Mapping genes and QTL in tilapias GI DEON HULATA 1 , AVNER CNAANI - - PowerPoint PPT Presentation

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Mapping genes and QTL in tilapias GI DEON HULATA 1 , AVNER CNAANI - - PowerPoint PPT Presentation

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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

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Mapping genes and QTL in tilapias

GI DEON HULATA1, AVNER CNAANI 1, 2, BO- YOUNG LEE2, WOO- JAI LEE3, AI MEE E. HOWE2, J. TODD STREELMAN2 & THOMAS D. KOCHER2

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

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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
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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.

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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.

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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

  • btained generally conf ormed that of O.

niloticus.

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Creation of the Mapping Family

Om

X

Oa ROn

X

Om Oa ROn ROn Oa

Agresti et al. , 2000. Aquaculture 185: 43- 56.

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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.

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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.

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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.

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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 F2 mapping f amily, resulted in the 2nd generation linkage map of tilapia. I t Spans 1311cM, in 24 LG, at average spacing of 2. 4 cM.

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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).

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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).

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

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.

First attempt at physical mapping in Nile tilapia

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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 F2 f amily. Other genes were mapped (using a bioinf ormatic approach) that are residing in sequences f lanking microsatellites.

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Table 1. Genes found by Blastn and Blastx comparisons between tilapia microsatellites and the GenBank database. Microsatellite name Matched gene Species Identity Similarity E value Tilapia linkage group Homology in the human genome HSA UNH142 Myeloid/lymphoid or mixed- lineage leukemia3 Homo sapiens 34/37 0.002 MLL3 (exon) (NM_021230) 7q36.2 UNH881 Immunoglobulin light chain variable region Seriola quinqueradiata

  • A. ruthenus, A. mino,

84/90 2e-28 Human mRNA (XM_033504) 2p11.2 UNH953 S164 protein Homo sapiens

  • M. musculus

88/104 4e-13 Human mRNA (AF109907) 14q24.2 UNH961 Phosphofructo-1-kinase Oryctolagus cuniculus

  • M. musculus, H. sapiens

118/142 4e-16 LG21 PFKP (exon) (NM_002627) 10p15.1 UNH1006 Cytochrom P450 Oryzias latipes

  • O. aries, M.musculus

43/48 2e-05 CYP3A5 (exon) (NM_000777) 7q22.1 UNH102 Myosin heavy chain Rattus norvegicus

  • H. sapiens

20/42 28/42 7e-04 LG16 MYO9A (exon) (NM_006901) 15q23 UNH110 GDNF family receptor alpha-1a Danio rerio

  • G. gallus, H. sapiens

22/38 26/38 5e-05 AR18 GFRA1 (exon) (NM_005264) 10q25.3 UNH178 Acetilecholine receptor delta chain precursor Xenopus laevis

  • G. gallus

20/25 23/25 4e-04 LG9 CHRND (exon) (NM_000751) 2q37.1 UNH179 Tyrosine phosphatase receptor type a Rattus norvegicus 25/53 36/53 6e-08 LG7 Human mRNA (XM_100850) 12q21.31 UNH208 Attractin Mus musculus

  • H. sapiens

35/40 39/40 1e-14 LG24 ATRN (exon) (NM_012070) 20p13 UNH303 KIAA 1694 protein Homo sapiens 23/25 24/25 1e-06 Human mRNA (AB051481) 16q24.1 UNH362 Transmembrane 4 superfamily member Homo sapiens 19/27 22/27 17/20 20/20 2e-13 LG10 NET-2 (2 exons) (NM_012338) 7q31.31 UNH735 Sodium bicarbonate cotransporter (NBC) Rattus norvegicus

  • M. musculus, H. sapiens

19/33 26/33 0.001 LG11 SLC4A7 (exon) (NM_003615) 3p24.1 UNH872 Sugar transporter Rattus norvegicus

  • M. musculus, H. sapiens

18/31 21/31 0.007 SLC2A9 (exon) (NM_020041) 4p16.1 UNH952 Cytokine-like nuclear factor n-pac Homo sapiens 52/63 57/63 5e-22 LG4 Human mRNA (AF326966) 16p13.3 Tmo M27 Guanine nucleotide release/exchange factor Rattus norvegicus

  • M. musculus, H. sapiens

25/26 25/26 2e-07 RASGRF1 (exon) (NM_002891) 15q25.1

Fishing Fishing in 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

Cnaani et al. , 2002. Animal Genetics, 33: 468

  • 4

85.

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

0.40 0.50 0.60 0.70 0.80 0.90 1.00 10 20 30 40 50 Position on LG3 ( cM( % g e n o t y p e s c o n s i s t e n t % males consistent % females consistent

Sex- linked region of LG3 in O. aureus.

0.60 0.70 0.80 0.90 1.00 5 10 15 20 25 30 35 40 Position on LG1 (cM) % g e n o t y p e s c o n s i s t e n t % males consistent % females consistent

Sex- linked region of LG1 in O. niloticus.

First genetic markers associated with sex in tilapia

identif ied by genome scan on pooled DNAs f rom males and f emales

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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

  • s

e gregant analysis. Animal Genetics, 34: 379

  • 3

83.

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

LG1 Z haplotype / Dominant male determiner LG3 W haplotype /

Dominant male repressor

Lee, B.

  • Y

. , Hulata, G. and Kocher, T. D. 2004. Two unlinked loci controlling the sex of blue tilapia (Oreochromis aureus). Heredity, 92: 543

  • 5

49.

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Z W

A locus associated with sex determination in Tilapia zillii was also localized to LG1.

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Red Color in Tilapia

  • Wild- type, red, and blotched phenotypes

in F2 of (O. aureus x Red O. niloticus)

  • Red color is dominant (McAndrew et al. ,

1988)

Howe, A. , 2004. The Genetic Basis of Red Color in Tilapia. M. S. thesis, Univ. of New Hampshire, Durham NH.

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

13/ 55 shot gun sequences hit Fugu scaf f old 18

0.0 26.0 33.8 41.6 57.4 59.8

Tilapia BAC Fugu Scaffold Tilapia LG3 Medaka LG18

(Naruse et al., 2000)

  • 0. 0
  • 22. 4
  • 37. 1
  • 56. 9
  • 80. 6
  • 98. 8

Red

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

BAC cont ig spanning 3cM

trp1 RFLP-1

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

RFLP Mapping of Trp1

  • 0. 0
  • 19. 7
  • 47. 4
  • 63. 7
  • 76. 3
  • 98. 5

9/ 156 recombinants between red phenotype and predicted genotype at Trp1 (5. 7 cM)

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Current Map LG3

An AFLP marker has been placed ~1 cM f rom t he red locus The RFLP marker maps ~3 cM f rom t he t rp1 locus

Sex

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Mapping Quant it at ive Trait Loci

The recent development

  • f

hundreds

  • f

microsatellite DNA markers enable coverage of the tilapia genome at ~2cM intervals

  • n

average, thus providing the inf rastructure f or systematic genome scans f or detection of QTL. The ability

  • f

tilapias to create viable interspecif ic hybrids makes them an ideal

  • rganism f or genetic studies, using backcrosses
  • r F2 intercrosses as a segregating population.

Following are results f rom two studies using an

  • O. mossambicus x O. aureus F2 hybrid.
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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

1 2 3 4 5 6 10 20 30 40 50 60 70 Position (cM) Standard length Body weight CDD

UNH907 UNH130 UNH180 UNH879 UNH898 UNH848

I nterval mapping f or cold tolerance (CDD), body weight (BW) and standard length (SL) in LG23. Arrows indicate the estimated map positions of microsatellite markers.

Cnaani et al. , 2003. Detection of a chromosomal region with two quantitative trait loci, af f ecting cold tolerance and f ish size, in an F2 tilapia hybrid. Aquaculture, 223: 117

  • 1

28.

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

UNH 848 UNH 898 UNH 216 UNH 876 GM 576 GM 378 UNH 180 UNH 130 GM 384 UNH 907 2 4 6 8 10 10 20 30 40 50 60 F CDD, male BW, female CDD, female BW, male

I nterval mapping of LG 23, with marker points indicated [(On x Sg) ♂ x (Oa x Om) ♀]

Moen et al. , 2004. A genome scan of a f our

  • w

a y cross tilapia f amily supports the existence of a QTL f or cold tolerance on UNH chromosome 23. Aquacult. Res. , 35: 893

  • 9

04 .

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Genetic variation in stress response

MHC- I and TF, related to the immune system, were previously mapped in the tilapia genome. Markers UNH881 and UNH208 are part of coding genes of the I gM light chain and Attractin, respectively. Mapping MHC- I I , Lysozyme and Ceruloplasmin has not succeeded yet. A genome scan to study genetic linkage between DNA markers and quantitative trait loci (QTL) f or innate immune response to stress, blood biochemical parameters and f ish size was perf ormed in an F2 population of an interspecif ic tilapia hybrid (O. mossambicus X O. aureus). A f amily of 114 f ish was scanned f or 40 polymorphic microsatellite DNA markers and two polymorphic genes, covering ~80% of the tilapia genome. These f ish were previously phenotyped f or seven immune response traits and six blood biochemical parameters. The genome scan analysis resulted in 35 signif icant marker- trait associations of 26 markers, in 16 linkage groups. I n a second experiment, nine markers were re- sampled in another f amily (n=79), of which seven, in f ive linkage groups, were associated with stress response

  • traits. Markers in three linkage groups - LG 1, 3 and 23 - were associated

with stress response, body weight and sex determination, conf irming the location of QTL reported by several other studies.

Cnaani et al. , 2004. Genome

  • scan analysis f or quantitative trait loci in an F2 tilapia hybrid. Molecular Genetics

and Genomics, in press (available on line).

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Sex Body wt . St ress

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Sex Body wt . St ress Cold t olerance

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Cichlid Genomic Resources

  • 1. Genet ic maps
  • 2. Physical map (in progress)
  • 3. ESTs/ microarrays
  • 4. I nf ormat ics (UNH

comparat ive mapping viewer)

http:/ / hcgs. unh. edu/ cichlid/

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

A whit epaper submit t ed t o t he t he J GI Communit y Sequencing compet it ion received a very posit ive review, BUT was not given high priorit y due t o t he large size of cichlid genome ~1000Mb.

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Acknowledgements

to colleagues contributing to research and to f unding agencies UNH

Karen Carleton, Justin Stern, Craig Albertson (FI )

ARO

Micha Ron, Eyal Seroussi, Joel Weller, Yniv Palti (NCCCWA)

UCD

Berney May, Jeremy Agresti, Thomas Moen (AKVAFORSK)

Funding Agencies USDA, UNH (TDK) NSF (TDK, JTS) NI H (JTS) BARD (GH, AC) I SF (GH)

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Gideon Hulata / Agricultural Research Organization / The Volcani Center / I srael

Mapping genes and QTL in tilapias / I STA6 2004

Thank you.