1. 1 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY Genetic Markers for Jatropha Biodiversity Evaluation and Breeding 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 HONG Yan, Group of Plant Biotechnology hongy@tll.org.sg

2. 2 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY “It has not been really domesticated…. will also require serious amounts of agronomic research” Nature editorial

3. 3 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY Breeding / domestication will increase productivity and improve other agronomic traits Wild plants vs domesticated ones Maize

4. 4 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY 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. 5 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY 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. 6 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY Comparison of Different genetic marker techniques Gustavo Caetano-Anolles and P.M.Gresshoff 1997 *ISSR as an variant

7. 7 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY 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. 8 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY Controlled pollination for breeding in TLL –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

9. 9 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY Interspecies jatropha hybrids Objectives: –Genetic mapping –Possible better traits F1 hybrid plants

10. 10 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY 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. 11 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY 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. 12 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY Low genetic diversity as detected by general AFLP analysis

13. 13 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY E2/H3-HpaII -methylation sensitive PZ14A SU6 MD42 MD44 E2/H3-MspI -methylation insensitive Methylation sensitive AFLP C C G G G G C C m5 C C G G G G C C m5 Cut X No cut Methylation sensitive AFLP analysis

14. 14 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY Segregation of methylation sensitive alleles

15. 15 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY 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. 16 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY DNA methylation in plant Best characterized epigenetic modification Implicated in may important biological processes Inheritance of methylation pattern Interference of DNA methylation in Arabidopsis by down regulate MET1, a methylase gene

17. 17 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY 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. 18 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY 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. 19 International Consultation on Pro-poor Jatropha Development, Rome, Apr 08HY Jatropha International Congress December 17 -18, 2008, Singapore First Circular Organized by Temasek Life Sciences Laboratory A True Event for Biofuel Research and Business 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