Reverse Breeding

Gives the basic information on reverse breeding and application of reverse breeding

1. WELCOME 1

2. Reverse Breeding Presented By, Shruthi H.B TAD/15-17

3. Contents Introduction Mechanism of Reverse breeding Mechanism of suppression of meiotic recombination RNAi and gene silencing. Application of Reverse Breeding Case studies MARB vs RMRB Consequence for food and environmental safety. Conclusion Future Thrust References 3

4. Introduction Reverse Breeding - Novel plant breeding technique designed to directly produce parental lines from any heterozygous plant. Proposed by Dirks et al. in 2009 (Erikson,2016). Reverse Breeding has not been commercialized yet. 4

5. Reverse Breeding vs. Traditional Breeding Heterozygous Heterogeneous Population Superior heterozygous plant of unknown parentage Engineered Meiosis Development of parental lines P1 P2 Traditional Breeding Reverse Breeding Subsequently, the obtained homozygous lines are hybridised, in order to reconstitute the original genetic composition of the selected heterozygous plants. 5

6. Why Reverse breeding? 1. Difficulty in maintaining hybrid stability. 2. To improve the hybrid performance first the parental lines has to be improved. 3. Inability to establish breeding lines for uncharacterized heterozygotes. 4. Clonal propagation (or apomixis) preserves the parental genotypes but prevents its further improvement through adapting parental lines. 6

7. To solve all these problems, REVESE BREEDING IS THE ANSWER. But How????????? 7

8. Selection of homozygous through Reverse Breeding MECHANISM INVOLVED Achaisma…?????? Gene silencing. 8

9. Selected Heterozygote Spores/gametes containing random combinations of maternally or paternally inherited chromosomes lines containing random combinations of maternally or paternally inherited chromosomes Crossing of complementary lines Suppression of meiotic recombination Doubled haploid Selection of complementary lines (parents) through marker assisted selection 1 2 3 Reverse Breeding Concept: Explanation 9

10. Step 1: Supression of Crossing over 10

11. 1. Produce gamete from heterozygote 2. Suppression of recombination during spore formation Suppressing gene required for meiotic recombination Complete knockout of gene by RNAi to knock down the function of DMC1 homologue to RecA, a meiosis specific recombinase essential for the formation of crossover. Exogenous application of chemical compounds that cause inhibition of recombination during meiosis would speed up the application of RB eg . Mirin (Dupree et al ., 2008) How to suppress meiotic recombination?? 11

12. How to suppress meiotic recombination RNAi knocks down the function of these genes during spore formation GENES RESPONSIBLE FOR MEIOTIC RECOMBINATION DMC1 gene RecA gene SPO11 gene 2. EXOGENOUS APPLICATION OF CHEMICAL COMPOUND THAT CAUSE INHIBITION OF RECOMBINATION For example, Mirin * Mirin causes G2 arrest and inhibits the phosphorylation of ATM Ataxia Telangiectasia Mutated (ATM) = serine/threonine protien kinase (Dupree et al ., 2008). 12

13. How ATM works? Ataxia telangiectasia mutated (ATM) = serine/ threonine protien kinase Serine/threonine protien kinase Activates the DNA double-strand breaks Before chiasmata formation Leads to crossing over ATM is arrested, it leads to NO FORMATION OF CHIASMATA and Cell continues to grow…..ultimately leads to formation of homozygous lines by producing double haploids. Mirin 13 Source: Dupree et al ., 2008

14. RNAi RNA interference (RNAi) is an evolutionally highly conserved process of post-transcriptional gene silencing (PTGS) by which double stranded RNA ( dsRNA ) causes sequence-specific degradation of mRNA sequences. It was first discovered in 1998 by Andrew Fire and Craig Mello in the nematode worm Caenorhabditis elegans and later found in a wide variety of organisms, including mammals.  14

15. Where do RNA interference occur?? homologue synapsis double strand break formation strand exchange RNA interference Achaisma 15

16. Silencing mechanism by RNAi Two-step model to explain RNAi. I. dsRNA is diced by an ATP-dependent ribonuclease (Dicer) into short interfering RNAs ( siRNAs ). II . siRNAs are transferred to a second enzyme complex, designated RISC for RNAi-induced silencing complex. The siRNA guides RISC to the target mRNA, Resulting target mRNA degradation 16

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18. Step 2: Production of Doubled Haploids Tissue culture of immature pollen Using tissue culture techniques referred to as “anther culture” and “isolated microspore culture”, immature pollen grains grow to produce colonies of cells. The colonies are transferred to media with different plant growth regulators and sugars to induce growth of shoots and then roots. Pollen colonies Shoots growth Root growth 18

19. Step 3: Selection of complementary lines (parents) through Marker Assisted Selection F 1 DOUBLE HAPLOIDS Step 4: Crossing appropiate DH lines on the basis of matching molecular markers to develop superior hybrids 19

20. 20 Wijnker and Jong (2008).

21. Comparison of end product reverse breeding and conventional bred crops The end product of reverse breeding will be similar to parental lines obtained by conventional breeding . The RNAi silencing is restricted only to meiotic crossover suppression but there will be no change in the DNA sequences of reverse bred plants. Thus resulting offspring can be regarded as non genetically modified. 21

22. APPLICATION 22

23. 1 . Reconstruction of heterozygous germplasm. 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 23

24. 24 Dirks et al. 2009

25. 2. Breeding on the single chromosome level Reverse Breeding explains how chromosome substitution lines can be obtained when RB is applied to an F 1 hybrid of known parents. These homozygous chromosome substitution lines provide novel tools for the study of gene interactions. Produce hybrids in which just one chromosome is heterozygous. 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. 25

26. 26 Dirks et al. 2009

27. 3. Reverse breeding and marker assisted breeding High throughput genotyping speeds up the process of identification of complementing parents in populations of DHs. The screening of populations that segregate for traits on a single chromosome allow the quick identification of QTLs, when genotyping is combined 27 Helps in the study of gene interaction in the Heterozygous inbred families. Aids in generation of chromosome specific linkage maps. Fine mapping of genes and alleles. Helps in studying nature of heterotic studies.

28. LIMITATION Development of RB is limited to those crops where DH technology is common practice eg . 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 28

29. 29

30. CASE STUDY :1 Year of Publication: 2012 Objective : generation of homozygous parental lines from a heterozygous plant. 30

31. Background Information. Plant Materials: A. thaliana plants were grown under standard conditions in a greenhouse. WTF 1 RBF 1 Ler-0(CS20) x Col-0 (ABRC stock CS600000) Col-0, Semi sterile RNAi Ler DMC1 transformant x (CS261) ( T39, T62) WTF 1 RBF 1 31

32. Cenh3-1 GFP- Tailswap x WTF 1, RBF 1 Haploids Diploids Anueploids Discarded Homozygous Genotype, Semi sterile smaller rosette size, narrow leaves 32

33. 2.Plant Transformation Method: RNAi Knock downs the function of RecA homolog 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. 33

34. RESULTS FIG 1 :In wild-type meiosis, chromosomes pair at pachytene stage after which five bivalents are formed in metaphase 1. This results in tetrads showing four regular nuclei. In RNAi:DMC1 transformants, tetrads are generally unbalanced, showing polyads, owing to unbalanced univalent segregation at metaphase 1. Suppression of DMC1 also disrupts pairing of chromosomes at pachytene . a) Suppression of crossing over 34

35. 3. Quantitative RT PCR : SYBR Green supermix on the RT PCR Detection system 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. 35

36. Outcome of the research. 21 reverse-breeding doubled haploids were identified out of the 36 possible genotypes, including the original Col-0 parent. Six sets of complementing parents—genotypes that would reconstitute the initial hybrid when crossed was identified. These complementing pairs are genetically distinct, and also differ from the original Col-0 and L er parents. To complete reverse breeding, crosses between three pairs of selected reverse-breeding doubled haploid progeny to reconstitute the starting heterozygous parent . These crosses gave rise to perfectly heterozygous plants that were genetically identical to the achiasmatic Col/Ler hybrid parent. 36

37. b)Development of chromosome substitution line 37

38. Case study: 2 38

39. Marker-Assisted Reverse Breeding (MARB), A simple and fast molecular breeding method, which will revert any maize hybrid to inbred lines with any level of required similarity to its original parent lines. Concept was first given by Yi- Xin et.al in the year 2015. No RNAi silencing is employed here. Instead chip based SNP genotyping is used. 39

40. MARB concept came up in Maize??? 40

41. Recently, with the whole-genome sequence of maize reference inbred line B73 and the fast advancement of high-throughput DNA sequencing technologies, scientists have successfully performed re-sequencing of many maize inbred lines with a huge number of SNP markers ( Chia et al. 2012) and produced High density genotyping chips produced. e.g., Illumina maize 50k array, a set of 57 838 SNPs designed by Ganal et al. (2011) and high density 600k SNP genotyping array composed of 616 201 variants (SNPs and small indels ) designed by Unterseer et al (2014). This formed basis for MARB. 41

42. Materials and Method Maternal parent : Pioneer SS inbred line PHG39 Paternal parent: Pioneer NSS inbred line PHH93 Method Parental lines’ genotypes were measured by an Infinium 50K high-density commercial chip. An Illumina low-density chip with 192 SNPs was designed to select offsprings similar to the two original parents. The 192 SNPs were selected following two rules: uniform distribution on 10 chromosomes polymorphic genotypes between the two parental lines. 42

43. General protocol of Marker-Assisted Reverse Breeding Extract DNA from seed embryo and pericarp of a selected elite hybrid separately. (2) Select genotyping platform and molecular markers that provide high density of genome coverage with high throughput genotyping available. (3) Genotype the seed embryo and pericarp DNA samples to derive the parental genotypes. (4) Select a subset of markers that are polymorphic between the parental genotypes for the following marker- assisted selection. 43

44. (5) Self the hybrid F 1 to generate F 2 seeds and genotype the F 2 seeds or plants with the subset markers to identify the progeny with the highest levels of similarity to their maternal and paternal genotypes, respectively. (6) Self the F 2 selected plants to get F 2 -derived F 3 families and continue with selection among F 3 seeds. (7 ) Self the selected F 3 plants to get F 3 -derived F 4 families and continue with selection among F 4 seeds or plants to identify the progeny with the highest levels of similarity to their maternal and paternal genotypes, respectively. 44

45. (8) Move to the next stage or continue with marker-assisted selection until the selected progeny reach a desirable level of similarity to the parental lines. (9) Use DH technology or continue with selfing to obtain fixed genotypes . 10) Scanning of the parental genotypes with an Infinium 50K high-density SNP chip. 11) Marker-assisted selection with an Illumina low-density SNP chip 45

46. FIG 3: Technical procedure of marker-assisted reverse breeding. The procedure involves using a 50k high-density SNP chip to identify markers that are polymorphic between the original parents and a low-density SNP chip containing 192 SNP markers to select progeny that are most similar to their two parents respectively. 46

47. FIG 4 : A traditional breeding procedure for development of new inbreds from an elite hybrid, which involves multiple cycles of selfing, yield testing and selection for combining ability (left) and takes six to seven years to develop new inbreds with genotypes improved or similarity to their original parents with fixed heterotic mode (right). SS, stiff stalk; NSS, non-stiff stalk. RESULTS 47

48. FIG 5 : Progress in marker-assisted reverse breeding (MARB) made in a year to differentiate an elite maize population into two distinct heterotic groups similar to its respective paternal and maternal parents. Increasing purity and similarity to the parents differentiated two heterotic groups in four crop seasons within a year (A, homozygosity and similarity revealed by marker-assisted selection. B, a profile to show the differentiation into two parental genotypes). 48

49. FIG 6 : The developed maternal and paternal inbreds phenotypically look very similar to those from two standard US heterotic groups, Lancaster (left) and Reid (right), respectively. 49

50. Outcome no of SNPs in high density illumina chip that share same alles between two lines Similarity = Total number of SNPs Similarity of MARB lines with maternal parent= 85.2% Similarity of MARB lines with paternal parent= 76.4% Similarity of MARB lines with common Commercial inbred line B73= 74% 50

51. MARB vs RMRB MARB No need of gene silencing 1- 1.5 years for the development of homozygous lines No limitation in crops with < 12 haploid chromosome no. Not Limited for crops where DH is not possible. RMRB Need of silencing 2-2.5 years for the development of homozygous lines limitation in crops with < 12 haploid chromosome no. Limited for crops where DH is not possible. Young technique, hence requires more research to supress crossover . 51

52. SAFE Consequence for food and Environmental Safety RNA-directed DNA methylation transmitted to the offspring will only have an effect on meiotic recombination and no genetic modification-related DNA sequences. Reverse bred crops are similar to those of parental lines and F 1 -hybrids obtained by conventional breeding. So said to be safe. . 52

53. Conclusion RB novel breeding approach which accelerates the breeding process. Increases the available genetic combinations. Facilitates selection of Superior plant hybrids. Large number of plants are generated, screened and regenerated without prior knowledge of their genetic constitution. Thus RB puts this century long endeavour upside down by starting with superior hybrid selection followed by recovery of parental lines. 53

54. Future Thrust 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. 54

55. References Anonymous.(2013). Reverse Breeding. Accelerating innovation. NBT Platform . Anonymous.(2014). New plant breeding techniques. Ages. Pp: 34- 40. Dirks, R., Dun1, K.V., Snoo , C. B., Berg1, M.V., Cilia,L.C ., Lelivelt , Woudenberg1, W.V., Wit, J.,Reinink, K., Schut,J.W, Zeeuw, W., Vogelaar, A., Freymark ,G.,Gutteling , W., Keppel, N.M.,Drongelen, P.N, Kieny , M., Ellul1,P., Touraev, M., Ma, H.,Jong , H.D. and Wijnker , E. (2009). Reverse breeding: a novel breeding approach based on engineered meiosis. Plant Biotechnology Journal. 7, pp. 837–8457. Erikkson.D and Schienmann , J.(2016). Reverse breeding ‘ Meet the Parents’ . Crop Genetic Improvement Techniques. Proceedings of European Plant Science Organisation . pp1-3. Yi- Xin , G.,Bao-hua1, W. and Yan, F., Ping,L .(2015). Development and application of marker-assisted reverse breeding using hybrid maize germplasm . Journal of Integrative Agriculture , 14(12): 2538–2546. Wijnker , E. and Jong H.D. (2008). Managing meiotic recombination in plant breeding.  Trends Plant Sciences. 3:640–646. Wijnker , K.V., Snoo , C.B.D., Lelivelt , C.L.C., Joost , K.B., Naharudin , N.S., Ravi, M., Chan, W.L., de Jong , H. and Dirks, R. (2012). Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant. Nature genetics . 55