Genetic Markers for Jatropha Biodiversity Evaluation and Breeding

1. Genetic Markers for Jatropha Biodiversity Evaluation and Breeding HONG Yan, Group of Plant Biotechnology hongy@tll.org.sg • Introduction to jatropha • Genetic markers, various techniques • Controlled pollination and genetic mapping • Our findings with SSR and AFLP on jatropha genetic diversity • Methylation sensitive diversity • Implication / discussion

2. “It has not been really domesticated…. will also require serious amounts of agronomic research” Nature editorial

3. Breeding / domestication will increase productivity and improve other agronomic traits Maize Wild plants vs domesticated ones

4. Genotype and environment • Morphology and agronomic traits are variable for jatropha • Between varieties, also within varieties • Variable performance of sibling plants under same growth conditions proves the importance of genotype • A few provenance trials* conducted in Africa found: • Genotype-environment interaction (GxE) • A great range of seed yield • Various crude fat content from 18.4-42.3% • Trait specific relative contribution remains to be further investigated *Heller 1996

5. Genetic Markers • A gene or DNA sequence having a known location on a chromosome and associated with a particular gene or trait • Advantages of genetic markers: • Profile of genetic markers will define a genotype • Uniform at all tissues at all ages of the organism • allows early detection • developmentally stable and not affected by the environment • highly specific pattern can be developed for each individual • Utilities: • For biodiversity study • To choose right parents for cross pollination • Marker assisted breeding • To differentiate plant varieties • Quality control of plantation

6. Comparison of Different genetic marker techniques *ISSR as an variant Gustavo Caetano-Anolles and P.M.Gresshoff 1997

7. Genetic marker development and biodiversity studies of jatropha • Few reports with RAPD and ISSR techniques • Potential problems of reliability and reproducibility • Generally very low genetic diversity reported in local populations analyzed • No report on global jatropha genetic diversity Basha 2007; Xiang 2007

8. Self pollination: for inbred lines and evaluating heredity of traits Cross pollination: to generate hybrid seeds between different varieties, also with other Jatropha species for hybrid vigor and new traits Controlled pollination for breeding in TLL

9. Interspecies jatropha hybrids • Objectives: • Genetic mapping • Possible better traits F1 hybrid plants

10. Genetic marker development in TLL • >500 SSR loci were cloned • >50 primer combinations of AFLP analysis • High throughput fluorescence marker analysis with ABI 3730xl DNA analyzer • Both techniques are used together for construction of genetic map of jatropha with BC populations Examples of SSR markers Example of SSR allele detection

11. SSR / AFLP evaluation of jatropha biodiversity • Work in progress to analyze 192 samples from Asia, South America and Africa • Preliminary findings: • SSR analysis showed very little diversity, most were homozygous • EcoRI / MseI AFLP showed very little diversity, too (polymorphic loci <5%, mean heterozygosity < 1%) Monomorphic band pattern shown for most SSR loci

12. Low genetic diversity as detected by general AFLP analysis

13. Methylation sensitive AFLP Methylation sensitive AFLP analysis PZ14A SU6 MD42 MD44 E2/H3-HpaII -methylation sensitive E2/H3-MspI -methylation insensitive X m5 m5 C C G G G G C C C C G G G G C C m5 m5 m5 Cut No cut

14. Segregation of methylation sensitive alleles

15. Epigenetic diversity in jatropha • Methylation sensitive AFLP • Much higher diversity observed in methylation sensitive AFLP analysis (polymorphic loci >60%, mean heterozygosity >14%) • Stable in various parts of a plant • Heritable, follows Mendelian segregation • Most diversity found within populations • Biological significance not known yet

16. DNA methylation in plant • Best characterized epigenetic modification • Implicated in may important biological processes Interference of DNA methylation in Arabidopsis by down regulate MET1, a methylase gene Inheritance of methylation pattern

17. Discussion • Reasons for the lack of biodiversity so far • Technical or inherent? • If low genetic diversity confirmed, issues to address: • Genetic basis for phenotype? • Breeding strategy? • Any significance role of epigenetic in jatropha? • Suggestions: • A global survey of genetic diversity is important and critical • International collaboration and verification is necessary

18. Temasek Life Sciences Laboratory (TLL) • A non-profit research institute owned by Temasek holding, NUS and NTU • With 30 research groups, we work on both basic and applied science • One center of excellence in the area of plant tissue culture, genetic modification, plant biology and development study • Experience and mechanism to collaborate and work with industry • A cluster with about 20 scientists working on jatropha: • Biodiversity and breeding • Genetic markers and mapping • Platform technology development • Phytochemistry • Genomics • Molecular and cell biology studies • Two experimental farms 1 Research Link, National University of Singapore, Singapore 117604 Tel: +65 6872 7000 Fax: +65 6872 7007 http://www.tll.org.sg

19. Jatropha International Congress December 17 -18, 2008, Singapore First Circular Themes of the congress 1. Genetic resources 2. Breeding technologies and strategies 3. Genetics and Genomics 4. Biosynthesis of fatty acids and toxins 5. Tissue culture, propagation and transformation 6. Plantation management and pest control 7. Oil processing 8. By-product utilization 9. Investment and business models 10. Government policies and incentives A True Event for Biofuel Research and Business Organized by Temasek Life Sciences Laboratory