Recent advances in tilapia genetics Prof Brendan McAndrew Institute of Aquaculture

1. Recent advances in tilapia genetics Prof Brendan McAndrew Institute of Aquaculture University of Stirling Scotland UK.

2. Tilapia genetics • Production now makes tilapia the second most important farmed fish species. • Growing proportion of the fish derived from selective breeding programs. • Significant gains in growth performance reported particularly with GIFT based strains. • Evidence for improvement in other commercial traits less obvious.

3. Developments in Genomics • Full sequence of O.niloticus has been available since 2011. About to be published in Nature. • Sequence based on XX clonal female produced from Stirling strain. • Despite a full sequence this has not been fully annotated so we still need high density maps to locate QTL associated with major commercial traits. • The nature of the sex-determination mechanism and the master gene are still unknown. • Until recently increasing the density of gene-map has been a costly and laborious process.

4. Genetic analyses Marker discovery (often laborious!) Genotyping Analysis Selection experiment Pedigree structure Selection Experiment Pedigree structure Marker discovery + genotyping Analysis (genotyping by sequencing)

5. Restriction-site associated DNA sequencing (RAD-seq) • This next-generation sequencing technique is capable of marker discovery and genotyping from sufficient individuals (in one or a few sequencing lanes) to generate enough data for population genetics, phylogenetic analyses, linkage mapping, QTL analysis, etc. • Massively parallel sequencing of a reduced representation of the genome with no prior knowledge of the genome required. • Basic steps: • Genome is fragmented with (rare cutter) restriction enzyme • An adapter (P1) is attached to the restriction site – this includes a barcode identifying individuals or pools of individuals (up to 50) • DNA is sequenced from P1 adaptor using Illumina Hi-Seq or Miseq • Hundreds of millions of sequence reads of 100 bp long • These are sorted into loci, SNPs and genotypes identified • Analysis can then proceed

6. Sex determination in Nile tilapia Commonly want all-male tilapia for aquaculture (via MT, YY/GMT, TSD, …) XX/XY in LG1, plus other loci (LG3, LG23?) and effects of temperature But is there any polymorphism in LG1 locus beyond “X” and “Y”, and/or interactions between loci? Fine mapping of LG1 locus Mapping of loci involved in departures from simple ratios predicted by XX/XY system

7. RAD tag sequencing Baird et al. (2008)

8. The problem in YY/GMT production Progeny testing of males from XY male x XY neofemale crosses – expect a mixture of XY and YY males Some fish give progeny sex ratios around 1:1 (nsd) – XY Mair et al. (1997)

9. The problem in YY/GMT production Progeny testing of males from XY male x XY neofemale crosses – expect a mixture of XY and YY males • Some fish give progeny sex ratios of 100% male or very close to this – YY Mair et al. (1997)

10. The problem in YY/GMT production Progeny testing of males from XY male x XY neofemale crosses – expect a mixture of XY and YY males • Uncertainty in identifying/defining YYs • Problems in GMT production (<100% males) Some fish give sex ratios between the two - ?? Mair et al. (1997)

11. Experimental crosses Series of males crossed to isogenic line* females to eliminate variation in female contribution *used for Nile tilapia genome sequence

12. Clonal line is all-female Series of males crossed to isogenic line females to eliminate variation in female contribution

13. Crosses analysed using RAD-seq Will focus on RAD-seq analysis of two highlighted groups

14. XY males • Phenotypic sex in all three progeny groups showed high association (>90%) with paternal alleles from LG1 marker UNH995 (as in earlier studies) • Concluded to be XY males: two of these families selected for RAD-seq analysis

15. 68 20

16. Genetic Map (R/Onemap-Tmap) Basic karyotype – 22 pairs Map has 22 LGs, but following notation for genome sequence, no #21!

17. Genetic Map Basic karyotype – 22 pairs

18. QTL analysis All SNPs showing genome-wide association with sex (in black) are in LG1 Plot of LOD scores for association with sex along LGs (LG1 in red) Palaiokostas et al. 2013 PLOS ONE 8(7):e68389

19. Verification of sex association in tilapia 136 random individuals were SNP genotyped using KASP assays for the 5 SNPs closest to the QTL. Comparison to draft genome region of 1.2mb and 10 annotated genes and 14 gaps – but SD gene may not be present in XX genome!

20. “Unknown” males χ2 (against expected ratio of 1:1 of each genotype in each sex): n.s. = not significantly different; ** = P<0.01; *** = P<0.001 One paternal UNH995 allele only associated with male progeny; the other paternal allele with both sexes (20-36% females in total); limited screening of other LGs with microsatellites showed no other associations a “strong” Y allele and an “ambivalent” allele?

21. Double-digest RAD-seq (ddRAD-seq) Size selection of DNA library excludes very small [a] or large [b] fragments Peterson et al. (2012) • ddRAD-seq v’s RAD-seq: • Reduced number of loci and markers • Two sets of barcodes (one for each restriction enzyme) • Allows more individuals to be run per sequencing lane (250?) • Reduces cost per individual (fish)

22. QTL scanning In the two crosses analysed, a QTL in LG20 accounts for the skewed sex ratio: many of the XX animals that have genotype AB at the LG20 locus are phenotypically male. (Palaiokostas et al unpublished)

23. RAD-seq sex determination analysis - summary XY To be analysed XY + LG20 QTL This now helps to explain many earlier results! We will sequence LG1 region in XY BAC library - Cichlid master sex-determination gene. We can also start to clean-up YY male sex ratios-applicable in Stirling based O. niloticus strains.

24. Application to breeding programmes • Some aquaculture breeding programmes use molecular markers for pedigree reconstruction (in core broodstock or sentinel populations) • Generally based on around 10 microsatellites, with moves to (100?) SNPs • Cost of developing and running SNP chips is still expensive • Cattle breeding programmes starting to use genome-wide selection (GWS) • One lane of ddRADseq costs minimum of $4000: we can genotype about 250 fish @ 1000 SNPs per fish for this • Can use such data for pedigree assignment, QTL analysis, … • < $20 per animal and costs of this technology still falling (already cheaper than running 10 microsatellites?)

25. Disadvantages/limitations • Most SNPs have only two alleles – higher chance of being non-informative than many microsatellites (compensated for by higher numbers of markers?) • After identifying a limited number of useful SNPs, it is not easy to multiplex these in a cost-effective way – single SNP assays OK (KASP assays), many SNPs on a chip OK (but expensive). Technology will improve (reducing cost)?

26. Summary • RAD-seq and variations allow large scale SNP discovery and genotyping by sequencing • This provides large genotype data sets suitable for a number of applications • Costs of sequencing/genotyping in this way have decreased dramatically and are still falling • Potential for application in breeding programmes as well as in underlying research We believe that this is the best technology for the foreseeable future and have bought the sequencing and computing needed to do this work inhouse.

27. Species-specific SNP markers Many tilapia strains with diverse genetic and management backgrounds. Development of species diagnostic markers will help to identify the genetic makeup of commercial strains. This should help future management and improvement programmes. Preliminary data, based on one RAD-seq run using 6-9 individuals from each of 6 tilapia species (each species from one source). *we expect the number of markers to decrease as we add in more individuals, populations and species.

28. Phylogeny based on initial RAD-seq data