Reverse Breeding: a tool to create homozygous plants from the heterozygous population.

Reverse Breeding: a tool to create homozygous plants from the heterozygous population.

1. Welcome

2. Credit Seminar on Reverse Breeding: A novel approach for development of homozygous plants Department of Genetics & Plant Breeding College of Agriculture S.K. Rajasthan Agriculture University, Bikaner Major Advisor Dr. Vijay Prakash Seminar Incharge Dr. A.K. Sharma By Sanjay Kumar Ph.D. (Ag.) GPB

3. Contents Introduction Concept of Reverse Breeding Why Reverse Breeding ? Steps of Reverse Breeding Applications of Reverse Breeding Case study Consequences for food and environmental safety Limitations Conclusion Future Thrust

4. Reverse Breeding - Introduction A novel plant breeding technique designed to directly produce homozygous parental lines from any heterozygous plant. It was proposed by Dirks et al. (2009) in Arabidopsis thaliana. Homozygous parental lines are produced from selected plants by suppressing meiotic recombination . Gametes are directly converted into adult plants, which after chromosome doubling are used as homozygous parental lines. The main objective of reverse breeding is to generate homozygous parental lines (complementing parents) that can be mated to recreate a desired heterozygous genotype (i.e. the initial hybrid; Wijnker et al. 2012) It has not been commercialized yet and very limited work has been done.

5. “Forward breeding” “Reverse breeding” Production of hybrid plants Selection of heterozygous plants Selection of “end product” at the start of breeding cycle Production of lines Selection of lines

6. To enhance the hybrid performance, first the parental lines have to be improved. Difficulty in maintaining hybrid stability. Breeders cannot produce parents of a hybrid. Clonal propagation (Apomixis) preserves the parental genotypes but prevents its further improvement. Moreover these techniques are restricted to limited crops. To solve all these problems, Reverse Breeding is the answer. Why Reverse Breeding ?

7. Reverse breeding includes the following Steps Reverse breeding comprises two essential steps: The suppression of crossover recombination in a selected plant. The regeneration of double haploids (DHs) from spores containing non-recombinant chromosomes.

8. Step 1: Suppression of crossing over

9. RNA interference RNA interference ( RNAi ) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translation or transcriptional repression. It was first discovered by Andrew Fire and Craig Mello (1998) in the nematode worm Caenorhabditis elegans .

10. The Gene Silencing Mechanism Two-step model to explain RNAi ds -RNA is diced by an ATP-dependent ribonuclease (Dicer) into short interfering RNAs (siRNAs). siRNAs are transferred to a second enzyme complex, designated as RISC (RNA induced silencing complex). The siRNA guides RISC to the target mRNA, leading to its destruction. The anti-sense strand of the siRNA is perfectly complementary.

11. Step 2. Production of Doubled Haploids Using tissue culture technique referred to as “Anther culture” and “Isolated microspore culture”, immature pollen grains grow to produce colonies of cells which are then transferred to different media to induce growth of shoots and then roots.

12. Step 3: Selection of complimentary parents through marker assisted selection F1 Step 4: Crossing appropriate DH lines on the basis of matching molecular markers.

13. 1. Reconstruction of heterozygote: For crops where an extensive collection of breeding lines is still lacking, RB can accelerate the development of varieties. In these crops, superior heterozygous plants can be propagated without prior knowledge of their genetic constitution. Examples – Arabidopsis thaliana , Maize etc. Applications (Dirks et al . 2009)

14. F 1 Segregating F 2 Starting hybrid Gametes Population double haploid Reconstituted hybrid

15. 2. Breeding at single chromosome level: Reverse Breeding explains how chromosome substitution lines can be obtained when RB is applied to a F1 hybrid of known parents . These homozygous chromosome substitution lines provide novel tools for the study of gene interactions. Offspring of plants in which just one chromosome is heterozygous, will segregate for traits present on that chromosome only. Development of improved breeding lines carrying introgressed traits. (Dirks et al. 2009)

16. 3. Reverse breeding and Marker assisted breeding: High throughput genotyping speeds up the process of identification of complementing parents in populations of DHs. Helps in the study of populations that segregate for traits on single chromosome allow the quick identification of QTLs, when genotyping is combined. Aids in generation of chromosome specific linkage maps. Fine mapping of genes and alleles.

17. Case study

18. Major objective: To produce homozygous parental lines from the heterozygous plant of Arabidopsis thaliana. ( Wijnker et al. 2012)

19. Materials and Methods Plant materials: 1. Arabidopsis thaliana plants were grown in glass house in standard conditions. 2. Plant transformation Method: RNAi knock downs the function of RecA , DMC1 (a meiosis-specific recombinase essential for the formation of crossovers). RNAi used – Brassica carinata DMC1 gene. Recombinase silenced- A. thaliana DMC1 gene. PCR amplified cDNA of Brassica carinata DMC1 gene was cloned to pKANNIBAL Hairpin RNAi vector. The vector was subsequently cloned into pART27 binary vector and transformed into Col-0

20. 3. Quantitative RT PCR: 4. Microscopy and FISH. 5. Genetic Analysis: SNP markers. 6. Marker segregation in WT and RB haploids. 7. Development of Homozygous diploids, each having half the genome of the original hybrid.

21. Fig: Meiosis in wild-type (WT; above) and RNAi:DMC1 transformants (below) in Arabidopsis thaliana.

22. Reverse bred crops are similar to those of parental lines and F1-hybrids obtained by conventional breeding. The parental lines produced by reverse breeding and the subsequent produced F1-hybrids do not contain any genetic modification-related DNA sequence and the RNA silencing signal itself will not be transmitted through seeds, so it is not covered under GMO regulations. So Reverse bred crops said to be safe. Consequences for food and environment safety

23. Development of RB is limited to those crops where DH technology is common practice e.g. cucumber, onion, broccoli, sugarbeet, maize, pea, sorghum. There are some exceptions such as soybean, cotton, lettuce and tomato where doubled haploid plants are rarely formed or not available at all. The technique is limited to crops with a haploid chromosome number of 12 or less and in which spores can be regenerated into DHs. Limitations

24. One important application is the production of complementary homozygous lines that can be used to generate specific F1 hybrids. Additionally, when RB is applied to F1 heterozygotes , it is possible to generate chromosome substitution lines that allow targeted breeding on the single chromosome scale. Conclusion

25. RNAi mediated Reverse Breeding is a young work, requires extensive study to overcome technical problems. Additional research is required to improve the efficiency of the DH production. Emphasis should be given for the production of hybrids in crops like cucumber, onion, broccoli, cauliflower where seed production is problematic. Future Thrust

26. Rob Dirks, Kees van Dun, C. Bastiaan de Snoo , Mark van den Berg, Cilia L. C. Lelivelt , William Voermans , Leo Woudenberg , Jack P. C. de Wit, Kees Reinink , Johan W. Schut , Eveline van der Zeeuw , Aat Vogelaar , Gerald Freymark , Evert W. Gutteling , Marina N. Keppel, Paul van Drongelen , Matthieu Kieny , Philippe Ellul , Alisher Touraev , Hong Ma, Hans de Jong and Erik Wijnker (2009). Reverse breeding: a novel breeding approach based on engineered meiosis. Plant Biotechnology Journal, 7: 837–845. Erik Wijnker , Kees van Dun, C Bastiaan de Snoo , Cilia L C Lelivelt , Joost J B Keurentjes , Nazatul Shima Naharudin , Maruthachalam Ravi, Simon W L Chan, Hans de Jong & Rob Dirks (2012). Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant. Nature Genetics, 1-5. Aude Dupré  , Louise Boyer- Chatenet , Rose M Sattler, Ami P Modi ,  Ji-Hoon Lee, Matthew L Nicolette, Levy Kopelovich , Maria Jasin , Richard Baer, Tanya T Paull , Jean Gautier (2008). Nature Chemical Biology , 4(2): 119-125. Referances