intact tissues or a sample containing tresh or frozen shrimp cells Hybridization can be in vitro or in situ The method of in situ hybridization is described by Haase, A , et al "Detection of viral nucleic acids by in situ hybridization", In- Methods in Virology (Eds K Maramorosch & H Koprowski) Vol 7, pp 189-226, Academic Press, New York, 1984, and Haase, A.T , et al , "Analysis of viral infections by in situ hybridization" , In In situ Hybridization-Applications to Neurobwlogy (Eds K Valentine, J Roberts & J Barchas), pp 197-219, Oxford University Press [Symposium Monograph! , Tairlawn, NJ, 1986

The method tor determining the presence and quantity of a specific label depends on the label employed Such methods are well known to those skilled in the art

It will be understood by those skilled in the art that other labels can also be used A review of nucleic acid labels can be found in the article by Landegren, et al , "DNA Diagnostics-Molecular Techniques and Automation" , Science, 242 229-237 (1988)

Methods for Amplification

The polymerase chain reaction (PCR) technique, described in U S Patent Nos 4,683, 195 and 4,683,202 to Mullis, the teachings ol which are incoφorated herein by reference, can be used to amplify a specific sequence form vanishingly small samples of DNA or RNA to produce easily visualized DNA fragments of characteristic size PCR, using either specifically defined or random primers, can therefore be used to identify species, strains and individual shrimp and to investigate population polymoφhisms PCR technology is described in PCR Protocols A Guide to

Methods and Applications by Michael A Innis, David H Geltand, John J Smnsky and Thomas J White, pp 39-45 and 337-385 (Academic Press, Inc . Harcourt Brace Jovanovich, Publishers, 1990) PCR technology is also descπbed by Marx, J L , Science 140 1408-1410 ( 1988) and in U S Patent Nos 4.683, 195 and 4,683,202, to Mullis, the teachings of which are also incoφorated herein by reference PCR using one primer is descπbed by Loh, E Y , et al . Sc ience

243 217 (1989) This technique is often used with cDNA (DNA derived from messenger RNA by reverse transcriptase) There are also asymmetric PCR systems and other methods that use one primer or vast excess of one primer These methods generate mostly single-stranded DNA, suitable for direct sequencing Single pπmers can also be used with random hexamers (a degenerate mixmre ot all or most of the possible DNA hexamers) so that at least one hexamer will act as a second primer by hybridizing somewhere along the sequence at a distance from the first primer PCR technology requires pairs of dissimilar DNA oligonucleotides

(short fragments of DNA sequence) which act as pπmers to initiate a controlled polymerase reaction which, in turn, amplifies the genomic sequence that lies between the two oligonucleotide binding sites The polymerase chain reaction employs a heat-stable polymerase (the Taq polymerase) which permits repeated heating and cooling of the leaction mixmre The amplification process is initiated by first heating the reaction mixmre to denature (dissociate) the two complementary strands of the double stranded DNA to be amplified Upon cooling, each single-stranded DNA oligonucleotide hybridizes to a specific region of one or the other ot the complementary DNA strands, and acts as a primer for the heat-stable polymerase The polymerase uses the oligonucleotide primers as starting points for the elongation of a DNA molecule complementary to the template DNA molecule to which each primer is hybridized Each of the elongating DNA chains grows towards and beyond the distal primer site of the other template strand By the end ot the first cycle two double stranded copies of the intervening genomic sequence lying between the primer binding sites are generated The cycle is repeated manyfold, exponentially doubling the number of copies each time In this fashion even a single copy of a specific DNA sequence can be amplified to detectable levels in a relatively short period of time

The polymerase chain reaction primer selection is limited by three factors First, the two oligonucleotides must be complementary to sequences found in the template DNA in order for the oligonucleotides to hybridize to the template DNA Without this initial hybridization step there would be no primer available for the DNA polymerase to use to initiate elongation, and no copy of the DNA sequence could be made Second, the primers should hybridize to discrete and unique regions of the template DNA If the primers hybridize to multiple different sites in the template sequence then the initiation site for elongation, and the DNA copy produced, would vary from cycle to cycle depending upon to which binding site the primer hybridized Third, the two primer binding sites must not be too distant from one another The elongation step optimally produces fragments up to approximately 2500 bases in length, and DNA sequences of greater length are amplified less efficiently or not at all If chain elongation terminates before the distal primer site is incoφorated into the sequence, the resultant incomplete DNA molecule will not participate in subsequent rounds of amplification

Examples ot other applicable amplification systems that currently exist or are being developed include PCR in situ, ligase amplification reaction (LAR), ligase hybridization, Qβ bacteriophage rephcase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS) and nucleic acid sequence-based amplification (NASBA)

PCR in situ is the use of PCR amplification on cells or tissue sections followed by detection using in situ hybridization This technique is described by Haase, A T , et al , "Amplification and detection of lentiviral DNA inside cells", Proc Natl Acad Sa (USA) 87 4971-4975 (July 1990) Ligase amplification reaction is described by Wu, D.Y and Wallace, R.B, Genomics 4:560-569 (1989) and Barπnger, K.J.. et al. . Gene 89: 117-122 (1990). Ligase hybridization is described by Landegren, U. , et ai . Science 241 : 1077-1080 (1988). The Qβ bacteriophage replicase system is described by Kramer,

F.R. and Lizardi, P.M. , "Replicatable RNA reporters" , Nature 339:401-402 (1989); Lizardi, P.M. , et al. , "Exponential amplification of recombinant-RNA hybridization probes" , Bio/Technology 6: 1197-1202 (1988); Lomeli, H. , et al. . "Quantitative assays based on the use of replicatable hybridization probes", Clin. Chem. 35: 1826-1831 (1989); and Chu, B.C.F, et al. , Nucl. Acids Res. 14:5591-5603 (1986).

TAS is described by Kwoh, D.Y. , et ai , Proc. Natl. Acad. Sci. USA 86: 1173-1177 (1989). GAWTS is described by Stotlet, E.S. , et al. . Science 239:491-494 (1988). NASBA is described by Compton, J. , Nature 350:91-92 (1991).

Methods for Detection and Analysis

Detection and analysis of the nucleotide fragments, amplified by one of the methods described above, are accomplished by standard methods including, for example, gel electrophoresis, dot blots, slot blots and colorimetry, as described in standard laboratory textbooks such as Sambrook, Frisch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed. , (Cold Spring Harbor Laboratory, NY 1989).

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT: WORCESTER POLYTECHNIC INSTITUTE (ii) TITLE OF INVENTION: DNA Markers for Shrimp Selection

(m) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Patrea L Pabst

(B) STREET: 2800 One Atlantic Center

1201 West Peachtree Street

(C) CITY: Atlanta

(D) STATE: Georgia

(E) COUNTRY: USA

(F) ZIP: 30306-3450

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE- Patentin Release #1 0, Version #1.25

(vi) CURRENT APPLICATION DATA

(A) APPLICATION NUMBER:

(B) FILING DATE.

(C) CLASSIFICATION.

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/570,751

(B) FILING DATE: 12 December 1995

(C) CLASSIFICATION:

(vm) ATTORNEY/AGENT INFORMATION:

(A) NAME: Pabst , Patrea L.

(B) REGISTRATION NUMBER: 31,284

(C) REFERENCE/DOCKET NUMBER: WPI102CIP2

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (404)873-8794

(B) TELEFAX: (404)873-8795

(2) INFORMATION FOR SEQ ID NO: 1 ^•

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 185 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. double

(D) TOPOLOGY: linear (n) MOLECULE TYPE: genomic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1.

GATCAAGTTT ATGTGAGAAA ACGAAATTTG GAGTCTCCTG GTTAAATTTT CGAGTACTAA 60

CCTAACCTAA CCTAACCTAA CC^TAACCTAA CCTAACCT? CCTA. 'CTAA CCAACGTAAC 120

CTAACCTAAC CTCCCCAAAT T JAGGTACT GTAACCTATC GTGG TAGA TGTGATTTAC 180

CGATC 185

(2) INFORMATION FOR SEQ ID NO: 2.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 base pairs

(B) TYPE: nucleic ac d

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

CCTAA 5

(2) INFORMATION FOR SEQ ID NO: 3 : d) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 448 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. double

(D) TOPOLOGY: linear (i ) MOLECULE TYPE: genomic

( i) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

GATCTTTTTA GTAGGTATCC AGTTATAATG CATCTAGCCC TTTTTCTCTA TTTATGTTTA 60

TAATTGTATA TTTCTGCATA TATGTGGCTT CTATTACCAT ATGTTTCTAT TTAGACACTT 120

CTGGTTAAAG AAAGCCACTG TCACATAAAC AAACATAATT TCAGATATGC TTTACTTTAA 180

CATAAGAAAA TGATAACATT TCTCTTAAAA TGGACTCACA CATGCACGCA TATGTATCGC 240

GTGCGCACAC ACACACACAC ACACACACAC ACACACACAC ACACACACAC ACACACACAC 300

ACACACACAC ACACACACAC ACACACACAC ACACACACAC ACACACACAC ACACACACAC 360

ACACACACAC ACACACACAC ACACACACTA CACACACACA CACACACACG GAAGAATATA 420

ACTCTTGAAT AGGTTTTGAT ATGTACTC 448

(2) INFORMATION FOR SEQ ID NO:4.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 90 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (n) MOLECULE TYPE: genomic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4.

GATCGCGGAA AAAACAAACA AAAAACTAAT TATCTCGACA CCAACAACAC CCCGACAGCC 60

TCTTCTTCTG GCCTCCGCCG AGCTAGGATC 90

(2) INFORMATION FOR SEQ ID NO: 5 :

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 205 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 :

GATCCGAGCG GGTCACTAGT TCTAGAGCGG CCGCGCACCG TGAGCTCCAA TTCGCCTATA 60

GTAGTCGTAT GTACAATTGC AGCTGTCCGT GTTGTACAAC GTCGTACTGG AAAACCTGCG 120

TTGACCAACT TGAATCGCTG CAGCACATCC CCTTTCGCAC TGCTAATAGC GAGAGCCTAC 180

GATCGCTTCC AACAGTTGCG CAGCA 205

(2) INFORMATION FOR SEQ ID NO: 6 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 497 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :

GATCGGAATG ACAGAAATGG ACACTAGGTT ACTAATTCAT TGAGAAAAAG AGAAAGAGTC 60

GATATATTTT AATTTATTCG TTGGTCGATT CTGGATATTG GTAAAGAGAT TTACTGTCTG 120

TCCTTTCATC TCTATCACAA GAAACAAATG GATTAACTGT GTAACACGCA TTGCAGTTAT 180

CATGAAAAAA TAGTTTCTCT GTGATTATTA TTATTAGTTG TTCGTTATAA TAATTATCCT 240

CATTAAGCAT CGATCAGTCA GTCAGTAGCG ATCAGCCATG CATCGACCAG TCACGATCAG 300

TTAGTCTAGT CATCGATCTG GTTAGTCAGT CACCGATCAG TCATCACATC ATTATGCTAT 360

CATCATCTAT TATCATCAGC ATCACCATTA TAATTGTGCA TCATCAAAAA CGTGACTAAA 420

GAGACGTAGG AATCGAAGAG TTTGACTAAT AAGAAATACG ATAGGATTAT TAAATGCGTT 480

ACATCCGTTC CTAACTC 497

(2) INFORMATION FOR SEQ ID NO: :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 124 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 :

GATCCGAGCC CCCTGAGTAG CCACATCCTA CAGGTAATCG CAGCTCATCC TTCCCTCACA 60

CACCGGCGTA CACAGCACAC ATGCCCCGCC CTCACTGCCC TCATAATTTG CACGCCCGGG 120

GATC 124

Claims

We claim

1 A marker for the selection of Penaeus shrimp having a predetermined genetically-transmitted characteristic comprising a repeating nucleotide repeat present in the genome of a marine shrimp in the Paenaeus genus

2 The marker of claim 1 selected from the group consisting of SEQ ID No 1 , 2, 3, 4, 5, 6 and sequences hybridizing thereto which are present as repeats in the genome of a marine shrimp in the Paenaeus genus

3 The marker of claim 1 wherein the marker is labelled with a detectable label

4 The marker ot claim 1 wherein the marker is a radioactively labelled probe

5 The marker ot claim 1 wherein the marker is a primer tor amplification of a portion of the nuclear nucleic acid molecule

6 The marker of claim 1 wherein the shrimp are a species of shrimp selected from the group consisting of Penaeus vannamei, Penaeus chinensis, Penaeus monodon, Penaeus styhrostris, Penaeus japomcus, Penaeus penicillatus, Penaeus merguiensis, Penaeus indicus, Penaeus subtilis, Penaeus paulensis, Penaeus setiferus, Penaeus brasdiensis, Penaeus duorarum, Penaeus occidentals, Penaeus schmitti, Penaeus calif orniensis, Penaeus semisulcatus, Penaeus latisulcatus, Metapenaeus monoceros, Metapenaeus dobsoni, Metapenaeus affinis, and Metapenaeus bnvicornis

7 The marker of claim 6 wherein the shrimp are ot the species Penaeus vannamei

8 The marker of claim 1 comprising an isolated Penaeus shrimp nucleic acid sequence having a length of 20 bases or greater

9 The marker of claim 1 wherein the sequence is between about 100 and 200 bases in length 10 The marker of claim 1 wherein the sequence is associated with or encodes a protein conferring a favorable characteristic on the shrimp

1 1 The marker of claim 1 wherein the sequence hybridizes to nucleic acid sequence of Penaeus shrimp selected from the group consisting of an internal transcribed spacer region of ribosomal RNA genes, a tandem repeat, a dispersed repeat, a restriction fragment length polymoφhism, a gene encoding homeostasis, a gene encoding a transcription factor, a gene encoding a peptide hormone, a gene encoding a peptide hormone receptor, a gene encoding a metabolic enzyme, a gene encoding a structural protein, and a gene encoding a component of exoskeleton

12 A method for characterizing shrimp comprising identifying the presence and patterns of a marker for the selection of Penaeus shrimp having a predetermined genetically-transmitted characteristic comprising a repeating nucleotide repeat present in the genome of a marine shrimp in the Paenaeus genus.

13 The method of claim 12 wherein the marker is selected from the group consisting of SEQ ID No 1 , 2, 3, 4, 5, 6 and sequences hybridizing thereto which are present as repeats in the genome ot a marine shrimp in the Paenaeus genus