Avner Cnaani

Breeding new strains of tilapia : development of an artificial center of origin and linkage map based on AFLP and microsatellite loci

Based on ideas from plant breeding and the opportunities offered by molecular biology, a program was initiated in 1995 to derive genetically superior tilapia from a synthetic stock .artificial center of origin, ACO produced by inter-crossing five groups of fish: Oreochromis w . . x

Detection of a chromosomal region with two quantitative trait loci, affecting cold tolerance and fish size, in an F2 tilapia hybrid

Abstract We searched for genetic linkage between microsatellite DNA markers and quantitative trait loci (QTL) for cold tolerance and fish size (body weight and standard length) in two unrelated F 2 families of interspecific tilapia hybrids ( Oreochromis mossambicus × Oreochromis aureus ). The first experiment was based on a family of 60 fish scanned for 20 microsatellites. A second experiment was conducted with a family of 114 fish scanned for 6 microsatellites in one linkage group, in order to test for QTL found in the first experiment. This two-step experimental design was used in order to protect against “false positive” associations. In both families, significant associations were found for two loci within the same linkage group. The two QTL, near UNH879 for cold tolerance, and near UNH130 for body size, were estimated to be 22 cM distant from each other, with no interaction found between the two traits. One of these loci, UNH879 , was also associated with sex determination. Distortion from the expected Mendelian genotypic ratio was observed for three markers: UNH130 , UNH180 and UNH907 , suggesting linkage with a QTL affecting survival. These results identify a chromosomal region in the tilapia genome harboring several QTL affecting fitness traits.

Amh and Dmrta2 Genes Map to Tilapia (Oreochromis spp.) Linkage Group 23 Within Quantitative Trait Locus Regions for Sex Determination

Recent studies have revealed that the major genes of the mammalian sex determination pathway are also involved in sex determination of fish. Several studies have reported QTL in various species and strains of tilapia, regions contributing to sex determination have been identified on linkage groups 1, 3, and 23. Genes contributing to sex-specific mortality have been detected on linkage groups 2, 6, and 23. To test whether the same genes might control sex determination in mammals and fishes, we mapped 11 genes that are considered putative master key regulators of sex determination: Amh, Cyp19, Dax1, Dmrt2, Dmrta2, Fhl3l, Foxl2, Ixl, Lhx9, Sf1, and Sox8. We identified polymorphisms in noncoding regions of these genes and genotyped these sites for 90 individuals of an F2 mapping family. Mapping of Dax1 joined LG16 and LG21 into a single linkage group. The Amh and Dmrta2 genes were mapped to two distinct regions of LG23. The Amh gene was mapped 5 cM from UNH879 within a QTL region for sex determination and 2 cM from UNH216 within a QTL region for sex-specific mortality. Dmrta2 was mapped 4 cM from UNH848 within another QTL region for sex determination. Cyp19 was mapped to LG1 far from a previously reported QTL region for sex determination on this chromosome. Seven other candidate genes mapped to LG4, -11, -12, -14, and -17.

Detection of genes with deleterious alleles in an inbred line of tilapia (Oreochromis aureus)

Abstract An increase in the viability of gynogenetic progeny has been recorded in Bar Ilan University (BIU) by following four successive generations of meiogynogenetic Oreochromis aureus . This augmentation is suggested to be the result of reduction in the number of deleterious alleles. The genome of progeny of the fourth successive generation of meiogynogenetic O. aureus was screened with 76 microsatellite DNA markers to identify polymorphic loci. Twenty polymorphic markers were tested for segregation distortion in four crosses of informative progeny to identify linkage to genes with recessive deleterious alleles. Significant distortion ( P UNH159 , UNH216 and UNH231 . A significant deleterious effect ( P UNH159 and UNH216 with significant epistatic interaction ( P ≤0.0001) between the two loci. In family 4, the onset was shown to occur by day 4 in early spawns and at a much later developmental stage in late spawns. The differences detected in marker effect among crosses and between spawns of the same cross indicate that complex mechanisms are involved. DNA markers linked to genes with deleterious alleles can aid in identification and elimination of those alleles from inbred lines of O. aureus .

Fine-mapping of a locus on linkage group 23 for sex determination in Nile tilapia (Oreochromis niloticus)

Genetic markers in tilapia species associated with loci affecting sex determination (SD), sex-specific mortality or both were mapped to linkage groups (LG) 1, 2, 3, 6 and 23. The objective of this study was to use these markers to fine-map the locus with the greatest effect on SD in Oreochromis niloticus. Our parental stock, full-sibs of Nile tilapia (Swansea origin), were divided into three groups: (i) untreated, (ii) feminized by diethylstilbestrol and (iii) masculinized by 17α-methyltestosterone. We analysed the first group for association of microsatellite markers representing these five LGs. The strongest association with gender was found on LG23 for marker UNH898 (χ(2) ; P=8.6×10(-5) ). Allele 276 was found almost exclusively in males, and we hypothesized that this allele is a male-associated allele (MAA). Sex-reversed individuals were used for mating experiments with and without the segregating MAA. Mating of individuals lacking the MAA resulted in all-female progeny. Mating of two heterozygotes for MAA gave rise to 81 males and 30 females. Analysis of association between gender and genotypes identified the MAA in 98.6% of males as opposed to 8.0% of females (χ(2) ; P=2.5×10(-18) ). Eight markers that flank UNH898 were genotyped to map the locus on LG23 within a confidence interval of 16-21 cM. Mating of homozygous individuals for MAA is underway for production of all-male populations.

Sex-linked markers and microsatellite locus duplication in the cichlid species Oreochromis tanganicae

Cichlid species of the genus Oreochromis vary in their genetic sex-determination systems. In this study, we used microsatellite DNA markers to characterize the sex-determination system in Oreochromis tanganicae. Markers on linkage group 3 were associated with phenotypic sex, with an inheritance pattern typical of a female heterogametic species (WZ–ZZ). Further, locus duplication was observed for two separate microsatellite markers on the sex chromosome. These results further advance our understanding of the rapidly evolving sex-determination systems among these closely related tilapia species.

Intestinal transcriptome analysis revealed differential salinity adaptation between two tilapiine species.

Tilapias are a group of freshwater species, which vary in their ability to adapt to high salinity water. Osmotic regulation in fish is conducted mainly in the gills, kidney, and gastrointestinal tract (GIT). The mechanisms involved in ion and water transport through the GIT is not well-characterized, with only a few described complexes. Comparing the transcriptome of the anterior and posterior intestinal sections of a freshwater and saltwater adapted fish by deep-sequencing, we examined the salinity adaptation of two tilapia species: the high salinity-tolerant Oreochromis mossambicus (Mozambique tilapia), and the less salinity-tolerant Oreochromis niloticus (Nile tilapia). This comparative analysis revealed high similarity in gene expression response to salinity change between species in the posterior intestine and large differences in the anterior intestine. Furthermore, in the anterior intestine 68 genes were saltwater up-regulated in one species and down-regulated in the other species (47 genes up-regulated in O. niloticus and down-regulated in O. mossambicus, with 21 genes showing the reverse pattern). Gene ontology (GO) analysis showed a high proportion of transporter and ion channel function among these genes. The results of this study point to a group of genes that differed in their salinity-dependent regulation pattern in the anterior intestine as potentially having a role in the differential salinity tolerance of these two closely related species.

Effects of salinity and prolactin on gene transcript levels of ion transporters, ion pumps and prolactin receptors in Mozambique tilapia intestine.

Euryhaline teleosts are faced with significant challenges during changes in salinity. Osmoregulatory responses to salinity changes are mediated through the neuroendocrine system which directs osmoregulatory tissues to modulate ion transport. Prolactin (PRL) plays a major role in freshwater (FW) osmoregulation by promoting ion uptake in osmoregulatory tissues, including intestine. We measured mRNA expression of ion pumps, Na(+)/K(+)-ATPase α3-subunit (NKAα3) and vacuolar type H(+)-ATPase A-subunit (V-ATPase A-subunit); ion transporters/channels, Na(+)/K(+)/2Cl(-) co-transporter (NKCC2) and cystic fibrosis transmembrane conductance regulator (CFTR); and the two PRL receptors, PRLR1 and PRLR2 in eleven intestinal segments of Mozambique tilapia (Oreochromis mossambicus) acclimated to FW or seawater (SW). Gene expression levels of NKAα3, V-ATPase A-subunit, and NKCC2 were generally lower in middle segments of the intestine, whereas CFTR mRNA was most highly expressed in anterior intestine of FW-fish. In both FW- and SW-acclimated fish, PRLR1 was most highly expressed in the terminal segment of the intestine, whereas PRLR2 was generally most highly expressed in anterior intestinal segments. While NKCC2, NKAα3 and PRLR2 mRNA expression was higher in the intestinal segments of SW-acclimated fish, CFTR mRNA expression was higher in FW-fish; PRLR1 and V-ATPase A-subunit mRNA expression was similar between FW- and SW-acclimated fish. Next, we characterized the effects of hypophysectomy (Hx) and PRL replacement on the expression of intestinal transcripts. Hypophysectomy reduced both NKCC2 and CFTR expression in particular intestinal segments; however, only NKCC2 expression was restored by PRL replacement. Together, these findings describe how both acclimation salinity and PRL impact transcript levels of effectors of ion transport in tilapia intestine.

Salinity-Dependent Shift in the Localization of Three Peptide Transporters along the Intestine of the Mozambique Tilapia (Oreochromis mossambicus)

The peptide transporter (PepT) systems are well-known for their importance to protein absorption in all vertebrate species. These symporters use H+ gradient at the apical membrane of the intestinal epithelial cells to mediate the absorption of small peptides. In fish, the intestine is a multifunctional organ, involved in osmoregulation, acid-base regulation, and nutrient absorption. Therefore, we expected environmental stimuli to affect peptide absorption. We examined the effect of three environmental factors; salinity, pH and feeding, on the expression, activity and localization of three PepT transporters (PepT1a, PepT1b, PepT2) along the intestine of the Mozambique tilapia (Oreochromis mossambicus). Quantitative real time PCR (qPCR) analysis demonstrated that the two PepT1 variants are typical to the proximal intestinal section while PepT2 is typical to the distal intestinal sections. Immunofluorescence analysis with custom-made antibodies supported the qPCR results, localized both transporters on the apical membrane of enterocytes and provided the first evidence for the participation of PepT2 in nutrient absorption. This first description of segment-specific expression and localization points to a complementary role of the different peptide transporters, corresponding to the changes in nutrient availability along the intestine. Both gene expression and absorption activity assays showed that an increase in water salinity shifted the localization of the PepT genes transcription and activity down along the intestinal tract. Additionally, an unexpected pH effect was found on the absorption of small peptides, with increased activity at higher pH levels. This work emphasizes the relationships between different functions of the fish intestine and how they are affected by environmental conditions.

Effects of the acclimation to high salinity on intestinal ion and peptide transporters in two tilapia species that differ in their salinity tolerance

Tilapiine species, widely distributed across habitats with diverse water salinities, are important to aquaculture as well as a laboratory model. The effects of water salinity on two tilapia species, that differ in their salinity tolerance, was evaluated. Oreochromis niloticus reared in brackish-water, showed a significant decrease in growth and feed efficiency, whereas O. mossambicus reared in seawater did not show any significant changes. The expression and activity of Na+/K+-ATPase (NKA), V-type H+-ATPase (VHA) and carbonic anhydrase (CA), as well as expression levels of genes encoding two HCO3- and three peptide transporters (nbc1, slc26a6, slc15a1a, slc15a1b and slc15a2) were measured in three intestinal sections of these two species, grown in freshwater and brackish/sea-water. Overall, the spatial distribution along the intestine of the genes examined in this study was similar between the two species, with the exception of tcaIV. The salinity response, on the other hand, varied greatly between these species. In O. mossambicus, there was a salinity-dependent increased expression of most of the examined genes (except slc26a6 and slc15a2), while in O. niloticus the expression of most genes did not change, or even decreased (tcaIV, nbc1 and slc15a1b). This study highlighted differences in the intestinal response to salinity acclimation between closely- related species that differ in their salinity tolerance. O. mossambicus, which has a high salinity tolerance, showed expression patterns and responses similar to marine species, and differed from the low-salinity-tolerance O. niloticus, which showed a response that differed from the accepted models, that are based on marine and diadromous fishes.