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Recent advances in tilapia genetics
Prof Brendan McAndrewInstitute of AquacultureUniversity of StirlingScotland UK.
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.
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.
Genetic analyses
Marker discovery (often laborious!)
Selection experimentPedigree structure
Genotyping Analysis
Selection ExperimentPedigree structure
Marker discovery+ genotyping Analysis
(genotyping by sequencing)
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
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
RAD tag sequencingBaird et al. (2008)
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)
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)
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 sex ratios between the two ‐ ??
Mair et al. (1997)
The problem in YY/GMT production
• Uncertainty in identifying/defining YYs
• Problems in GMT production (<100% males)
Male type Abbreviated PIT tag id
Crosses to clonal females Crosses to outbred females No. of female
progeny
No. of male
progeny
Proportion of males
(%)
No. of female
progeny
No. of male
progeny
Proportion of males
(%) Clonal line neomale (XX)
FD8F 39 0 0.0 28 0 0.0 E607 14 0 0.0 49 0 0.0 EBEE 35 0 0.0 - - -
Putative XY
D9E3 75 71 48.6 194 189 49.3 FBE2 57 61 51.7 26 34 56.7 46A8 62 60 49.2 32 33 50.8
Putative YY
315C 36 404 91.8 15 112 88.2 E167 15 206 93.2 13 167 92.8 F8BB 4 76 95.0 1 277 99.6
Unknown sexual genotype
44F0 49 87 64.0 - - - F99B 46 120 72.3 - - - F5FC 21 86 80.4 - - -
Experimental crosses
Series of males crossed to isogenic line* females to eliminate variation in female contribution
*used for Nile tilapia genome sequence
Clonal line is all‐female
Male type Abbreviated PIT tag id
Crosses to clonal females Crosses to outbred females No. of female
progeny
No. of male
progeny
Proportion of males
(%)
No. of female
progeny
No. of male
progeny
Proportion of males
(%) Clonal line neomale (XX)
FD8F 39 0 0.0 28 0 0.0 E607 14 0 0.0 49 0 0.0 EBEE 35 0 0.0 - - -
Putative XY
D9E3 75 71 48.6 194 189 49.3 FBE2 57 61 51.7 26 34 56.7 46A8 62 60 49.2 32 33 50.8
Putative YY
315C 36 404 91.8 15 112 88.2 E167 15 206 93.2 13 167 92.8 F8BB 4 76 95.0 1 277 99.6
Unknown sexual genotype
44F0 49 87 64.0 - - - F99B 46 120 72.3 - - - F5FC 21 86 80.4 - - -
Series of males crossed to isogenic line females to eliminate variation in female contribution
Crosses analysed using RAD‐seq
Will focus on RAD‐seq analysis of two highlighted groups
Male type Abbreviated PIT tag id
Crosses to clonal females Crosses to outbred females No. of female
progeny
No. of male
progeny
Proportion of males
(%)
No. of female
progeny
No. of male
progeny
Proportion of males
(%) Clonal line neomale (XX)
FD8F 39 0 0.0 28 0 0.0 E607 14 0 0.0 49 0 0.0 EBEE 35 0 0.0 - - -
Putative XY
D9E3 75 71 48.6 194 189 49.3 FBE2 57 61 51.7 26 34 56.7 46A8 62 60 49.2 32 33 50.8
Putative YY
315C 36 404 91.8 15 112 88.2 E167 15 206 93.2 13 167 92.8 F8BB 4 76 95.0 1 277 99.6
Unknown sexual genotype
44F0 49 87 64.0 - - - F99B 46 120 72.3 - - - F5FC 21 86 80.4 - - -
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
68 20
Genetic Map (R/Onemap‐Tmap)
Map has 22 LGs, but following notation for genome sequence, no
#21!
Basic karyotype – 22 pairs
Genetic Map
Basic karyotype – 22 pairs
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
Verification of sex associationin tilapia
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!
136 random individuals were SNP genotyped using KASP assays for the 5 SNPs closest to the QTL.
“Unknown” malesSire
(abbreviated PIT tag no)
LG1 marker
Sire genotype
Clonal dam genotype
ProgenyGenotype No. of
female progeny
No. of male progeny
44F0 UNH995 236/252 184/184 184/236 184/252
0 23
***
12 11 n.s.
UNH104 190/210 147/147 147/190 147/210
0 23
***
12 11 n.s.
F99B UNH104 147/190 147/147 147/147 147/190
23 0
***
11 12 n.s.
F5FC UNH995 236/252 184/184 184/236 184/252
0 20 **
8 10 n.s.
UNH104 190/210 147/147 147/190 147/210
0 20 **
8 10 n.s.
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?
χ2 (against expected ratio of 1:1 of each genotype in each sex): n.s. = not significantly different; ** = P<0.01; *** = P<0.001
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)
0
2
4
6
8
10
Linkage Group
LOD
1 2b 4 7 8b 10 13 16 18 20 22b2a 3 6 8a 9 11 14 17 19 22a 23
QTL scanning
LG1: AA LG1:AB
LG20:AA Female:38 Male:0 Female:0 Male:17
LG20:AB Female:8 Male: 20 Female:0 Male:13
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)
RAD‐seq sex determination analysis ‐ summary
Male type Abbreviated PIT tag id
Crosses to clonal females Crosses to outbred females No. of female
progeny
No. of male
progeny
Proportion of males
(%)
No. of female
progeny
No. of male
progeny
Proportion of males
(%) Clonal line neomale (XX)
FD8F 39 0 0.0 28 0 0.0 E607 14 0 0.0 49 0 0.0 EBEE 35 0 0.0 - - -
Putative XY
D9E3 75 71 48.6 194 189 49.3 FBE2 57 61 51.7 26 34 56.7 46A8 62 60 49.2 32 33 50.8
Putative YY
315C 36 404 91.8 15 112 88.2 E167 15 206 93.2 13 167 92.8 F8BB 4 76 95.0 1 277 99.6
Unknown sexual genotype
44F0 49 87 64.0 - - - F99B 46 120 72.3 - - - F5FC 21 86 80.4 - - -
XY
XY + LG20 QTL
To be analysed
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.
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?)
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)?
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.
Species‐specific SNP markers
Preliminary data, based on one RAD‐seq run using 6‐9 individuals from each of 6 tilapia species (each species from one source).
Species Number of species‐specific markers*
O. niloticus 276
O. mossambicus 355
O. aureus 512
O. hornorum 376
O. karongae 538
T. zillii 3447
Groups (markers specific to at least two species)
763
*we expect the number of markers to decrease as we add in more individuals, populations and species.
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.
Phylogeny based on initial RAD‐seq data