Mapping genes and QTL in tilapias / I STA6 2004 Mapping genes and QTL in tilapias GI DEON HULATA 1 , AVNER CNAANI 1, 2 , BO- YOUNG LEE 2 , WOO- JAI LEE 3 , AI MEE E. HOWE 2 , J. TODD STREELMAN 2 & THOMAS D. KOCHER 2 1 I nstitute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan 50250 I srael 2 Hubbard Center f or Genome Studies, University of New Hampshire, Durham , NH 03824 USA 3 GenoMar ASA, Oslo Research Park, Gaustadalleen 21 Oslo, 0349 Norway Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Tilapia (Cichlid) Genome Fact s • 22 chromosome pairs • ~1 billion base pairs • < 2000 cM • ~500kb/ cM • ~45Mb/ chromosome Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Genet ic Maps Lee & Kocher (1996) developed >130 microsatellite markers (MS); 12 more developed f or other cichlids; 62 out of those were heterozygous in a Nile tilapia ( Oreochromis niloticus ) f emale whose haploid embryos were used f or mapping. – Kocher et al. (1998) (62 MS, 112 AFLP) Lee & Kocher, 1996. Microsatellite DNA markers f or genetic mapping in Oreochromis niloticus . J. Fish Biol. 49: 169 - 1 71. Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 First generation Nile tilapia genetic linkage map (24 LG / n=22) Kocher et al. , 1998. A genetic linkage map of a cichlid f ish, the tilapia (Oreochromis niloticus). Genetics 148: 1225- 1232. Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Genet ic Maps – Kocher et al. (1998) (62 MS, 112 AFLP) – Agresti et al. (2000) (60 MS, 154 AFLP) – McConnell et al. (2000) (28 MS) MS developed f or O. niloticus at UNH were used successf ully f or mapping other tilapias ( O. aureus x Red O. niloticus x O . mossambicus ; O. aureus ). Linkage maps obtained generally conf ormed that of O. niloticus . Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Creation of the Mapping Family ROn X Oa ROn X Om Oa ROn Om Oa Agresti et al. , 2000. Aquaculture 185: 43- 56. Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Male ( O. aureus x Red O. niloticus ) Agresti et al. , 2000. Aquaculture 185: 43- 56. Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Female ( O. mossambicus ) Agresti et al. , 2000. Aquaculture 185: 43- 56. Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 A comparison between O aureus and O. niloticus genetic linkage maps, showing extensive segment homologies. McConnell et al. , 2000. Microsatellite marker based genetic linkage maps of Oreochromis aureus … . Animal Genetics 31: 214- 218. Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Genet ic Maps – Kocher et al. (1998) (62 MS, 112 AFLP) – Agresti et al. (2000) (60 MS, 154 AFLP) – McConnell et al (2000) (28 MS) – Lee et al. ( submitted ) (525 MS, 21 genes) Extensive MS development by both UNH and GenoMar, and work at UNH using an O. aureus x Red O. niloticus F 2 mapping f amily, resulted in the 2 nd generation linkage map of tilapia. I t Spans 1311cM, in 24 LG, at average spacing of 2. 4 cM. Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Lee et al. , 2004. A second generation genetic linkage map of tilapia ( Oreochromis spp. ). Genetics (submitted). Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Lee et al. , 2004. A second generation genetic linkage map of tilapia ( Oreochromis spp. ). Genetics (submitted). Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 First attempt at physical mapping in Nile tilapia Martins et al. , 2004. Physical mapping of the Nile tilapia ( Oreochromis niloticus ) genome by f luorescent in situ hybridization of repetitive DNAs to metaphase chromosomes—a review. Aquaculture , 231: 37–49. Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Mapping Type I markers (genes) Polymorphisms in known genes were identif ied by several approaches. Some loci already deposited in GenBank contained simple- sequence motif s which were polymorphic in our crosses ( c- ski class 1; RasGRF2 ; Clc 5 ; TH/ I gf 2 ; I nsulin ; UVopsin ; Blue opsin ; Prolactin ). For Bmp4, Dlx2, Dmo, Dmrt1, Mhc , Otx2 , Pax9 , Pmch, Pomc, Tf , Trp1 and WT1_1 , degenerate primers were used to amplif y genomic DNA or pooled cDNAs derived f rom tilapia embryos. Resulting amplicons were cloned, sequenced and verif ied by BLAST. Gene- specif ic primers were designed and SNPs distinguishing O. niloticus and O. aureus were identif ied. PCR- RFLP was then used to genotype the F 2 f amily. Other genes were mapped (using a bioinf ormatic approach) that are residing in sequences f lanking microsatellites. Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 Fishing in Fishing in silico silico: searching f or tilapia genes using sequences of : searching f or tilapia genes using sequences of microsatellite DNA markers microsatellite DNA markers Table 1. Genes found by Blastn and Blastx comparisons between tilapia microsatellites and the GenBank database. Microsatellite Matched gene Species Identity Similarity E value Tilapia linkage Homology in the HSA name group human genome UNH142 Myeloid/lymphoid or mixed- Homo sapiens 34/37 0.002 MLL3 (exon) 7q36.2 lineage leukemia3 (NM_021230) UNH881 Immunoglobulin light chain Seriola quinqueradiata 84/90 2e-28 Human mRNA 2p11.2 variable region A. ruthenus, A. mino, (XM_033504) UNH953 S164 protein Homo sapiens 88/104 4e-13 Human mRNA 14q24.2 M. musculus (AF109907) UNH961 Phosphofructo-1-kinase Oryctolagus cuniculus 118/142 4e-16 LG21 PFKP (exon) 10p15.1 M. musculus, H. sapiens (NM_002627) UNH1006 Cytochrom P450 Oryzias latipes 43/48 2e-05 CYP3A5 (exon) 7q22.1 O. aries, M.musculus (NM_000777) UNH102 Myosin heavy chain Rattus norvegicus 20/42 28/42 7e-04 LG16 MYO9A (exon) 15q23 H. sapiens (NM_006901) UNH110 GDNF family receptor 22/38 26/38 5e-05 AR18 GFRA1 (exon) 10q25.3 Danio rerio alpha-1a G. gallus, H. sapiens (NM_005264) UNH178 Acetilecholine receptor delta Xenopus laevis 20/25 23/25 4e-04 LG9 CHRND (exon) 2q37.1 chain precursor G. gallus (NM_000751) UNH179 Tyrosine phosphatase Rattus norvegicus 25/53 36/53 6e-08 LG7 Human mRNA 12q21.31 receptor type a (XM_100850) UNH208 Attractin Mus musculus 35/40 39/40 1e-14 LG24 ATRN (exon) 20p13 H. sapiens (NM_012070) UNH303 KIAA 1694 protein Homo sapiens 23/25 24/25 1e-06 Human mRNA 16q24.1 (AB051481) UNH362 Transmembrane 4 Homo sapiens 19/27 22/27 2e-13 LG10 NET-2 (2 exons) 7q31.31 superfamily member 17/20 20/20 (NM_012338) UNH735 Sodium bicarbonate Rattus norvegicus 19/33 26/33 0.001 LG11 SLC4A7 (exon) 3p24.1 cotransporter (NBC) M. musculus, H. sapiens (NM_003615) UNH872 Sugar transporter Rattus norvegicus 18/31 21/31 0.007 SLC2A9 (exon) 4p16.1 M. musculus, H. sapiens (NM_020041) UNH952 Cytokine-like nuclear factor Homo sapiens 52/63 57/63 5e-22 LG4 Human mRNA 16p13.3 n-pac (AF326966) Tmo M27 Guanine nucleotide Rattus norvegicus 25/26 25/26 2e-07 RASGRF1 (exon) 15q25.1 release/exchange factor M. musculus, H. sapiens (NM_002891) Cnaani et al. , 2002. Animal Genetics , 33: 468 - 85. 4 Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 First genetic markers associated with sex in tilapia identif ied by genome scan on pooled DNAs f rom males and f emales 1.00 1.00 0.90 0.90 % g e n o t y p e s c o n s i s t e n t % g e n o t y p e s c o n s i s t e n t 0.80 0.80 0.70 0.60 0.70 % males consistent % males consistent % females consistent % females consistent 0.50 0.60 0.40 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 Position on LG3 ( cM ( Position on LG1 (cM) Sex- linked region of LG1 in O. niloticus . Sex- linked region of LG3 in O. aureus . Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael
Mapping genes and QTL in tilapias / I STA6 2004 LG1 Lee, B. - Y . , Penman, D. J. and Kocher, T. D. 2003. I dentif ication of a sex - d etermining region in Nile tilapia ( Oreochromis niloticus ) using bulked Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael - s e gregant analysis. Animal Genetics , 34: 379 - 3 83.
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