1. DEPARTMENT OF GENETICS AND PLANT BREEDING
DOCTORAL SEMINAR ON
MOLECULAR MECHANISM OF SELF INCOMPATIBILITY AND IT'S USE IN CROP IMPROVEMENT
Presented By
PARMESHWAR SAHU
Ph.D. SCHOLAR
DEPARTMENT OF GENETICS AND PLANT BREEDING
IGKV, RAIPUR (C.G.)

2. Introduction

3. Self Incompatibility B A
First reported by Koelreuter (1761) in Tobacco crop and term given by: Stout (1917).
SI is the inability of plants having functional gametes to set seed when self-pollinated.
Self-incompatibility (SI) is a genetically controlled cell – cell recognition system that acts as a barrier to self-pollination in a wide range of flowering plants.
Important out breeding mechanism which prevent self pollination.
Maintains high degree of heterozygosity due to cross pollination.
Can operate at any stage between pollination and fertilization of flowering plants.
May be due to morphological, genetic, physiological and biochemical causes. B A
SAHU

4. Classification Self Incompatibility Complementary system
Oppositional system
Heteromorphic system
Homomorphic system
Distyly
Tristyly
Gametophytic system
Sporophytic system
Pin
Thrum
SAHU

5. Gametophytic Self Incompatability
Explained by East and Mengelesdorf (1925) in Nicotiana sinderae.
Outcome of the interaction between the pollen tube and the style is determined by the genotype of the pollen (gamete)
S-locus products are synthesized after completion of meiosis. So allele may shows their individual effect. (Co-dominanace like action)
Growth of the pollen tube arrests in the style but sometimes also in stigmatic region
Three types of crosses are produced:
1. Fully Incompatible (S) 2. Partial Compatible (PF) 3. Fully Compatible (F)
Found in:
Solanaceous crops
Papavarecea crops
Clove
Rye
S1=S2=S3=S4…= Sn
Pollen
Style
Overy
Fully Incompatible
Partial Compatible
Fully Compatible

6. Sporophytic Self Incompatability
Explained by Huges and Babcock (1950) in Crepis foetida.
Outcome of the interaction between the pollen tube and the style is determined by the genotype of the sporophyte (diploid tissue)
S-locus products are synthesized before completion of meiosis so alleles may not shows their individual effect. So that dominent one shows their effect on all. (Dominant effect)
Growth of the pollen tube arrests at the surface of the stigma.
Two types of crosses are produced:
1. Fully Incompatible (S) Fully Compatible (F)
Found in:
Brassica spp.
S1>S2>S3>S4… >Sn
Fully Incompatible
Fully Compatible
Pollen
Stigma
Overy

7. Molecular Mechanism of
Self Incompatibility

8. Self Incompatibility Mechanism
Different types SI mechanisms are found in different plant species. Some of them are as:
SI mechanism in Cruciferae family SSI
SI mechanism in Solanaceae family GSI
SI mechanism in Papaveraceae family GSI
SI mechanism in Poacaea family GSI

9. Basic Terms Ligand: Haplotype:
An ion, a molecule, or a molecular group that binds to another chemical entity to form a larger complex.
Haplotype:
A set of closely linked genetic markers present on one chromosome which tend to be inherited together (not easily separable by recombination)
It is collection of specific alleles (particular DNA sequence) in a cluster of tightly linked genes on a chromosome, that are likely to inherited togather.
Example- S locus haplotype of SI species

10. The S-locus
With classical genetics S-locus was assumed to be a single gene but after 1987 molecular studies revealed S-locus to be much more complex
In dicots, self-incompatibility maps to a single genetic locus (grasses have 2 unlinked loci; S and Z)
Molecular dissection of the S-locus in several plant species has shown that:
The S-locus consists of multiple, tightly-linked genes, encoding male and female compatibility determinants
Divergent mechanisms of self-incompatibility are encoded by the S-loci of different plant species
Expression of these genes are temporally (anthesis) and spatially (Stigma)
regulated.

11. S-locus in Different SI Systems (S- haplotypes)
Cruciferaea
Solanaceae
Papaveraceae

12. List of Identified Male and Female Determinant Genes in Different SI System
Family
Types of SI
Male Determinant
Female Determinant
Brassicaceae
SSI
SP11/SCR
SRK
Solanaceae,
Rosaceae,
Scrophulariaceae
GSI
SLF/SFB
S-RNase
Papaveraceae
Unknown
S-protein
Poacaea
S-Z System
(Takayama et al.,2005)

13. 1. SSI in Brassicaceae Family
1. SLG (S-locus glycoproteins):
The first S-linked gene identified in Brasssica.
Soluble cellwall localized protein.
Secreted to exterior or papillar protein
Encodes a stigma specific glycoprotein localized to cellwall of papillar cell
It shares as much as 98% nucleotide sequence identity with SRK
SLG is not necessary for SI response, but enhances the activity of SRK
2. SRK (S-locus receptor kinase)
The female determinant of SSI
Encodes allelic forms of a receptor serine/ threonine receptor domain
Expressed in the epidermal cells (papillae) of the stigma
It directly interact with pollen ligand/ SCR protein to initiate the SI reaction
High similarity to SLG protein
Transgenic gain of function mutation experiments showed that SRK alone determine SI specificity and its ability is enhanced by SLG .
(Takayama et al.,2005)

14. 1. SSI in Brassicaceae Family
3. SP11/ SCR (S-locus protein 11/
S-locus Cysteine-Rich protein ):
Male determinant of SSI (Pollen Ligand)
Secreted from pollen cellwall and interact directly with SRK protein
Tightly linked with SLG and SRK protein
Induces incompatible reactions in stigma papilla cells (SP11/SCR of matching S-locus haplotype induced autophosphorylation of SRK in stigma plasma membrane)
4. Pollen Coat Proteins (PCP):
Small molecules located in the pollen coating i.e. cellwall of pollen
Have high affinity to bind with SLG protein
PCP-SLG complex is bind with SRK protein and enhances their affinity with SCR/ S-11.

15. 1. SSI in Brassicaceae Family
5. ARC1 (Armadillo Repeat Containing -1):
Positive effectors molecule
Helps in signal transduction of SSI
Specific function is unknown
6. MOD protein/ MLPK protein
Found in stagmatic cell/ papillar cell
It is aquaprotein like protein which form a channel in the plasma membrane and transport water into pollen grains for their germination and growth.
Transport water until the pollen grain become fully turgid.
This is checkpoint for pollen grain growth and their interaction with stigmatic cell

16. Molecular model of the SI in Brassicaceae
(Takayama et al.,2005)

17. 2. GSI in Solanaceae Family
Male Determinant : SLF/SFB (S locus F box protein)
Female determinant: S-RNase (Ribonuclease activity )
S-RNase is expressed in the pistil and is localized mostly in extracellular matrix in the upper region of style, where its concentration may range from 10 to 50 mg/ml.
S-RNase are glycoproteins of 30 KDa; which function as highly specific cytotoxin that inhibits the growth of incompatible pollen tubes.
SLF/SFB are known to function as ubiquitin ligases which degrade the non self S-RNases so that pollen tubes from compatible pollen would grow normally.
Pollen tube growth is inhibited in stylar region
S-RNase enters into both compatible and incompatible pollen tubes but they degrade the RNA of only incompatible pollen tubes.
However the exact mechanism is unknown.
Tobacco (Nicotiana), Petunia, tomato (Lycopersicon), roses .
Two models proposed: Receptor model and Inhibitor model
Sahu 30-11

18. Molecular model of the SI in Solanaceae

19. i. Receptor or Gatekeeper Model
The gatekeeper model assumes that the pollen S product (SFB) is a membrane receptor that allows haplotype-specific uptake of cognate S-RNase. The S-RNase that is taken up, exerts its cytotoxic effects in the pollen tube to stop pollen tube growth.
-Tao and Iezzoni, 2010

20. ii. Inhibitor Model Tao and Iezzoni, 2010
The inhibitor model assumes that the pollen S product (SFB) is an RNase inhibitor that inactivates all but not the cognate S-RNases.
All S-RNases are taken up by the pollen tube, but S-RNases of non-matching S-haplotypes are inhibited/ degraded by pollen S products, S-Rnase inhibitor.
The pollen S product specifically interacts with its cognate S-RNase to leave it active to stop pollen tube growth.
Tao and Iezzoni, 2010

21. 3. GSI in Papaveraceae Family
SI under gametophytic control (GSI)
Pollen germination and pollen tube growth can be inhibited by the recombinant S-protein in an S-haplotype specific manner in the stigmatic surface.
S- protein acts as female determinant
Male determinant: is not yet identified
However a receptor SBP (S protein binding protein) present in pollen plasma membrane and binds with S- protein.
In the Papaveraceae, the only identified female determinant induces a Ca2+-dependent signaling network that ultimately results in the death of incompatible pollen.

22. 3. GSI in Papaveraceae Family
As the incompatible pollen tube is growing along the stigmatic surface, the stigmatic S protein is predicted to interact with the pollen S protein in an S allele specific manner.
The unidentified pollen S product is thought to be a membrane receptor in the pollen tube and may in fact be SBP; nevertheless, SBP is involved in the interaction. Binding of the stigmatic S protein to the pollen S receptor then triggers a signal transduction cascade in the pollen tube.
The signal transduction pathway is thought to involve two phases, a calcium-dependent signaling pathway followed by a calcium-independent signaling pathway.
In the first step, there is a rapid increase in calcium levels which may be mediated by IP3. This is then followed by an increase in phosphorylation of specific proteins, such as p26, by calcium dependent protein kinases.
During this early phase, rearrangements of the actin cytoskeleton are also occurring, though it is not known if this is caused by the calcium signaling (Geitmann et al., 2000). Subsequently, the calcium independent signaling pathway occurs and there is an increase in phosphorylation of other proteins.
These events may lead to gene activation and ultimately may result in programmed cell death and the inhibition of pollen tube growth.

23. SI Response in the Papaveraceae
Shank region of pollen

24. 4. GSI in Poaceae Family
The genetics of SI in the Poaceae remained unknown until studies in Secale cereale by Lundqvist (1954) and Phalaris oerulescens by Hayman (1956). Both researchers proposed that self-incompatibility in grasses was under the gametophytic control of two multiallelic, unlinked genes, named S and Z, which segregated independently. A pollen grain is incompatible when both its S and Z alleles are matched in the recipient pistil.
However, in diploid ryegrass, pollen grains that have at least one different allele from the S or Z alleles in the stigmata are compatible (Yang et al., 2008).
Fig in next page: Pollen–pistil interactions between plants heterozygous at the S and Z alleles results in a degree of compatibility. For example, pollination of a S1S2 Z1Z2 plant with pollen from a S1S3 Z1Z4 plant is 75% compatibility. The S1Z1 pollen is incompatible because both the S1 and Z1 alleles are present in the pistil.
Sophia L. Stone & Daphne R. Goring, 2001

25. 3. GSI in Poaceae Family
Sophia L. Stone & Daphne R. Goring, 2001

26. 3. GSI in Poaceae Family
Sophia L. Stone & Daphne R. Goring, 2001

27. Applications of Self Incompatibility

28. Utilization of SI in Plant Breeding
Production of hybrids
Combining desirable genes
Multiplication of inbred lines
Creation of variability
Testing the combining ability
-B.D. Singh, 2014

29. Production of Hybrids

30. Chaozhi et al. (2009) explained that how the self incompatibility is utilized in hybrid seed production in B. napus and how the self incompatibility is overcome by salt solution spray.
B. rapa and B. oleracea are naturally self-incompatible majorly used as vegetables. On the contrast, B. napus is naturally self-compatible.
They developed one self-incompatible B. napus line ‘S- 271’ by introgressing an S-haplotype of B. rapa called Xishuibai into a B. napus line through interspecific hybridization.
Crossing the self incompatible line (S-271 or S-1300) with restorer lines can produces hybrids.
The hybrid with highest seed yield had very high positive mid-parent heterosis of 115% . Its seed yield was 15% over the check, Zhongyouza2.
They used 3-5 % NaCl solution with hand or bees pollination to overcome the SI. So that they can multiply the S- 271 line.

31. Self incompatible inbred lines of B. oleracea and B
Self incompatible inbred lines of B. oleracea and B. compestris with different S alleles were used in the experiment. Two different S alleles (Sx and Sy) derived from plant material described by Esch (1994). They did single cross and double cross to produce hybrids.
Self­incompatibility was characterised by observing pollen tube growth by fluorescent microscopy (Kho and Baër, 1968) and self seed set.
Best hybrids showed yield performance 10 to 20 % higher than standard cultivars. Hybridity levels were influenced by environmental factors and genotype; nevertheless, in some combinations levels over 90 % have been achieved. Significant heterosis for seed yield exists in oilseed rape.
To overcome the SI; they used 3-5% NaCl solution spray directly on stigma surface and 6% atmospheric CO2 increased after selfing.

32. Single cross hybrids
Population hybrids

33. Molecular markers based on analysis of SLG I, II, SCR I and SRK I can be successfully used for selection of SI plants and undesirable SC plants are eliminated at early stage from breeding population.

34. Continued……

35. Havlickova et al. (2014) showed that the use of a molecular marker based selection of SI plants leads to elimination of undesirable SC plants at an early stage and to favorable reduction of the number of plants available for subsequent experiments.
A particular technique that involved two pairs of primers (SLGa and SCO2) was used to manage the breeding population. L

36. Achievement of Self Incompatibility Crop Variety
Cauliflower
Pusa Hybrid-2, Snow Queen, Snow King, White Contessa
Cabbage
BRH-5, H-44,H-43, Pusa Synthetic, Meenakshi
Chinese Cabbage
Hamburg-3

37. Measures to Overcome SI

38. Temporary Suppression of SI
Temporary suppression of SI is essential for development and multiplication of inbred lines and self incompatible lines. So that they can be used in hybrid development.
Bud pollination (Lilium spp)
Surgical techniques (Brassica spp, Petunia)
High temperature [Trifolium(320C), Lycopersicon, Brassica, Oenothera]
Increased CO2 concentration (Brassica spp)
High humidity (Brassica spp)
Salt (NaCl) spray (Brassica spp) (5-10% for 3-5 days)
Irradiation (Solanaceous crops) (X-rays, γ-rays)
Delayed pollination

39. Two inbred lines of B. rapa; a CO2-sensitive line (HA-11621) and a CO2-insensitive line (HA-11623), were used in this study. Both lines were self-incompatible under normal conditions (control).
Flowers were self-pollinated by hand pollination and incubated in a CO2 incubator (4.5% CO2) for 4 h. They showed significantly different responses to CO2 gas treatment. Specifically, in the CO2-sensitive line, many pollen tubes were seen to penetrate into papilla cells after CO2 treatment. This pollination test confirmed that the CO2-sensitive line had high sensitivity to CO2 while the CO2-insensitive line hardly responded to 4.5% CO2.
In contrast, massive Ca2+ accumulation was observed at the pollen–stigma interface specifically in CO2 -sensitive plants under high CO2 conditions.
Brewbaker and Kwack (1963) were the first to describe the need for a high concentration of Ca2+ for pollen germination and pollen tube growth.
The high concentration of Ca2+ could be needed for activating pectinase to loosen the papilla cell wall, allowing the pollen tube to penetrate (Black and Charlwood, 1995), or for keeping the pollen tube cell wall rigid enough not to burst (Hepler and Winship, 2010).

40. Application of heat during self pollination of open flowers by means of an electric mini soldering iron resulted in seed set in self incompatible Brassica oleracea varieties.
Temperatures of 60, 70 and 80°C were tested.
Compared with bud pollination, in some cases the so called thermally aided pollination (TAP) method, gave a considerably higher seed yield at 70 and 80°C.
In view of the ease and rapidity of TAP it should be ascertained how far this method can replace bud pollination in maintaining inbred lines.

41. Limitations of SI Production and maintenance of inbred lines
Cost of hybrid seed production
Environmental factors sometimes reduce or totally overcome SI
Preferential visit of Bees
Transfer of S-allele is tedious and complicated process.

42. Future Prospects
We don’t know what S-alleles are prevalent in our germplasm of different crops so there is a need to identify and characterize precisely the S-alleles in our germplasm of various crops and utilize the strong alleles to develop stable S.I. parents
Greater understanding of S.I. expression and our ability to clone and transfer S.I. related gene and subsequent exploitation of heterosis by producing hybrid seeds
Can be used preferably for hybrid seed production in various vegetable crops
However, further molecular analyses on the function of S-locus-related genes are necessary, because several reports show that other unknown factors might be involved in SI response.

43. Thank you…

44. References

45. References

46. References

47. References

48. Plant Breeding: Principles and Methods Kalyani Publishers, Ludhiana
References
Plant Breeding: Principles and Methods
B.D.Singh
Ninth Edition, 2012
Kalyani Publishers, Ludhiana

49. References