FRESHWATER PRAWN MACROBRACHIUM ROSENBERGII

Field of Invention

The invention relates to a method for identifying parentage in Macrobrachium rosenbergii. More particularly, the invention relates to a method of assigning parents to respective progeny by comparing the amplicon sizes of expressed sequence tag- simple sequence repeat (EST-SSR) loci for each allele.

Background of The Invention

Macrobrachium rosenbergii or commonly known as giant freshwater prawn is ranked as the sixth largest aquaculture species in Asia based on volume. It is one of the commercially important food sources and has been cultured in many countries where M. rosenbergii is not indigenous. For the past fifteen years, M. rosenbergii has been the focus of the aquaculture study upon realizing its commercial value. An extensive study has been conducted in order to improve the genetic trait of M. rosenbergii in hopes of ensuring long term sustainability, improving disease resistance, increasing genetic gain rate and lowering cost of production of M. rosenbergii.

Genealogical information of brood stock is essential in selective breeding program and genetic improvement program. Physical tags have been conventionally used to track the genealogy of cultured individuals and access their growth and health status. However, the application of physical tags on prawns is labor-intensive and has caused the tagged individuals to become unmarketable. Therefore, the necessity to develop markers that are suitable for aquaculture genetic improvement program is increasing in recent days. Advances in molecular biology provide new tools for genetic research. Molecular markers are developed as substitution for physical tags and commonly used to identify individuals of interest. Examples of molecular markers include expressed sequence tag (EST) and simple sequence repeat (SSR). The China Patent No. 101921850 (A) claims a process of screening largemouth black bass parent with high hatchability using a molecular marker derived from a recessive lethal deletion mutant locus flanking sequence on the promoter sequence of Growth Hormone Releasing Hormone gene. This screening method allows for differentiation between two genotypes of largemouth black bass according to the number of DNA bands formed on agarose gel after electrophoresis. However, the molecular marker disclosed in China Patent No. 101921850 (A) is not used in parentage identification. Instead, it is used to identify and remove incompetent largemouth black bass from the brood stock in order to produce offspring with better genetic traits.

The use of polymorphic simple sequence repeat (SSR) or microsatellite in parentage identification was disclosed by Selvamani et al. in 2001. The study revealed the efficiency of using microsatellite markers in parentage identification whereby three or fewer microsatellite loci are sufficient to identify the parentage of all offspring. However, the microsatellite markers used in the mentioned study were developed from genome instead of expressed sequence tag (EST). SSR derived from EST (EST- SSR marker), as compared to genomic SSR marker, incurs lower cost of development and has lower frequency of null alleles.

EST-SSR serves as a transferable molecular marker whereby EST provides gene identification and SSR expresses high level of polymorphism. EST-SSR marker has several advantages such as wide distribution in genome, high level of polymorphism, locus-specific co-dominant inheritance, transferability across related species, and repeatability and clarity of scoring; these advantages enable EST-SSR marker to be applied in various ways to improve conventional breeding of aquaculture. Furthermore, EST-SSR marker is useful in assigning progeny to respective family, given the parental genotypes are known. Mohanty et al. (2012) disclosed the development of a set of twenty-three EST-SSR markers for M. rosenbergii that could be used in genetic studies of that species. The study revealed that majority of the respective EST-SSR loci had successfully amplified in closely related species such as M. malcolmsonii, M. gangeticum and M. lamarrei. Nevertheless, the study did not disclose the use of these markers in parentage identification in M. rosenbergii.

It is desirable to invent a method of identifying parentage in M. rosenbergii using molecular markers that are highly conserved and multi-allelic. Conserved molecular markers lower the frequency of null alleles that rise from mutation at targeted site. Number of alleles for each targeted locus is useful in identifying the parentage in M. rosenbergii. Summary of The Invention

One of the objects of the invention is to provide a method of amplifying the nucleic acid sequences of a set of EST-SSR loci of Macrobrachium rosenbergii that are conserved, and thus are stable and applicable to all individuals of the species and/or the closely related species of Macrobrachium rosenbergii, and to provide a method of identifying the parentage in Macrobrachium rosenbergii by comparing the amplicon sizes of each allele from respective set of locus obtained for parental and progeny.

Another object of the invention is to provide a method of detecting the amplicons by stimulating the fluorochrome of the forward primer incorporated in the amplicons during the process of nucleic acid amplification through polymerase chain reaction to emit light using laser beam.

Still another object of the invention is to provide a method of separating amplicons using capillary electrophoresis that is able to separate the amplicons according to size whereby the separated amplicons are subsequently sized and genotyped. Capillary electrophoresis saves time, requires smaller amount of sample and can be automated.

Yet another object of the invention is to provide a method of assigning parentage based on a likelihood-based statistical approach in which the probability of the candidate paternal and maternal parents to be the true parents are assessed by looking at the percentage of similarity in allele frequency and genotype of amplicons in parental and progeny. At least one of the preceding objects is met, in whole or in part, by the invention, in which the embodiment of the invention describes a method of identifying parentage in Macrobrachium rosenbergii prawn comprising the steps of amplifying targeted EST- SSR nucleic acid sequences from a biological sample of Macrobrachium rosenbergii through a polymerase chain reaction using primer pairs with nucleic acid sequences as set forth in SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, SEQ ID NO. 5 ans SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12 to produce respective amplicons; determining fragment size of the produced amplicons; regarding the fragment size of the amplicons corresponding to predetermined alleles and identifying parentage of the prawn based on the regarded allele.

In a preferred embodiment of the invention, the primers of nucleic acid sequences SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 11 are fluorescently-labeled at the 5' end. The amplicons produced through polymerase chain reaction are denatured prior to separation. Preferably, the amplicons are separated by capillary electrophoresis and then sized and genotyped.

In the preferred embodiment of the invention, parentage of Macrobrachium rosenbergii prawn is identified using a likelihood-based statistical approach. The fragment size and genotype of the amplicons obtained from set of paternal, maternal and progeny are required to conduct the identification step. This likelihood-based statistical approach further comprising a combination of allele frequency analysis, simulation of parentage analysis and parentage analysis (with sexes known). With the aid of the innovative EST-SSR primer sets, the parentage identification in Macrobrachium rosenbergii prawn can be conducted with higher efficiency as the frequency of null alleles that rise from mutation is minimized by using polymorphic molecular markers that has high non-exclusion probability value. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention. Brief Description Of The Drawings

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

Figure 1 shows the exemplary results of allele frequency analysis for the set of targeted EST-SSR loci: the available number of alleles presents in each locus (K), heterozygosity observed (H _0bs ), heterozygosity expected

(H _ex p) and Polymorphic Information Content (PIC). For illustration purpose, the exclusion probability exhibited by this set of EST-SSR loci equals to 0.97565, the remainder after deducting combined non- exclusion probability (second parent) from 1. Figure 2 shows the exemplary results of simulation of parentage analysis that is performed based on the allele frequency calculated: the critical Delta value that serves as criterion for further parentage analysis. Figure 3 shows the exemplary results of parentage analysis: five

Macrobrachium rosenbergii progeny whose parentage have been successfully identified.

Detailed Description of The Invention

The invention relates to a method for identifying parentage in Macrobrachium rosenbergii. More particularly, the invention relates to a method of assigning parents to respective progeny by comparing the amplicon sizes of expressed sequence tag- simple sequence repeat (EST-SSR) loci for each allele.

Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

The invention discloses a method of identifying parentage in Macrobrachium rosenbergii prawn comprising the steps of amplifying targeted EST-SSR nucleic acid sequences from a biological sample of Macrobrachium rosenbergii through a polymerase chain reaction using primer pairs with nucleic acid sequences as set forth in SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12 to produce respective amplicons; determining fragment size of the produced amplicons; regarding the fragment size of the amplicons corresponding to predetermined alleles and identifying parentage of the prawn based on the regarded allele.

According to the preferred embodiment of the invention, the targeted EST-SSR nucleic acid sequences are polymorphic simple sequence repeat (SSR) derived from the expressed sequence tag (EST). The targeted EST-SSR loci are amplified from Macrobrachium rosenbergii genomic DNA. In the preferred embodiment, genomic DNA is extracted using tissue DNA extraction and modified CTAB method. However, it can also be extracted using other methods including but not limiting to phenol/chloroform extraction, anion exchange chromatography, cesium chloride density gradient centrifugation, silica-gel column-based DNA extraction and magnetic bead-based genomic DNA extraction.

In the preferred embodiment, six primer sets are used to amplify six EST-SSR loci. Each primer set flanks a particular EST-SSR locus of Macrobrachium rosenbergii and consists of a forward primer and a reverse primer that hybridizes to the antisense strand and sense strand of the corresponding EST-SSR locus, respectively. The 5' end of the forward primers is labeled with fluorochrome, preferably 6-FAM ^® , HEX ^® and NED ^® . The forward primers can be labeled with fluorochromes including but not limiting to JOE™, TET™, TAMRA™, Cy3 ^® , Cy5 ^® , Cy5.5 ^® , Cal Fluor® Gold 540, Cal Fluor Orange 560, Cal Fluor Red 590, Quasar ^® 570, Quasar 670, ROX™, and Texas Red ^® .

The first set of primers containing two primers of nucleic acid sequences SEQ ID NO. 1 and SEQ ID NO. 2. SEQ ID NO. 1 is TCATGTCTCTTGAGTCTTTC, is the forward primer. SEQ ID NO. 2 is GTTATCTGATCGTCACAGTT, is the reverse primer.

The second set of primers containing two primers of nucleic acid sequences SEQ ID NO. 3 and SEQ ID NO. 4. SEQ ID NO. 3 is CTATTGCCAGGCCAAAAA, is the forward primer. SEQ ID NO. 4 is TACCACCAAACTCCACTAC, is the reverse

The third set of primers containing two primers of nucleic acid sequences SEQ ID NO. 5 and SEQ ID NO. 6. SEQ ID NO. 5 is CCAGTGATTGAAATCGTC, is the forward primer. SEQ ID NO. 6 is AAGAAGAGGCCACGACAG, is the reverse primer.

The fourth set of primers containing two primers of nucleic acid sequences SEQ ID NO. 7 and SEQ ID NO. 8. SEQ ID NO. 7 is CTTGTGCAAGAGATTATCC, is the forward primer. SEQ ID NO. 8 is GGTGATGCCTTTGTTATAC, is the reverse primer.

The fifth set of primers containing two primers of nucleic acid sequences SEQ ID NO. 9 and SEQ ID NO. 10. SEQ ID NO. 9 is GGGAAAAGCGACACATATAA, is the forward primer. SEQ ID NO. 10 is ACTAGTAGCTGCTCTTTGTG, is the reverse primer.

The sixth set of primers containing two primers of nucleic acid sequences SEQ ID NO. 11 and SEQ ID NO. 12. SEQ ID NO. 11 is GAGCATGACATTGTGAAGA, is the forward primer. SEQ ID NO. 12 is GAGTAAAGTGCCCAGGAC, is the reverse primer.

Polymerase chain reaction amplifies EST-SSR loci flanked by their respective sets of forward and reverse primers. The amplification reaction is performed in a volume of 10-20 uL containing 1.5-1.7 mM MgC , 10 μΜ dNTPs, 5-7 pmoles of each primer, 0.5-1.0 unit of Taq DNA polymerase, and 1-2 uL of genomic DNA. The DNA amplification is performed with the following thermocycling conditions: 5 minute initial denaturation at 94 °C, followed by 40 cycles of 30-40 second denaturation at 94 °C, 30-40 second annealing at 50-60 °C and 30-40 second extension at 72 °C, followed by a 7-10 minute final extension at 72 °C.

In the preferred embodiment, amplicons are amplified nucleic acid sequences of EST- SSR loci. The EST-SSR loci each has two alleles whose sizes may or may not be identical. The amplicons are regarded to corresponding alleles of each locus based on their fragment sizes. The allele fragment sizes for each locus of the offspring is then compared to those of all the candidate paternal and maternal parents. The higher the similarity between the offspring to a candidate maternal and/or paternal parent, the higher the likelihood that the candidate maternal and/or paternal parent is/are the true parent(s).

Fragment size determination can be conducted using variable techniques such as agarose gel electrophoresis and capillary electrophoresis. Preferably, fragment size determination is performed by passing the amplicons through a capillary electrophoresis. The amplicons are denatured prior to passing through the capillary electrophoresis. In the preferred embodiment, the amplicons are denatured using HiDi Formamide ^® and heat. Other denaturing agents including but not limiting to urea, hydrochloric acid (HCl) and sodium hydroxide (NaOH). In the preferred embodiment, capillary electrophoresis is used to separate the denatured amplicons. The capillary electrophoresis separates the denatured amplicons by sizes whereby the smaller amplicons reaches the detector near the outlet earlier than larger amplicons. It detects the fluorochrome in the forward primer by detecting the light emitted by the fluorochrome after stimulated by a laser beam at a particular wavelength. The intensity of the fluorescence emitted is recorded as a function of time and wavelength. The genotypes and sizes of the separated amplicons are preferably determined using GeneMapper ^® v.4.0. A fluorescently-labeled size standard is used as a fragment size reference. This size standard should be labeled with a fluorochrome that is different from that in the forward primer. Preferably, the parentage identification step is conducted using a likelihood-based statistical approach. The preferred computer program to perform this approach is CERVUS Version 2.0. The likelihood-based statistical approach further comprising a combination of allele frequency analysis, simulation of parentage analysis and parentage analysis. In simulation of parentage analysis, the rate of genotyping error genotyping error is default to 0.01 or 1% on the basis that laboratory accuracy rarely exceeds 99%. Other computer programs that can be used in parentage analysis including but not limiting to COLONY, FaMoz, KinlnFor, MER and PARENTE.

As described in the foregoing description, the set of primers can be used to identify parentage in Macrobrachium rosenbergii and/or its closely related species. The highly polymorphic EST-SSR loci are conserved and applicable on all individuals of the Macrobrachium rosenbergii species and probably individuals of the closely related Macrobrachium species. The present disclosure includes as contained in the appended claims, as well as that of the foregoing description. Although this invention has been described in its preferred form with a degree of particularity, it is understood that the present disclosure of the preferred form has been made only by way of example and that numerous changes in the details of construction and the combination and arrangements of parts may be resorted to without departing from the scope of the invention.

Example

An example is provided below to illustrate different aspects and embodiments of the invention. The example is not intended in any way to limit the disclosed invention, which is limited only by the claims.

Example 1 The genomic DNA of the parental breeding family is extracted from pleopods using tissue DNA extraction method whereas the genomic DNA of the progeny is extracted from larvae using modified CTAB method. The concentration of the extracted DNA is estimated by comparing band intensity with lambda DNA digests of known concentrations under UV light, after running a 0.8% (w/v) agarose gel electrophoresis using the extracted DNA and the lambda DNA digests and staining the agarose gel with ethidium bromide.

After the concentration of the DNA samples are optimized to a workable range, the EST-SSR loci in the DNA samples are amplified using polymerase chain reaction (PCR). The sequences of primers used in the PCR reaction are shown in Table 1. Each forward primer is fluorescently-labeled with 6-FAM ^® , HEX ^® and NED ^® . The amplification reaction is performed in a volume of 10 uL containing 1.5 mM MgCl ₂ , 10 uM dNTPs, 5 pmoles of each primer, 1 unit of Taq DNA polymerase, and 2 uL of genomic DNA. The DNA amplification is performed with the following thermocycling conditions: 5 minute initial denaturation at 94 °C, followed by 40 cycles of 40 second denaturation at 94 °C, 40 second annealing at 50-60 °C and 40 second extension at 72 °C, followed by a 7-minute final extension at 72 °C. The annealing temperature (T _m ) for each primer set is shown in Table 1. The amplicons are size fractionated by electrophoresis in 2% agarose gels stained with ethidium bromide and the gel is then visualized under UV light.

Table 1

Primer Locus Forward Sequence Reverse Sequence PIC MgCh T„ Set Name Value (°C)

1 MR4 SEQ ID NO. 1 : SEQ ID NO. 2: 0.777 1.0 53.9

TCATGTCTCTTGAGTCTTTC GTTATCTGATCGTCACAGTT

2 MR6 SEQ ID NO. 3: SEQ ID NO. 4: 0.615 1.0 58.0

CTATTGCCAGGCCAAAAA TACCACCAAACTCCACTAC

3 MR8 SEQ ID NO. 5: SEQ ID NO. 6: 0.638 1.0 57.3

CCAGTGATTGAAATCGTC AAGAAGAGGCCACGACAG

4 MR20 SEQ ID NO. 7: SEQ ID NO. 8: 0.622 1.5 55.3

CTTGTGCAAGAGATTATCC GGTGATGCCTTTGTTATAC

5 MR29 SEQ ID NO. 9: SEQ ID NO. 10: 0.534 1.5 55.3 GGGAAAAGCGACACATATAA ACTAGTAGCTGCTCTTTGTG

6 MR39 SEQ ID NO. 11: SEQ ID NO. 12: 0.610 1.2 51.4

GAGCATGACATTGTGAAGA GAGTAAAGTGCCCAGGAC

The amplicons are diluted 1 :10 in sterile water. 1 uL of diluted amplicons and 9 uL of a mixture of HiDi Formamide ^® and ROX™ 400HD ^® size standard are loaded to 96-well PCR plate. The samples are denatured at 95 °C for 2 minutes and immediately placed on ice. The plates are kept covered during processing as the standards and primers are light-sensitive. The processed amplicons are then sized and genotyped using ABI 3730 Genetic Analyzer equipped with GeneMapper ^® v.4.0 software. The amplicon sizes are analyzed from the sizing curve generated by the size standard. Table 2 and Table 3 show the fragment sizes of two alleles of each targeted EST-SSR locus of paternal and maternal Macrobrachium rosenbergii prawns, respectively. On the other hand, Table 4 shows the fragment sizes of two alleles of each targeted EST- SSR locus of Macrobrachium rosenberii progeny. The allele sizes obtained using respective set of primers for both the parental and progeny are then analyzed using CERVUS for the purpose of parentage assignment.

Table 2

Population ID MR4A R4B MR6A MR6B MR8A MR8B MR20A MR20B MR29A MR29B MR39A MR39B

Popl Fl 311 311 197 201 274 280 244 247 197 209 200 200

Popl F2 311 311 197 201 274 277 247 250 200 209 191 200

Popl F3 309 311 189 197 271 271 235 247 200 209 162 200

Popl F4 307 311 197 201 280 280 247 250 200 209 148 200

Popl F5 317 327 197 201 265 274 235 247 200 212 162 200

Popl F6 295 311 197 201 262 265 247 247 200 209 200 213

Popl F7 313 315 189 197 280 283 235 235 209 209 162 200

Popl F8 307 311 197 201 271 280 247 247 200 209 200 200 Table 3

Population ID MR4A MR4B MR6A MR6B MR8A MR8B MR20A MR20B MR29A MR29B MR39A MR39B

Popl

Ml 311 313 197 280 280 201 235 247 209 209 162 200

Popl

M2 327 327 197 271 274 201 235 247 209 212 148 148

Popl

M3 309 311 197 277 280 201 244 247 197 197 162 200

Popl

M4 311 313 197 271 271 201 235 247 209 212 162 200

Popl

M5 311 313 183 271 280 197 244 250 212 212 162 200

Popl

M6 309 311 201 280 280 205 226 232 200 212 162 200

Popl

M7 313 327 197 280 280 205 244 247 209 209 200 200

Popl

M8 309 311 197 271 271 201 235 247 209 212 162 200

Table 4

Population ED MR4A MR4B MR6A MR6B MR8A MR8B MR20A MR20B MR29A MR29B MR39A MR39B

Popl PI 315 317 197 201 274 280 235 247 209 212 200 215

Popl P2 315 317 197 201 280 280 235 247 209 212 148 200

Popl P3 309 311 189 201 280 280 235 247 209 212 200 215

Popl P4 307 311 197 201 277 280 244 250 209 212 200 215

Popl P5 309 311 193 201 280 280 232 250 209 212 200 215

Popl P6 309 311 197 201 280 280 247 247 209 212 149 200

Popl P7 309 311 201 205 0 0 238 238 209 212 200 215

Popl P8 317 317 197 201 280 280 238 247 209 212 200 215

Popl P9 309 311 197 201 280 280 235 247 209 212 149 200

Popl P10 309 311 197 205 280 280 235 247 209 212 149 200

Popl Pl l 311 313 197 201 271 280 235 247 200 212 149 200

Popl P12 311 313 197 201 271 280 247 247 209 212 149 200

Popl P13 311 313 197 201 262 280 235 247 209 212 149 200

Popl P14 307 311 201 205 277 280 0 0 200 212 149 200

Popl P15 295 311 197 201 262 280 226 247 209 212 149 200

Popl P16 295 311 193 197 262 280 235 250 209 212 149 200

Popl P17 313 327 197 201 262 280 235 247 209 212 200 215

Popl P18 309 311 197 201 277 280 226 247 209 212 149 200

Popl P19 309 311 197 201 265 280 235 247 209 212 200 215

Popl P20 309 311 185 201 271 280 235 247 209 209 149 200

Popl P21 311 327 197 201 265 280 235 247 209 212 200 215

Popl P22 311 327 197 201 262 280 244 244 209 212 149 200

Popl P23 313 313 197 201 280 280 229 247 209 212 200 215

Popl P24 309 315 185 197 277 280 247 247 209 212 200 200

Popl P25 311 313 197 201 280 283 235 247 209 212 200 215

Popl P26 313 315 197 201 280 280 235 247 209 212 191 200 Popl P27 311 313 197 201 262 280 235 247 209 212 149 200

Popl P28 311 313 197 201 265 280 235 247 209 212 200 215

Popl P29 309 311 185 189 280 280 235 247 209 212 149 200

Popl P30 313 327 197 201 262 280 244 244 209 221 200 215

Popl P31 315 317 201 205 280 280 235 235 209 212 200 215

Popl P32 309 311 201 205 280 280 235 247 209 212 200 215

Popl P33 311 317 201 205 277 280 235 247 209 212 200 215

Popl P34 311 327 197 201 262 280 0 0 209 212 191 200

Popl P35 311 311 197 201 280 280 235 247 209 212 200 215

Popl P36 311 313 185 201 0 0 235 247 200 212 149 200

Popl P37 309 311 197 201 274 280 235 247 197 209 200 215

Popl P38 311 311 197 201 265 280 244 244 209 212 200 215

Popl P39 315 317 197 201 280 280 0 0 209 212 200 215

Popl P40 313 315 197 201 280 280 247 247 209 212 191 200

Popl P41 313 327 197 201 262 280 235 247 209 212 200 215

Popl P42 307 311 197 205 277 280 235 247 209 212 200 215

Popl P43 309 311 197 201 265 280 235 247 209 212 200 215

Popl P44 309 311 197 201 274 280 235 247 197 209 191 200

Popl P45 313 327 197 201 262 280 0 0 209 212 191 200

Popl P46 311 317 197 201 280 280 229 247 209 212 200 215

Popl P47 309 311 197 201 277 280 250 250 197 212 200 215

Popl P48 311 313 197 201 271 277 247 250 0 0 149 200

Popl P49 309 311 197 201 280 280 235 247 209 212 149 200

Popl P50 309 311 197 201 280 280 0 0 209 212 200 215

Popl P51 307 311 185 189 277 280 247 250 200 212 200 215

Popl P52 315 317 197 201 271 280 235 247 200 209 149 200

Popl P53 307 307 197 201 265 277 235 247 209 212 191 200

Popl P54 311 317 197 205 280 280 0 0 209 212 200 215

Popl P55 311 311 201 205 280 280 235 247 209 212 149 200

Popl P56 309 311 201 205 280 280 229 247 209 212 200 215

Popl P57 315 317 197 201 274 280 235 247 209 209 200 215

Popl P58 315 317 197 201 271 280 235 247 209 212 149 200

Popl P59 307 311 201 205 280 280 244 250 200 212 200 215

Popl P60 313 327 197 201 265 280 235 247 209 212 200 215

Popl P61 311 317 197 201 280 280 235 247 209 212 191 200

Popl P62 309 311 201 205 277 280 235 235 209 221 200 215

Popl P63 315 317 193 201 280 280 247 250 209 212 200 215

Popl P64 309 311 201 205 280 280 235 247 209 212 200 215

Popl P65 311 317 201 205 280 280 235 247 209 212 200 215

Popl P66 309 311 0 0 280 280 235 250 200 212 200 215

Popl P67 309 311 193 197 280 280 247 250 209 212 200 215

Popl P68 309 311 197 201 280 280 232 250 200 221 200 215 Popl P69 309 311 201 205 280 280 235 247 209 212 191 200

Popl P70 311 313 197 205 280 283 232 247 209 212 191 200

Popl P71 309 311 201 205 280 280 235 247 209 212 200 215

Popl P72 315 317 197 205 274 280 247 247 197 209 200 215

Popl P73 309 311 201 205 277 280 244 250 209 209 200 215

Popl P74 307 311 193 201 271 277 247 250 200 209 149 200

Popl P75 311 317 197 201 280 280 235 247 209 212 200 215

Popl P76 311 327 197 205 262 280 235 247 209 212 149 200

Popl P77 307 307 197 201 280 280 235 247 209 212 191 200

Popl P78 311 317 197 205 280 280 0 0 209 212 191 200

Popl P79 315 317 193 201 280 280 226 247 209 212 200 215

Popl P80 315 317 197 201 280 280 235 247 209 212 200 215

Popl P81 307 311 197 201 271 280 235 247 212 212 149 200

Popl P82 315 317 197 201 280 280 235 247 209 212 200 215

Popl P83 307 307 197 201 277 280 226 229 200 200 191 200

Popl P84 311 311 197 201 283 286 235 247 0 0 200 215

Popl P85 317 313 197 201 271 280 247 250 200 212 200 215

Popl P86 317 317 197 201 274 280 244 265 197 209 191 200

Popl P87 309 311 197 205 280 280 235 247 209 212 200 215

Popl P88 309 311 197 205 280 280 226 247 209 212 200 215

Popl P89 317 319 197 201 271 280 235 250 197 209 200 215

Popl P90 311 327 197 201 262 280 235 247 209 212 191 200

Popl P91 309 311 201 205 277 280 244 247 200 212 149 200

Popl P92 309 311 197 201 280 280 235 247 209 212 200 215

Popl P93 315 317 197 201 280 280 244 244 209 212 200 215

Popl P94 307 311 197 201 277 280 244 247 209 227 200 215

Popl P95 315 317 197 201 280 280 244 247 209 212 200 215

Popl P96 311 311 197 201 262 280 235 247 0 0 200 215

Popl Bl 309 311 197 201 0 0 235 247 209 212 148 200

Popl B2 0 0 189 197 0 0 0 0 209 212 148 200

Popl B3 317 319 197 197 0 0 235 247 212 212 148 200

Popl B4 311 313 197 197 0 0 235 247 209 212 148 200

Popl B5 0 0 189 197 271 280 235 247 209 209 200 200

Popl B6 311 319 185 197 271 277 235 247 209 212 148 191

Popl B7 285 311 197 201 274 280 235 247 209 209 200 213

Popl B8 313 319 197 205 271 280 244 250 206 212 148 200

Popl B9 311 313 189 197 271 280 235 247 212 212 148 200

Popl BIO 311 319 189 197 277 280 235 247 212 212 148 200

Popl B l l 311 313 197 201 271 277 235 247 212 212 148 200

Popl B12 0 0 197 201 0 0 250 250 212 212 200 200

Popl B13 317 319 197 201 0 0 235 247 212 212 148 200

Popl B14 319 319 197 201 0 0 247 247 209 212 200 200 Popl B15 317 313 197 201 0 0 226 229 212 212 200 200

Po l B16 313 313 197 201 0 0 247 250 212 212 200 200

Popl B 17 315 319 189 197 271 277 235 247 212 212 162 200

Popl B18 317 319 189 197 271 277 235 247 212 212 148 200

Popl B19 311 313 189 197 271 277 235 247 209 212 162 200

Popl B20 311 315 197 201 274 280 235 247 209 209 191 200

Popl Al 309 311 197 201 277 280 247 247 212 212 200 200

Popl A2 309 311 185 201 277 280 237 247 209 212 148 200

Popl A3 0 0 197 201 280 280 247 247 212 212 200 200

Popl A4 0 0 197 201 0 0 247 247 209 212 200 200

Popl A5 0 0 193 201 0 0 0 0 0 0 200 200

Popl A6 0 0 197 201 0 0 0 0 0 0 200 200

Popl A7 311 317 197 201 0 0 0 0 0 0 200 200

Popl A8 311 313 197 201 0 0 247 247 209 212 200 200

Popl A9 311 313 197 201 262 280 247 247 212 212 200 200

Popl A10 311 317 197 201 274 280 247 247 209 209 191 200

Popl All 311 311 197 201 274 280 0 0 209 212 200 200

Popl A12 317 317 197 205 280 280 247 247 209 212 191 200

Popl A13 309 311 197 201 262 280 247 247 209 212 200 200

Popl A14 311 313 197 201 280 280 247 247 0 0 200 200

Popl A15 309 311 197 201 280 283 247 247 209 212 200 200

Popl A16 311 311 197 201 280 280 247 247 209 212 200 200

Popl A17 311 311 197 201 0 0 0 0 209 212 191 200

Popl A18 315 317 197 201 0 0 247 247 209 212 191 200

Popl A19 311 311 197 201 280 280 247 247 209 212 200 200

Popl A20 311 311 197 201 274 280 235 247 209 209 148 200

Popl 2AB1 315 319 197 201 274 280 235 247 209 212 200 213

Popl 2AB2 309 311 197 201 271 274 235 247 206 209 200 213

Popl 2AB3 311 319 189 197 277 280 235 247 209 212 148 200

Popl 2AB4 317 319 189 197 277 280 235 247 209 212 148 200

Popl 2AB5 311 315 189 197 277 280 235 247 209 212 200 213

Po l 2AB6 311 311 189 197 271 274 0 0 209 212 148 200

Popl 2AB7 315 319 189 197 271 277 247 247 209 212 148 200

Popl 2AB8 311 315 197 201 271 277 235 247 206 209 148 200

Popl 2AB9 311 319 189 197 277 280 235 247 209 212 148 200

Popl 2AB10 317 319 189 197 271 277 235 247 209 212 148 200

Popl 2AB11 315 319 197 201 277 280 235 247 209 212 148 200

Popl 2AB12 313 319 197 201 271 280 235 247 212 212 200 213

Popl 2AB 13 311 315 189 197 277 280 229 247 206 212 148 200

Popl 2AB 14 313 319 197 201 271 280 244 247 206 212 191 200

Popl 2AB 15 285 311 189 197 277 280 226 247 209 209 191 200

Popl 2AB16 311 315 189 197 274 280 235 247 206 209 200 213 Popl 2AB17 311 315 189 197 277 280 247 247 206 212 148 200

Popl 2AB 18 317 319 197 201 274 280 235 247 206 209 200 213

Popl 2AB 19 311 315 189 197 274 280 235 247 209 212 200 213

Popl 2AB20 315 319 197 201 277 280 235 242 209 209 148 200

Popl 2AB21 313 315 197 201 262 265 247 250 212 212 148 200

Po l 2AB22 317 319 189 197 0 0 247 247 212 212 200 200

Popl 2AB23 313 313 189 197 0 0 226 247 209 212 148 200

Popl 2AB24 311 311 197 201 271 271 235 247 209 209 191 200

Popl 2AB25 311 315 197 201 262 283 235 247 212 212 191 200

Po l 2AB26 317 319 197 201 271 277 235 247 206 209 200 213

Popl 2AB27 311 313 197 201 271 277 238 250 212 212 148 200

Popl 2AB28 311 315 197 201 262 265 247 250 212 212 148 200

Popl 2AB29 311 313 197 201 271 280 247 250 212 212 200 200

Popl 2AB30 311 313 197 201 280 283 229 247 197 212 191 200

Popl 2AB31 311 313 197 201 271 277 247 250 0 0 200 213

Popl 2AB32 311 315 197 201 265 280 235 247 0 0 200 200

Popl 2AB33 313 315 197 201 280 283 235 247 209 212 162 200

Popl 2AB34 311 315 197 201 271 280 235 247 200 212 200 200

Popl 2AB3 ₅ 311 313 197 201 262 265 238 250 200 212 191 200

Popl 2AB36 0 0 189 197 271 280 247 250 200 212 200 213

Popl 2AB37 315 319 189 197 271 274 235 247 209 212 200 200

Popl 2AB38 307 313 189 197 271 271 235 247 212 212 148 200

Popl 2AB39 311 313 197 201 262 265 232 250 212 212 148 200

Popl 2AB40 311 313 189 197 277 280 235 247 209 212 191 200

The genotypes recorded are then used to determine the Hardy- Weinberg equilibrium using a computer program GENEPOP. Following that, a computer program CERVUS Version 2.0 is used to assess null allele frequency and practicability of the EST-SSR loci as tools to identify parentage in Macrobrachium rosenbergii. Input of genotypes of the both the parental and progeny are required. A null allele frequency greater than 5% is deemed significant. Allele frequency analysis, simulation of parentage analysis and final parentage analysis are performed using CERVUS. The rate of genotyping error used in simulation of parentage analysis is 0.01 or 1%.

Figure 1 shows the results of allele frequency analysis. The allele frequency (κ) calculated and the number of parental Macrobrachium rosenbergii prawns are then used for simulation of parentage analysis. Figure 2 shows the results of simulation of parentage analysis. The critical Delta values serve as critical value for this level of confidence for further parentage analysis wherein any candidate parent with a Delta score exceeding the critical value with 95% confidence is assigned to be true parent pair. Figure 3 shows the results of parentage analysis. Five pairs of parental Macrobrachium rosenbergii prawns are successfully assigned to their progenies when using a critical Delta value of 1.01. The Macrobrachium rosenbergii progenies whose parentage are successfully identified are shown in Table 5 together with the identities of their respective parents.

Table 5. Identities of parental prawns of filial Macrobrachium rosenbergii prawns whose parentage are successfully identified.