Development of Molecular Markers for MAS in Lettuce (Lactuca sativa)

Transcription

1 Development of Molecular Markers for MAS in Lettuce (Lactuca sativa) Application of High-Resolution DNA Melting Analysis Ivan Simko

2 Lettuce production Fresh vegetables in 2008 (mil. $): Lettuce. 1,979 omato. 1,414 Onions. 865 antaloupes 776 Peppers 763 Sweet corn Broccoli 708 arrots % 20% alifornia 71% Arizona 27% 53% Economic Research Service, USDA

3 Why do we need markers? onventional breeding is time consuming and depends on environmental conditions. Breeders are interested in technologies that could make this procedure more efficient. Molecular marker technology offers improved selection strategies in breeding

4 Marker-assisted selection Phenotypic selection Marker-assisted selection Desired material Segregating material Not phenotyped accurately Desired material Segregating material Not phenotyped accurately

5 Marker-assisted selection is more efficient than selection based on phenotype If evaluation is: ime consuming Difficult ostly Performed off season If used for: Gene pyramiding Early stages of development Several selections simultaneously Low heritability traits

6 MAS in lettuce Downy mildew (Dm3) ornell University Dm3 is defeated by new races of Bremia lactucae

7 MAS in lettuce orky root (cor1) 60-80% accurate U Davis

8 MAS in lettuce LMV (mo1) INRA alleles mo1 1 & mo1 2

9 Development of molecular markers for marker-assisted selection of diebackresistant lettuce

10 Lettuce dieback Mottling Yellowing Necrosis Stunting Plant death

11 Lettuce dieback Stem Leaf Romaine Romaine

12 Material & Methods ombination of linkage and association mapping 192 RILs (Valmaine x Salinas 88) x Salinas - linkage mapping set 68 accessions from all cultivated types of lettuce - association mapping set 132 accessions from all cultivated types of lettuce - validation set ES-based SNP markers High resolution DNA melting curve analysis Sequencing 61 ES-SSR markers for assessment of population structure Replicated field trials

13 Workflow Linkage mapping set 20 markers on LG cm apart vr1 Linkage mapping set 15 markers vr1 region Fine Linked markers Sequencing Detect SNPs Validation set HRM markers Development of High resolution DNA melting curve analysis markers SNP-trait association Markers ready for marker-assisted selection 61 ES-SSR Population structure

14 Workflow Linkage mapping set 20 markers on LG cm apart vr1 Linkage mapping set 15 markers vr1 region Fine Linked markers Sequencing Detect SNPs Validation set HRM markers Development of High resolution DNA melting curve analysis markers SNP-trait association Markers ready for marker-assisted selection 61 ES-SSR Population structure

15 Linkage mapping of vr1 Linkage 1 cm LK1457 ntg10044 QGG19E03 Highly significant linkage (< 0.001) with nine markers. ntg4252 ntg10192 LSM9959 LSZ1525 QG11N03 Each marker explains over 90% of the total phenotypic variation. 100 R 2 % 90 Marker effect ntg11275 A perfect match with two markers.

16 Workflow Linkage mapping set 20 markers on LG cm apart vr1 Linkage mapping set 15 markers vr1 region Fine Linked markers Sequencing Detect SNPs Validation set HRM markers Development of High resolution DNA melting curve analysis markers SNP-trait association Markers ready for marker-assisted selection 61 ES-SSR Population structure

17 Association mapping of vr1 Linkage 1 cm LK1457 ntg10044 Association QGG19E03 ntg4252 ntg10192 LSM9959 LSZ1525 QG11N ntg R 2 % R 2 % Marker effect

18 Haplotypes of ntg SNP position Res/ Sus # of acc. L. sativa G G A A Res 17 L. sativa G G A A Res 19 L. serriola G G A A Res 1 L. sativa & L. serriola G G A A Sus 33 L. saligna A G G Res 1 L. virosa A A Res Dissimilarity

19 Workflow Linkage mapping set 20 markers on LG cm apart vr1 Linkage mapping set 15 markers vr1 region Fine Linked markers Sequencing Detect SNPs Validation set HRM markers Development of High resolution DNA melting curve analysis markers SNP-trait association Markers ready for marker-assisted selection 61 ES-SSR Population structure

20 DNA Melting urves

21 DNA Melting urves

22 Workflow Linkage mapping set 20 markers on LG cm apart vr1 Linkage mapping set 15 markers vr1 region Fine Linked markers Sequencing Detect SNPs Validation set HRM markers Development of High resolution DNA melting curve analysis markers SNP-trait association Markers ready for marker-assisted selection 61 ES-SSR Population structure

23 Haplotypes of ntg10192

24 Workflow Linkage mapping set 20 markers on LG cm apart vr1 Linkage mapping set 15 markers vr1 region Fine Linked markers Sequencing Detect SNPs Validation set HRM markers Development of High resolution DNA melting curve analysis markers SNP-trait association Markers ready for marker-assisted selection 61 ES-SSR Population structure

25 MAS in lettuce Dieback - BSV (vr1) ested on 2,000+ accessions so far so good

26 Acknowledgement U Davis R. Michelmore L. McHale M. ruco O. Ochoa USDA-ARS Salinas D. Pechenick A. Atallah L. Lai A. Folck USDA-ARS Stoneville B. Scheffler

27 hank you