Reverse breeding is a novel plant breeding technique that allows the development of parental lines directly from any superior heterozygous plant. It involves suppressing meiotic recombination to produce gametes with whole parental chromosome sets, followed by doubling of haploids to generate parental lines. Two case studies demonstrate using RNAi to silence meiotic genes in Arabidopsis thaliana, producing parental lines that reconstitute the original hybrid when crossed. A second technique, marker-assisted reverse breeding, uses high-density SNP genotyping instead of gene silencing to select maize lines similar to original parents within one year. Reverse breeding techniques accelerate breeding and facilitate hybrid improvement without prior knowledge of parental lines.

1. 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. Heterozygous
Heterogeneous
Population
Superior heterozygous plant of unknown
parentage
Engineered Meiosis
Development of
parental lines
P1 P2
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. RNAi knocks down the function of these genes
during spore formation
1. GENES RESPONSIBLE FOR MEIOTIC RECOMBINATION
1. DMC1 gene
2. RecA gene
3. SPO11 gene
2. EXOGENOUS APPLICATION OF CHEMICAL COMPOUND THAT CAUSE
INHIBITION OF RECOMBINATION
1. 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. 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

14. Where do RNA interference occur??
homologue synapsis
double strand break
formation
strand exchange
RNA
interference
Achaisma15

15. 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

16. 17

17. 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

18. Step 3: Selection of complementary
lines (parents) through Marker Assisted Selection
F1 DOUBLE HAPLOIDS
Step 4: Crossing appropiate DH lines on the basis of matching molecular
markers to develop superior hybrids 19

19. 20Wijnker and Jong (2008).

20. 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

21. APPLICATION
22

22. 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

23. 24Dirks et al. 2009

24. 2. Breeding on the single chromosome level
 Reverse Breeding explains how chromosome substitution lines can
be obtained when RB is applied to an F1 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

25. 26Dirks et al. 2009

26. 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.

27. 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

28. 29

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

30. Background Information.
Plant Materials:
1. A. thaliana plants were grown under standard conditions in a
greenhouse.
WTF1 RBF1
Ler-0(CS20) x Col-0 (ABRC
stock CS600000)
Col-0,Semi sterile RNAi Ler
DMC1 transformant x (CS261)
( T39, T62)
WTF1 RBF1
31

31. • Cenh3-1 GFP- Tailswap x WTF1, RBF1
Haploids Diploids Anueploids
Discarded
Homozygous Genotype, Semi sterile smaller
rosette size, narrow leaves
32

32. 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

33. 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

34. 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

35. 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 Ler 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

36. b)Development of
chromosome
substitution line
37

37. Case study: 2
38

38. 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

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

40. • 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

41. 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

42. General protocol of Marker-Assisted Reverse Breeding
(1) 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

43. (5) Self the hybrid F1 to generate F2 seeds and genotype the F2 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 F2 selected plants to get F2-derived F3 families and continue
with selection among F3 seeds.
(7) Self the selected F3 plants to get F3 -derived F4 families and
continue with selection among F4 seeds or plants to identify the
progeny with the highest levels of similarity to their maternal and
paternal genotypes, respectively.
44

44. (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

45. 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

46. 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

47. 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

48. 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

49. 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

50. 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

51. 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 F1-hybrids obtained by conventional breeding.
• So said to be safe.
.
52

52. 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

53. 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

54. 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

55. Charles Darwin...
“It is not the strongest species
that survive, nor the most
intelligent, but the ones most
responsive to change”
THANK YOU 56