Application of Molecular markers for identification of restorers basis and case studies.

1. APPLICATION OF MOLECULAR
MARKERS FOR
IDENTIFICATION OF
RESTORERS
Speaker :
Samrath Baghel
Ph.D. , IInd Sem
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DOCTORAL SEMINAR I - GP 691

2. CONTENT
• HYBRID AT A GLANCE
• WHY MOLECULAR MARKERS NEEDED FOR Rf GENE SCREENING
• THE TEST CROSS NURSERY (TCN).
• CGMS MALE STERLITY
• CONCEPTS AND MECHANISM OF CYTOPLASMIC MALE STERLITY & FERTILITY RESTORATION .
• TYPES OF MALE STERLITY SYSTEMS
• IDENTIFICATION OF FERTILITY RESTORATION (Rf) GENE .
• CASE STUDY I & II
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3. HYBRID , At a Glance …
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MALE STERILITY
HYBRID VIGOUR
HYBRIDS
RESTORER
MAINTAINERS
TEST CROSS
NURSERY
POLLEN FERTLITY TEST
SPIKELET FERTILITY TEST
MALE FERTILITY HETEROSIS

4. WHY MOLECULAR MARKERS NEEDED FOR Rf GENE SCREENING
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• MOLECULAR MARKER: Differences in the nucleotide DNA at the
corresponding site on homologous chromosome which follow mendelian pattern of
inheritance or follow maternal inheritance.
• The molecular markers aid the selection procedure of Restorers through MAS
(Marker Aided Selection) .
• The nature of genes involved in fertility restoration is precisely understood through
Molecular Markers
• The discovery of new genes responsible for fertility restoration is identified .

5. THE TEST CROSS NURSERY (TCN)
• The test cross nursery used to identify Restorers/Maintainers/Partial restorers/Promising F1’s
over there parents and standard checks.
• TCN is field validation of restorers ( Rf gene) by using two methods
a. Pollen Fertility Test.
b. Spikelet Fertility Test.
• Spikelet fertility (SF) - Observe the open pollinated panicles of F1s for seed setting in
comparison to the corresponding male parent
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6. • POLLEN FERTILITY/STERILITY - the ratio of fertile/sterile pollen grains to the total pollen
grains counted in 3-4 fields under a microscope, and expressed in percentage. Fertility/sterility of
pollen grains is decided by their stainability with 1% IKI stain.
Pollen fertility/sterility gradation.
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PHENOTYPIC MALE STERLITY GENOTYPIC MALE STERLITY
CHEMICALLY INDUCED MALE
STERLITY

8. CONCEPTUALIZING MALE STERLITY & FERTILITY RESTORATION
• German botanist Joseph Gottlieb Kolreuter 1763 reported failure to produce/dehiscent
anthers/functional pollen and viable male gametes.
• Defective development of the male reproductive organ, the anthers, causes male
sterility
• Male sterility mutants caused abnormal development either of Sporophytic or
Gametophytic anther tissues.
• Sporophytic male sterile mutants effects mainly tapeta or meiocytes which leads to
pollen abortion/ pollenless sterility.
• Gametophytic male sterile mutants mainly affects development of microspores / pollen
grain.
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Chen, L. and Lui, Y.G. (2014)

9. FERTILITY RESTORATION
• Fertility restoration gene (Rf) is functional in cytoplasmic male sterility system, regulated by both
cytoplasmic and nuclear gene interaction.
• There are two kind of cytoplasms , N (Normal) and S (Sterile) and Restorer of fertility genes (Rf).
• The (Rf) genes do not have any expressions of there own but expresses in presence of sterile (S)
cytoplasm only.
• (Rf) genes only restore fertility in sterile (S) cytoplasm.
• A Line =
• B Line =
• R Line =
• Any mutation in (Rf),results in no fertility restoration.
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rd
rd
rf rfS
N
S/N
rf rf
Rf Rf

10. (A) THREE LINE SYSTEM
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CGMS MALE STERLITY SYSTEM
CYTOPLASM (Rf) GENE RESULT
Fertile (F) rfrf / Rfrf FERTILE
Sterile (S) Rf FERTILE
Sterile (S) rfrf STERILE

11. PROCEDURE TO SCREEN FERTILITY RESTORATION (Rf) GENES
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F1
CMS X RESTORER LINE (BASED OF POLLEN FERTLITY TEST)
F2
F6, Mapping Population constituted ( Select only Fertile)
DNA EXTRACTION , CANDIDATE GENE BASED MARKERS AND
FUNCTIONAL MARKERS WERE USED FOR SCREENING Rf locus .
RESTORER LINE USED AS CHECK TO IDENTIFY RESTORATION ALLELE
& CMS LINE USED TO IDENTIFY STERLITY INDUCING ALLELE.
SCREENING PROCEDURE WHEN THE FERTILITY RESTORATION (Rf) GENE IS UNIDENTIFIED.

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(B) SCREENING PROCEDURE WHEN THE FERTILITY RESTORATION (RF) GENE IS IDENTIFIED.
DNA extraction from the various Restorers
Running the known markers for fertility restoration
Lines showing the alleles are identifies as restorers

13. Characterized cytoplasmic male sterility (CMS)/restorer (Rf ) gene systems in major crops
13Chen, L. and Lui, Y.G. (2014)

14. CASE STUDY-I :
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• This is marker‐cum‐phenotype‐based restorer selection using TRJ‐derived lines.Combined
phenotypic and molecular screening using gene‐based and functional markers identified
42 lines that carried Rf3 and/or Rf4 genes.
• All the selected lines produced fertile F1s when crossed to a WA‐CMS. Gene‐based
(DRRM‐Rf3‐5 and DRRM‐Rf3‐10) and functional marker (RMS‐SF21‐5) targeted Rf3
locus, while gene linked (RM6100) and functional marker (RMS‐PPR9‐1) targeted the Rf4
locus.
INTRODUCTION

15. PUSA 44 X 29 diverse Tropical japonica (TRJ)
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MATERIAL AND METHODS
F2 (NPT SELECTED)
F6 (310 NPT DEVELOPED)
DNA extraction by CTAB Method
Candidate gene based markers DRRM‐Rf3‐5, DRRM‐Rf3‐10 and a functional
marker RMS‐SF21‐5 were used for screening the Rf3 locus.
&
Gene linked marker RM6100 and a functional marker RMS‐PPR9‐1 were
used for identifying the Rf4 locus
• Two checks used : PRR 78 for Rf gene and Pusa 6A for sterility inducing gene.
The gel scoring was done based on the amplicon size of the restorer, “PRR
78” and a WA‐based CMS line, “Pusa 6A”.

16. DISTRIBUTION OF FERTILITY RESTORER GENE(S) IN THE TRJ‐DERIVED LINES.
• The marker alleles were identified based on the amplicon size produced by test lines, “PRR 78” for the restorer allele and
“Pusa 6A” for the non‐restorer/ maintainer allele .
• Different markers produced different amplicon sizes and in most of the cases, the fertility restoring (F) allele was longer than
its alternate non‐restorer allele (M) in the male sterile line.
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RESULTS

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18. Case study II
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INTRODUCTION
• C-type cytoplasmic male sterility (CMS-C) is widely utilized for hybrid maize there is a
divergence on the number of fertility restorer genes in maize inbred line A619 for CMS-C.
• CMS-C has stable male sterlity and has positive effect on grain yield.
• Fertility restoration of CMS-C is controlled by two dominant genes, Rf4 and Rf5, which located
on chromosomes 8 and 5 and Rf6 and some quantitative trait loci (QTLs) involved in the partial
restoration of male fertility for CMS-C have also been identified.
• Cloning and genetic complementation experiments indicating that GRMZM2G021276 is a
candidate gene for CMS-C fertility restoration

19. MATERIAL & METHODS
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• To identify restorer mechanism of A619 gene in CMS-C , Segregating F2 (C48-2 X A619) used as mapping population as
restorer gene segregated in 15:1 ratio, two dominant restorer genes in A619 for CMS-C, i.e., Rf4 and a novel gene named
Rf*-A619 are responsible.
• Based on the sequence differences between Rf4 and its recessive allelic rf4, a novel dominant marker F2/R2 was developed
and validated to genotyping Rf4 in the F2 population, Results concluded that inbred line A619 one additional restorer gene
for CMS-C ,except Rf4.
• To identify the specificity of primers F2/R2, we got its amplifying band sequence from A619 by direct sequencing and
searched it with maize GDB BLAST program in the maize genome.
• Tightly linked SSR marker used for dominant restorer gene Rf5 which differentiated those fertile plants without Rf4 in the
F2 population and PCR amplification showed that Rf*619 is not allelic to Rf5 but is noval restorer gene for CMS-C.

20. RESULTS
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• Two Fertility Restorer Genes namely Rf4 and Rf*- A619 reported in the background of A619 Inbred
though other alleles may also be found but those involve highly complicated mechanisms. Rf4 is
located on Chromosome 8
• The hybrid of (C48-2 A619)F1 was totally fertility restored, then self pollinated to obtain its F2
• Plant male fertility was graded on a scale of I to V as follows I
• Plants with scores of I or II were viewed as sterile, while scores of III, IV, and V were recorded as fertile
plants.

21. • Pollen fertility was rated on a scale of 1 to 3 according to the pollen staining ability using 1% (w/v)
KI-I2
• A total of 165 F2 plants in 2014 and 150 F2 plants in 2015 were used for Rf4 genotyping
• The presence of an amplification fragment for Rf4 and no amplification indicated the rf4 genotype
using F2/R2 in this experiment.
• The plants without Rf4 in the (C48-2 A619)F2 population were analyzed using the SSR marker
bnlg1346 (50-CATCATGAAGCAATGAAGCC-30, 50-CCGCGCCATTATCTAGTTGT- 30), which is
tightly linked with the Rf5 gene . bnlg 1346 is located on 5.07 bin position.
• The amplification fragment by F2/R2 represented (550bp) for Rf4_ genotype and the lack of
amplification indicated the genotype of rf4rf4 in this experiment.

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