Marker-assisted selection in enhancing genetically male Nile tilapia (Oreochromis niloticus L.) production

All-male fry are preferred to prevent uncontrolled reproduction before harvest in intensive Nile tilapia (Oreochromis niloticus) aquaculture. Males also grow faster than females. An alternative approach to direct hormonal masculinisation of tilapia fry is to produce fry that are genetically male. However, sex determination system in tilapia is fairly complex. Recent developments have resulted in a linkage map and genetic markers that can be used to analyse the sex determination system. To analyse the genetic sex determination mechanism and to develop marker-assisted selection in the Stirling Nile tilapia population, a fully inbred line of clonal females (XX) was verified using test crosses and DNA markers (mostly microsatellites) to use as a standard reference line in sex determination studies. A series of crosses were performed involving this line of females and a range of males. Three groups of crosses were selected (each group consisted of three families) from progeny sex ratio distributions, and designated as type ‘A’ (normal XY males x clonal XX females), type ‘B’ (putative YY males x clonal XX females) and type ‘C’ (unknown groups of males x clonal XX females), for sex linkage study. For type ‘A’, inheritance of DNA markers and phenotypic sex was investigated using screened markers from tilapia linkage group 1 (LG1) to confirm the LG1-associated pattern of inheritance of phenotypic sex and the structure of LG1. Screened markers from LG1, LG3 and LG23 were used to investigate the association of markers with sex in families of type ‘B’ and ‘C’. In addition, a genome-wide scan of markers from the other 21 LGs was performed to investigate any association between markers and sex, in only families of cross type ‘B’. LG1 associated pattern of inheritance of phenotypic sex was confirmed by genotype and QTL analyses in families of cross type ‘A’. Analyses of genotypes in families of type ‘B’ and ‘C’ showed strong association with LG1 markers but no association with LG3 and LG23 markers. Genome wide scan of markers from all other LGs did not show any significant association between any markers and the sex. The allelic inheritance of two tightly linked LG1 markers (UNH995 and UNH104) in families of type ‘B’ and ‘C’ identified polymorphism in the sex determining locus: one of the alleles was associated mostly with male offspring whereas another allele was associated with both progeny (mostly males in type ‘B’ families, and approximately equal numbers in type ‘C’ families). This knowledge was used to identify and separate supermales (‘YY’ males) that should sire higher proportions of male progeny, reared to become sexually mature for use as broodstock. Two of them were crossed with XX females (one clonal and one outbred) to observe the phenotypic expression of the strongest male-associated allele in progeny sex. The observations of 98% male (99 males out of 101 progeny) and 100% male (N=75) from these two crosses respectively, suggest that a marker-assisted selection (MAS) programme for genetically male Nile tilapia production could be practical. This study also suggests that the departures from the sex ratios predicted using a “simple” XX/XY model (i.e., YY x XX should give all-male progeny) were strongly associated with the XX/XY system, due to multiple alleles, rather than being associated with loci in other LGs (e.g., LG3, LG23). This study also tentatively names the allele(s) giving intermediate sex ratios as “ambivalent” and emphasizes that the presence and actions of such allele(s) at the same sex-determining locus could explain departures from predicted sex ratios observed in some earlier studies in Nile tilapia

Validation of clonal line females for sex determination studies in Nile tilapia Oreochromis niloticus L.

Sex determination in the Nile tilapia Oreochromis niloticus is more complex than a simple XX-XY sex determining mechanism, as evidenced from fairly frequent unexpected sex ratios in progeny. The production of uniform, homozygous experimental material is particularly advantageous for studying sex determining mechanism as well as for the genetic mapping and genome sequencing studies in which interpretations are facilitated by homozygosity. To better understand the genetic mechanism of sex determination, a fully inbred line of clonal females (XX) was verified in controlled environmental conditions using test crosses and microsatellite DNA markers from the tilapia linkage map. A total of successfully amplified 87 microsatellite DNA markers covering all 24 linkage groups were selected for screening sexually mature females from this line. 67 markers were found polymorphic in outbred individuals screened. Markers from LG1, LG3 and LG23 were given more emphasis because sex determining genes have been mapped on these LGs in different species of tilapia. The verification and validation of this clonal line of females made them an important resource to use as a ‘standard reference line' in genomics, sex determination studies and other studies in Nile tilapia

Marker-assisted selection in enhancing genetically male Nile tilapia (Oreochromis niloticus L.) production

All-male fry are preferred to prevent uncontrolled reproduction before harvest in intensive Nile tilapia (Oreochromis niloticus) aquaculture. Males also grow faster than females. An alternative approach to direct hormonal masculinisation of tilapia fry is to produce fry that are genetically male. However, sex determination system in tilapia is fairly complex. Recent developments have resulted in a linkage map and genetic markers that can be used to analyse the sex determination system. To analyse the genetic sex determination mechanism and to develop marker-assisted selection in the Stirling Nile tilapia population, a fully inbred line of clonal females (XX) was verified using test crosses and DNA markers (mostly microsatellites) to use as a standard reference line in sex determination studies. A series of crosses were performed involving this line of females and a range of males. Three groups of crosses were selected (each group consisted of three families) from progeny sex ratio distributions, and designated as type ‘A’ (normal XY males x clonal XX females), type ‘B’ (putative YY males x clonal XX females) and type ‘C’ (unknown groups of males x clonal XX females), for sex linkage study. For type ‘A’, inheritance of DNA markers and phenotypic sex was investigated using screened markers from tilapia linkage group 1 (LG1) to confirm the LG1-associated pattern of inheritance of phenotypic sex and the structure of LG1. Screened markers from LG1, LG3 and LG23 were used to investigate the association of markers with sex in families of type ‘B’ and ‘C’. In addition, a genome-wide scan of markers from the other 21 LGs was performed to investigate any association between markers and sex, in only families of cross type ‘B’. LG1 associated pattern of inheritance of phenotypic sex was confirmed by genotype and QTL analyses in families of cross type ‘A’. Analyses of genotypes in families of type ‘B’ and ‘C’ showed strong association with LG1 markers but no association with LG3 and LG23 markers. Genome wide scan of markers from all other LGs did not show any significant association between any markers and the sex. The allelic inheritance of two tightly linked LG1 markers (UNH995 and UNH104) in families of type ‘B’ and ‘C’ identified polymorphism in the sex determining locus: one of the alleles was associated mostly with male offspring whereas another allele was associated with both progeny (mostly males in type ‘B’ families, and approximately equal numbers in type ‘C’ families). This knowledge was used to identify and separate supermales (‘YY’ males) that should sire higher proportions of male progeny, reared to become sexually mature for use as broodstock. Two of them were crossed with XX females (one clonal and one outbred) to observe the phenotypic expression of the strongest male-associated allele in progeny sex. The observations of 98% male (99 males out of 101 progeny) and 100% male (N=75) from these two crosses respectively, suggest that a marker-assisted selection (MAS) programme for genetically male Nile tilapia production could be practical. This study also suggests that the departures from the sex ratios predicted using a “simple” XX/XY model (i.e., YY x XX should give all-male progeny) were strongly associated with the XX/XY system, due to multiple alleles, rather than being associated with loci in other LGs (e.g., LG3, LG23). This study also tentatively names the allele(s) giving intermediate sex ratios as “ambivalent” and emphasizes that the presence and actions of such allele(s) at the same sex-determining locus could explain departures from predicted sex ratios observed in some earlier studies in Nile tilapia.EThOS - Electronic Theses Online ServiceCommonwealth Scholarship CommissionGBUnited Kingdo

Mapping and Validation of the Major Sex-Determining Region in Nile Tilapia (Oreochromis niloticus L.) Using RAD Sequencing

Sex in Oreochromis niloticus (Nile tilapia) is principally determined by an XX/XY locus but other genetic and environmental factors also influence sex ratio. Restriction Associated DNA (RAD) sequencing was used in two families derived from crossing XY males with females from an isogenic clonal line, in order to identify Single Nucleotide Polymorphisms (SNPs) and map the sex-determining region(s). We constructed a linkage map with 3,802 SNPs, which corresponded to 3,280 informative markers, and identified a major sex-determining region on linkage group 1, explaining nearly 96% of the phenotypic variance. This sex-determining region was mapped in a 2 cM interval, corresponding to approximately 1.2 Mb in the O. niloticus draft genome. In order to validate this, a diverse family (4 families; 96 individuals in total) and population (40 broodstock individuals) test panel were genotyped for five of the SNPs showing the highest association with phenotypic sex. From the expanded data set, SNPs Oni23063 and Oni28137 showed the highest association, which persisted both in the case of family and population data. Across the entire dataset all females were found to be homozygous for these two SNPs. Males were heterozygous, with the exception of five individuals in the population and two in the family dataset. These fish possessed the homozygous genotype expected of females. Progeny sex ratios (over 95% females) from two of the males with the "female" genotype indicated that they were neomales (XX males). Sex reversal induced by elevated temperature during sexual differentiation also resulted in phenotypic males with the "female" genotype. This study narrows down the region containing the main sex-determining locus, and provides genetic markers tightly linked to this locus, with an association that persisted across the population. These markers will be of use in refining the production of genetically male O. niloticus for aquaculture

Genetic variation and differentiation in the Stinging catfish, Heteropneustes fossilis (Bloch), populations assessed by heterologous microsatellite DNA markers

85-90Microsatellite DNA markers have been increasingly used in genetic diversity studies. The present study reports on the characterization of genetic variation and differentiation in four different natural populations of the stinging catfish, Heteropneustes fossilis (Bloch), in Bangladesh, viz., Mymensingh, Netrakona, Narsingdi and Rangpur, using cross-species microsatelllite DNA markers developed from the walking catfish, Clarias batrachus. Eighteen polymorphic alleles were found in the 128 diploid individuals (32 from each population), with nine alleles at each of the two loci analyzed. The Netrakona and Rangpur population deviated from the Hardy-Weinberg proportion at one locus. The population differentiation (FST) value between the Narsingdi and Netrakona population was found to be insignificant, while the values between all the other population pairs were found to be significant. The genetic distance values ranged between 0.165 and 0.626. The UPGMA dendrogram based on genetic distance resulted in two clusters: the Mymensingh population alone was in one cluster and the three other populations in the second cluster. This study revealed a fairly high level of genetic variation in the microsatellite loci within and between the four populations, and identified existence of distinct population groups of H. fossilis

Cyclic variations of gonad development of an air-breathing fish, Channa striata in the lentic and lotic environments

Abstract The study was conducted to know the cyclic changes in gonadal maturation and to investigate the developmental stages of oocytes and testicular germ cells of an air-breathing fish, Channa striata. Fish were sampled monthly from lentic and lotic environments of three geographical locations of Bangladesh from December to November and the histological analysis of their gonad was done to evaluate the objectives. The highest mean GSI was 5.95 ± 0.20 for female in July and 0.14 ± 0.01 for male also in July showing that the gonadal development reached its peak during this month. The highest mean oocyte diameter was 1257.50 ± 24.17 μm observed in July implying that the oocyte reached maturity in this month. Histological study of ovary revealed the evidence of early yolk granule stage and late yolk granule stage from April to July. In case of male four stages of spermatogenesis were distinguished and spermatozoa were highly abundant in June and July. So the monthly pooled values of GSI and the analysis of gonadal histology indicated that the peak breeding season of C. striata occurred in July in the lentic and lotic environments. Samples collected from lentic and lotic habitats are suggestive of no difference in the development of the gonad. The results of the present study will be useful for selective breeding programme, conservation and sustainable fishery management of C. striata in its natural habitat

Diallel Cross Application and Histomolecular Characterization: An Attempt to Develop Reference Stock of <i>Labeo ariza</i>

The objective of the present study was to evaluate the growth performance and genetic variation in diallel crosses of Ariza labeo (Labeo ariza) originating from three geographically separated rivers (Atrai, Jamuna and Kangsha) in Bangladesh. Intra (G1K♀K♂, G2J♀J♂, and G3A♀A♂) and inter (G4K♀A♂, G5K♀J♂, G6A♀K♂, G7A♀J♂, G8J♀K♂, and G9J♀A♂) stocks were produced following diallel cross (sex ratio—1:1 and n = 48; 16 from each river). Reproductive and growth performance, muscle cellularity and genetic variation following genotyping of eight microsatellite markers (Lr1, Lr2, Lr3, Lr22, Lr24, Lr27, Lr28 and Lr29) and analysis of all crossbreeds was performed. The fertilization (95% ± 2.11%), hatching (88% ± 1.03%), and survival rates (82% ± 1.88%) of G4K♀A♂ were higher compared to other groups. With respect to length and weight gains (2.67 ± 0.4 cm and 3.39 ± 0.2 g), SGR (3.23% ± 0.20%), and heterosis (8.87% and 24.74%) G4K♀A♂ was the superior group. A higher number of hyperplastic muscle fibers, mean number of alleles (2.75) and mean observed heterozygosity (0.417) from G4K♀A♂ could be interpreted to mean that G4K♀A♂ comprise better performance efficiency compared to others and are considered for continuing the L. ariza stock improvement program