Sigrid A. Lehnert

Genetic mapping of the black tiger shrimp Penaeus monodon with amplified fragment length polymorphism

Abstract We report construction of an initial genetic linkage map for the black tiger shrimp, Penaeus monodon. Mapping was carried out using polymorphic markers derived from 23 Amplified Fragment Length Polymorphism (AFLP) primer pairs. These were analysed on three reference families of known pedigree. A total of 673 polymorphic AFLP loci that conformed to expected Mendelian segregation ratios were scored in three families, and these were used to construct separate male and female linkage maps for each family. AFLP markers that consisted of a segregating fragment of the same size, amplified with the same primer pair in two or more of the reference families, were considered to be common markers. 116 such common AFLP markers were used to construct a common linkage map across the three families. This linkage map has 20 linkage groups covering a total genetic distance of 1412 cM. Future directions for genetic mapping in P. monodon are discussed in light of these initial data.

Genetic mapping of the kuruma prawn Penaeus japonicus using AFLP markers

Abstract Amplified fragment length polymorphism (AFLP) analysis is a rapid, efficient technique for detecting large numbers of DNA markers for linkage analysis. We have used AFLP markers in a two-way pseudo-testcross strategy to generate genetic maps of a Penaeus japonicus family. A two-stage selective genetic mapping strategy was applied in this study. The initial stage involved the linkage mapping on 46 progeny from two tails (top and bottom 6%) of the size distribution of an intermediate cross family (F1 from HH×LL and F2 from HL×LH) using 54 pairs of AFLP primer combinations. The second stage of linkage mapping involved genotyping an additional 56 progeny (top and bottom 8%) of the same family with the same 54 primer combinations based on the framework map from the first stage. Of 535 polymorphic fragments scored in 46 progeny of the first stage, 355 segregated in a 1:1 ratio, corresponding to DNA polymorphisms heterozygous in one parent and null in the other. The other 154 markers followed a 3:1 Mendelian segregation ratio. Of 502 bands scored in 56 progeny of the second stage, 359 segregated in a 1:1 ratio and 138 in a 3:1 ratio. When the markers with a 1:1 segregating ratio were combined from both stages (401 in total), 217 markers were ordered into 43 linkage groups (1780 cM) of the paternal map and 125 markers in 31 linkage groups (1026 cM) of the maternal map. The average density of markers was approximately 1 per 10 cM. To investigate the homologies between two parental maps, we included 182 markers segregating 3:1 in the analysis. One homologous linkage group was recognised. The sex marker initially mapped on the maternal parent map was also confirmed in the second-stage mapping with more progeny information. The linkage data developed in these maps will be used to detect loci controlling commercially important traits.

Isolation of a cDNA encoding a putative cellulase in the red claw crayfish Cherax quadricarinatus.

Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1, 4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.

Tissue-Specific Expressed Sequence Tags from the Black Tiger Shrimp Penaeus monodon

Abstract: Expressed sequence tag data were generated from complementary DNA libraries created from cephalothorax, eyestalk, and pleopod tissue of the black tiger shrimp (Penaeus monodon). Significant database matches were found for 48 of 83 nuclear genes sequenced from the cephalothorax library, 22 of 55 nuclear genes from the eyestalk library, and 6 of 13 nuclear genes from the pleopod library. The putative identities of these genes reflected the expected tissue specificity. For example, genes for digestive enzymes were identified from the cephalothorax library and genes involved in the visual and neuroendocrine system from the eyestalk library. A few sequences matched anonymous EST or genomic sequences, and others contained mini-satellite or microsatellite repeat sequences. The remainder, 31 from the cephalothorax library, 25 from the eyestalk library, and 5 from the pleopod library, were sequences of high nucleotide complexity with no matches in any database searched and thus may represent novel genes.

The Penaeus monodon Chitinase 1 Gene Is Differentially Expressed in the Hepatopancreas During the Molt Cycle

Abstract: We have isolated a full-length chitinase complementary DNA from the tiger shrimp Penaeus monodon that encodes a 621 amino acid protein possessing the functional domains of the chitinase protein family. The Penaeus monodon chitinase 1 (PmChi-1) gene product is 81.8% identical to a chitinase 1 protein expressed in the hepatopancreas of Penaeus japonicus. Analysis by reverse transcription–polymerase chain reaction (RT-PCR) indicates that PmChi-1 messenger RNA is detectable in the hepatopancreas and the gut. PmChi-1 expression during the molt cycle fluctuates markedly, with lowest mRNA levels at stages A1, C, and D3; there is a dramatic increase in transcript abundance at the D2 stage. Using the same tissues and molt stages, RT-PCR analyses of genes encoding other digestive enzymes (trypsin, chymotrypsin, and cathepsin L), a muscle structural protein (tropomyosin II), and housekeeping proteins (elongation factor II and GTP-binding protein) indicate that PmChi-1 is expressed in a distinct tissue-specific and stage-specific manner. The other digestive enzyme genes are expressed in a similar spatiotemporal pattern, but none exhibited a dramatic increase in transcript abundance at stage D2. Increased expression of PmChi-1 at D2 suggests that hepatopancreas-expressed chitinase is involved in the degradation of endogenous chitin in the gut peritrophic membrane prior to molting.

Prospecting major genes in dairy buffaloes

BackgroundAsian buffaloes (Bubalus bubalis) have an important socio-economic role. The majority of the population is situated in developing countries. Due to the scarce resources in these countries, very few species-specific biotechnology tools exist and a lot of cattle-derived technologies are applied to buffaloes. However, the application of cattle genomic tools to buffaloes is not straightforward and, as results suggested, despite genome sequences similarity the genetic polymorphisms are different.ResultsThe first SNP chip genotyping platform designed specifically for buffaloes has recently become available. Herein, a genome-wide association study (GWAS) and gene network analysis carried out in buffaloes is presented. Target phenotypes were six milk production and four reproductive traits. GWAS identified SNP with significant associations and suggested candidate genes that were specific to each trait and also genes with pleiotropic effect, associated to multiple traits.ConclusionsNetwork predictions of interactions between these candidate genes may guide further molecular analyses in search of disruptive mutations, help select genes for functional experiments and evidence metabolism differences in comparison to cattle. The cattle SNP chip does not offer an optimal coverage of buffalo genome, thereafter the development of new buffalo-specific genetic technologies is warranted. An annotated reference genome would greatly facilitate genetic research, with potential impact to buffalo-based dairy production.