Molecular control of male fertility for crop hybrid breedingSuresh Antre
Harnessing hybrid vigor or heterosis is a promising approach to tackle the current challenge of sustaining enhanced yield gains of field crops. More than half of the production of major crops such as maize, rice, sorghum, rapeseed, and sunflower comes from hybrid varieties.
this presentation intends to familiarize students with the basic concept of male sterility. this is deemed essential to proceed with the cytoplasmic male sterility.
Molecular control of male fertility for crop hybrid breedingSuresh Antre
Harnessing hybrid vigor or heterosis is a promising approach to tackle the current challenge of sustaining enhanced yield gains of field crops. More than half of the production of major crops such as maize, rice, sorghum, rapeseed, and sunflower comes from hybrid varieties.
this presentation intends to familiarize students with the basic concept of male sterility. this is deemed essential to proceed with the cytoplasmic male sterility.
Association mapping, also known as "linkage disequilibrium mapping", is a method of mapping quantitative trait loci (QTLs) that takes advantage of linkage disequilibrium to link phenotypes to genotypes.Varioius strategey involved in association mapping is discussed in this presentation
Introduction:
Proposed by Meuwissen et al. (2001)
GS is a specialized form of MAS, in which information from genotype data on marker alleles covering the entire genome forms the basis of selection.
The effects associated with all the marker loci, irrespective of whether the effects are significant or not, covering the entire genome are estimated.
The marker effect estimates are used to calculate the genomic estimated breeding values (GEBVs) of different individuals/lines, which form the basis of selection.
Why to go for genomic selection:
Marker-assisted selection (MAS) is well-suited for handling oligogenes and quantitative trait loci (QTLs) with large effects but not for minor QTLs.
MARS attempts to take into account small effect QTLs by combining trait phenotype data with marker genotype data into a combined selection index.
Based on markers showing significant association with the trait(s) and for this reason has been criticized as inefficient
The genomic selection (GS) scheme was to rectify the deficiency of MAS and MARS schemes. The GS scheme utilizes information from genome-wide marker data whether or not their associations with the concerned trait(s) are significant.
GEBV: Genomic Estimated Breeding Values-
The sum total of effects associated with all the marker alleles present in the individual and included in the GS model applied to the population under selection
Calculated on a single individual basis
Gene-assisted genomic selection:
A GS model that uses information about prior known QTLs, the targeted QTLs were accumulated in much higher frequencies than when the standard ridge regression was used
The sum total of effects associated with all the marker alleles present in the individual and included in the GS model applied to the population under selection
Calculated on a single individual basis
Population used:
Training population: used for training of the GS model and for obtaining estimates of the marker-associated effects needed for estimation of GEBVs of individuals/lines in the breeding population.
Breeding population: the population subjected to GS for achieving the desired improvement and isolation of superior lines for use as new varieties/parents of new improved hybrids.
Training population-
large enough: must be representative of the breeding population: max. trait variance with marker : by cluster analysis
should have either equal or comparable LD, LD decay rates with breeding populations
Updated by including individuals/lines from the breeding population
Training more than one generation
Low colinearity between markers is needed since high colinearity tends to reduce prediction accuracy of certain GS models. (colinearity disturbed by recombination)
Hybrid rice breeding problems, prospects and future strategies by Deepak SharmaDeepak Sharma
The presentation describes all the constraints worldwide regarding hybrids in rice and potential solutions. The material includes all the findings and the researches going on in the world. Material collection is surely going to be very helpful from conventional and molecular point of view and having all the recent achievement and work done .
Association mapping, also known as "linkage disequilibrium mapping", is a method of mapping quantitative trait loci (QTLs) that takes advantage of linkage disequilibrium to link phenotypes to genotypes.Varioius strategey involved in association mapping is discussed in this presentation
Introduction:
Proposed by Meuwissen et al. (2001)
GS is a specialized form of MAS, in which information from genotype data on marker alleles covering the entire genome forms the basis of selection.
The effects associated with all the marker loci, irrespective of whether the effects are significant or not, covering the entire genome are estimated.
The marker effect estimates are used to calculate the genomic estimated breeding values (GEBVs) of different individuals/lines, which form the basis of selection.
Why to go for genomic selection:
Marker-assisted selection (MAS) is well-suited for handling oligogenes and quantitative trait loci (QTLs) with large effects but not for minor QTLs.
MARS attempts to take into account small effect QTLs by combining trait phenotype data with marker genotype data into a combined selection index.
Based on markers showing significant association with the trait(s) and for this reason has been criticized as inefficient
The genomic selection (GS) scheme was to rectify the deficiency of MAS and MARS schemes. The GS scheme utilizes information from genome-wide marker data whether or not their associations with the concerned trait(s) are significant.
GEBV: Genomic Estimated Breeding Values-
The sum total of effects associated with all the marker alleles present in the individual and included in the GS model applied to the population under selection
Calculated on a single individual basis
Gene-assisted genomic selection:
A GS model that uses information about prior known QTLs, the targeted QTLs were accumulated in much higher frequencies than when the standard ridge regression was used
The sum total of effects associated with all the marker alleles present in the individual and included in the GS model applied to the population under selection
Calculated on a single individual basis
Population used:
Training population: used for training of the GS model and for obtaining estimates of the marker-associated effects needed for estimation of GEBVs of individuals/lines in the breeding population.
Breeding population: the population subjected to GS for achieving the desired improvement and isolation of superior lines for use as new varieties/parents of new improved hybrids.
Training population-
large enough: must be representative of the breeding population: max. trait variance with marker : by cluster analysis
should have either equal or comparable LD, LD decay rates with breeding populations
Updated by including individuals/lines from the breeding population
Training more than one generation
Low colinearity between markers is needed since high colinearity tends to reduce prediction accuracy of certain GS models. (colinearity disturbed by recombination)
Hybrid rice breeding problems, prospects and future strategies by Deepak SharmaDeepak Sharma
The presentation describes all the constraints worldwide regarding hybrids in rice and potential solutions. The material includes all the findings and the researches going on in the world. Material collection is surely going to be very helpful from conventional and molecular point of view and having all the recent achievement and work done .
MOLECULAR APPROACHES IN RICE PEST MANAGEMENTsubhashree1994
SUBHASHREE PRIYADARSHINI, DEPT. OF ENTOMOLOGY, Ph.D IInd Year, ID No; RAD/2017-14, PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, HYDERABAD., TELANGANA, 500030
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Access to large-scale omics datasets i.e. genomics, transcriptomics, proteomics, metabolomics, phenomics, etc. has revolutionized biology and led to the emergence of systems approaches to advance our understanding of biological processes. With decreasing time and cost to generate these datasets, omics data integration has created both exciting opportunities and immense challenges for biologists, computational biologists, biostatisticians and biomathematicians. Genomics, transcriptomics, proteomics, and metabolomics together they help to bring out the best of characters in plants.
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Molecular characterization of rice (Oryza sativa L.) genotypes using target r...Innspub Net
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Seminar on male sterility and fertility restoration 50026 5 01-2018
1. The Genetic and Molecular Basis of
Cytoplasmic Male Sterility and
Fertility Restoration in Rice
Presented by:
Sonu Shekhawat
Roll No 50026
Division of Genetics
2. Outline
1) Introduction
2) Hybrid rice and its overview
3) Male Sterility; manifestation and its classification
4) CGMS system of hybrid rice production
5) Genes for sterility and fertility restoration in rice and some other crops.
6) Genetic and molecular basis of cytoplasmic male sterility and fertility restoration
7) Case study
8) Future thrust
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
3. Introduction
• Monocotyledonous diploid(2n=24) angiosperm.
• Staple food of more than half of the world’s
population.
• Asia alone accounts for 90% of global rice
consumption.
• Lower Glycemic Index(64) so beneficial for
health too.
• Important plant for genetic & genomic studies.
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
4. Over View of Hybrid Rice
During 2016, hybrid rice was planted in an area of 2.7-3 m.ha. and an additional
rice production of 3.5 to 4.0 m.t. was added.
More than 80 % of the total hybrid rice area is in eastern Indian states.
Totally 91 hybrids(58-private sector, 33-public sector) have been released for
commercial cultivation.
In China, Hybrids account for more than half of the area under rice cultivation in
the country (17 mha out of 30 mha).
Wild Abortive (WA) system is widely used CMS source for hybrid production.
5. Male Sterility
• What is Sterility?
An inability of a living organism to effect sexual reproduction.
• What is Male Sterility?
It is the failure of plants to produce functional anthers, pollen, or male
gametes. Occurs mainly in bisexual plants.
• K. Koelreuter (1763) observed anther abortion within species & species
hybrids.
• More prevalent than female sterility.
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
6. Male Sterility
Phenotypic male sterility Genotypic male sterility Chemically induced male sterility
Structural or staminal MS
Pollen MS
Functional MS
Genic MS
Cytoplasmic MS
Cytoplasmic Genetic MS
Environmental Sensitive MS Environmental Insensitive
Temperature sensitive Photoperiod sensitive
Transgenic MS
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
7. Manifestation of male sterility
Absence or malformation of male organs.
Failure to develop normal microsporogenous tissue anther.
Abnormal microsporogenesis deformed or inviable pollen.
Abnormal pollen maturation
Non dehiscent anthers but viable pollen, sporophytic control.
Barriers other than incompatibility preventing pollen
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
10. CMS lines Restorer genes Sterility transcripts Chromosome
WA-CMS Rf3 and Rf4 WA-352/orf284 or orf288 1 and 10
BT-CMS Rf1 B-atp6/orf79 10
HL-CMS Rf5 and Rf6 atp6/orfH79 10
LD-CMS Rf2 L-atp6/orf79 2
CW-CMS Rf Unknown 4
• Male sterility controlled by interaction of nuclear genes and cytoplasmic
genes.
• All theses genes are dominant except rf17 restoration of fertility
• Rf4 gene : Encoding “pentatricopeptide repeat” (PPR) protein it block the
expression of mitochondrial genes induce CMS.
Fertility Restorer gene in Rice
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
Huang et al. Rice (2014)
11. Genetic basis of cytoplasmic male sterility and fertility restoration
Huang et al. Rice (2014)
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
12. Developed in indica rice cultivars from the wild rice
Oryza rufipogon.
1. Sterility Mechanism
• Pollen abortion at uninucleate stage of microspore
development and
• Determined by genotype of sporophytic tissue.
• WA352 gene is the CMS gene which is composed
of orf284,orf224,orf288 and an unknown
segment is also identified.
2. Fertility Restoration
WA-cms can be restored by Rf3 or Rf4 ,located on
chromosome no. 1 and 10,respectively.
Wild Abortive
Huang et al. Rice (2014)
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
13. Hong Lian
1. Sterility mechanism
A chimeric ORF defined as atp6-orfH79 is gene
governing male sterility.
2. Fertility mechanism
• MS of HL-CMS can be restored by either Rf5 or Rf6.
• Rf5 gene is equivalent to Rf1a and Rf1,encoding for
PPR protein PPR791.
Huang et al. Rice (2014)
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
14. Boro Tai -II
1. Sterility mechanism
• orf79 was first identified and cloned in rice .
Co-transcribed with a duplicated atp6
,called as B-atp6-orf79.
2. Fertility mechanism
• Two fertility restoration genes ,Rf1a and Rf1b,
both encoding proteins containing
pentatricopeptide repeat motifs (PPR) are there
in BT-CMS .Both genes are located on Rf1 locus.
Arose from a wide cross based on the cytoplasm
of Chinsurah Boro II (O. sativa subsp indica) and
the nucleus of Taichung 65 (subsp. japonica)
Huang et al. Rice (2014)
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
15. Lead rice-CMS
1.Sterility mechanism-
Yet to be determined.But , a B-atp6-orf79 like
structure L-atp6-orf79 was identified.
2. Fertility mechanism
Pollen fertility of LD-CMS can be restored by
either Rf1 or Rf2.
The Rf2 gene encodes a mitochondrial glycine
rich protein ,replacement of isoleucine by
threonine at amino acid 78 of the RF2 protein
causes functional loss of the rf2 allele.
Huang et al. Rice (2014)
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
16. MMC MEIOTIC PROPHASE 1 DYAD TETRAD
MIDDLE MICROSPORELATE MICROSPOREBICELLULAR POLLENTRICELLULAR POLLEN
A SCHEMATIC REPRESENTATION OF ANTHER AND POLLEN DEVELOPMENT IN WT(wild type) RICE (Zhang and Wilson,2009)
BP-Binucleated pollen; E-Epidermis; En-Endothecium; ML-Middle layer; T-tapetum; MMC-Microspore Mother Cell;
MC-Meiotic Cell; DY-Dyad; Td-Tetrad; MP-Mature pollen.
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
17. MMC MEIOTIC PROPHASE DYAD TETRAD
MIDDLE MICROSPORELATE MICROSPOREBICELLULAR POLLENTRICELLULAR POLLEN
A SCHEMATIC REPRESENTATION OF ANTHER AND POLLEN DEVELOPMENT IN WA-CMS(wild abortive) (Luo et al,2013)
AP-Aborted pollen; BP-Binucleated pollen; E-Epidermis; En-Endothecium; ML-Middle layer; T-tapetum; MMC-
Microspore Mother Cell; MC-Meiotic cell; DY-Dyad; Td-Tetrad; MP-Mature pollen.
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
18. Molecular Basis of CMS and its Restoration
Cytotoxicity and detoxification:
• CMS is caused by a cytotoxic peptide, ORF79, encoded by mitochondrial dicistronic gene B-
atp6 orf79.
• ORF79 is a transmembrane protein, accumulated in microspore.
• Molecular mechanism is still unknown.
Restoration:
• By blocking ORF79 production through mRNA silencing
• Endonucleolytic cleavage of the dicistronic B-atp6-orf79 mRNA by RF1A and degradation
byRF1B leading to reduced orf79 expression
1). BT-CMS
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
Huang et al. Rice (2014)
19. Fertility Restoration
• A RF5’s partner protein ,GRP162 ,can only bind to atp6-orfH79
through an RNA recognition motif. Interaction between these
protein is called restoration of fertility complex (RFC) cleave atp6-
orfH79.
• RF6 gene can also restore the fertility of HL-CMS.
2.) HL-CMS (Hong lian )INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
Huang et al. Rice (2014)
orfH79 + P61 subunit
of ETC complex III
ETC activity
reduced
Reduced ATP
production
Increase in ROS and
orfH79 accumulation.
Programmed Cell Death in
Microspores.
Mitochondrial Dysfunction
20. 3) WA –CMS (wild abortive)
A protein causing premature tapetal PCD:
• The interaction of WA352 with OsCOX11,
prevents the normal function of OsCOX11 in
H2O2 degradation.
• COX11 protein is essential for the assembly of
cytochrome oxidase.
Restoration mechanism:
• Restoration by either by Rf3 or Rf4 .
• Rf4 causes 20-25% reduction in WA352
transcripts, but were not affected in the Rf3
carrying genes.
• RF4 functions like RF1B that mediates the
degradation of atp6-orf79mRNA.
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
Huang et al. Rice (2014)
21. CMS genes are unknown.
Restorer genes cloned:
• Fertility of LD-CMS is restored by either RF1 or RF2.
• Although it possesses chimeric atp6-orf79 dicistronic gene , L-atp6-orf79, CMS is
not due to accumulation of ORF79.
• In LD-CMS rf17 is the fertility restorer gene .
• SNP in promoter region is speculated to restore compatibility between nucleus
and mitochondria ,leading to male fertility.
4) LD-CMS and CW-CMS
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
Huang et al. Rice (2014)
22. CASE STUDY-1
Objective-
• Determination of chromosomal location of two rf loci by linkage to molecular marker .
• To characterize the genetic effect of genes on fertility restoration.
Material and Methods-
• Zhenshan 97A , Zhenshan 97B and Minghui63 .
• Southern blot hybridization , QTL analysis , RAPD and RFLP markers .
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
23. 1.) Fertility segregation in F2 fits in 15;1 ration ,
indicating that fertility restoration is controlled by
two major dominant gene.
The recombination frequencies suggested that the Rf
locus on chromosome 1 is located closest to RG532.
Results
2.)
However, the distances calculated based on the highly
sterile individuals did not add up to those in the local
maps constructed using the random sample (Fig.1),
suggesting that the highly sterile individuals are not
necessarily all homozygous for the recessive allele.
Fig.1 Molecular marker linkage map showing
Rf loci
3.) Recombination frequencies
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
24. Continued…
The estimates indicated that both loci had major effects
of almost complete dominance on fertility restoration
and the locus on chromosome 10 conferred a larger
effect than did the one on chromosome 1 .
It is also clear from Table 3 that the two loci acted
as a pair of classical duplicate genes; a single
dominant allele at one of the two loci would
suffice to restore the fertility to normal.
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
25. Discussion
Rf locus on chromosome 10 corresponds to the one determined by
Bharaj et al. (1995) using trisomic analysis.
We further infer that the Rf locus on chromosome 1 detected in the
present study is the same as the one identified by Zhang et al. (1997).
No RF locus detected on chromosome no.7 , as suggested by Bharaj
et al. (1995) although the polymorphic markers obtained along this
chromosome would allow for the detection of such a locus.
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
26. CASE STUDY 2 The Plant Journal (2016)
Objective-
To provide a model for a molecular mechanism of fertility restoration of RF2 gene and
the difference in pollen abortion level between the BT-CMS and LD-CMS line and
different fertility restoration effect of RF2 in these two lines.
Materials and Methods-
BT-CMS line of cultivar Taichung 65 and LD-CMS line Taichung 65
Western blotting, Southern blotting, Immunoblot analysis.
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
29. Discussion
1. Accumulation of ORF 79 in the mitochondria causes LD –CMS and BT-
CMS.
2. Orf 79 gene of both CMS line has the same sequence except for a single
SNP in LDS –CMS , which leads to an amino acid substitution in LD type
mitochondria.
3. RF2 , a glycine rich domain containing protein, facilitate the degradation
of atp6-orf79 in LD-CMS and BT-CMS line.
INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
30. INDIANAGRICULTURALRESEARCHINSTITUTE,NEWDELHI
Future Thrust
1) Need of commercialization of cytoplasm other than WA.
2) The genetic and molecular basis is still not clearly understood for all the
cytoplasm, this area need to be focused.
3) Their should be expansion of germplasm use to widen the genetic base for
hybrid rice production.
4) With the increasing population it’s a challenge to secure high heterosis levels in
hybrid rice.
