Molecular Plant Breeding.

1. 1
Presented by
Balaji S. Thorat
M. Sc. (Scholar)
Department of Agril. Botany
Dr. B. S. K. K. V., Dapoli

2. A new tool for an old scienceA new tool for an old science
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NATIONAL SEMINAR

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5. INTRODUCTION
Rice (Oryza sativa L.) is staple food of more than 60% of
Indian population. Rice occupies pivotal place in Indian
Agriculture.
Cytoplasmic male sterility (CMS) is a cornerstone of hybrid
production in rice.
In three-line hybrid systems, use of CMS, maintainer, and
fertility restorer lines is necessary for production of hybrid
seeds.
Limited sources and low variation of CMS lines cause
genetic vulnerability to pathogens.
Therefore, diversifying the CMS sources, maintainer lines
and restorers is indispensible for a sustainable production
system of hybrid seed. 5

6. Indirect selection for a desired
plant phenotype based on banding
pattern of linked molecular (DNA)
markers
Marker2
Marker1
chromosome
Gene of no interestGene of interest
Marker1 Marker2
Gene itself
Marker
3
Marker3
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7. 7
MARKER-ASSISTED
BACKCROSSING

8. Parental line improvement in rice by Marker-
Assisted Backcrossing (MAB)
crop Character Gene/QTL Marker Assisted Seletion
Forground Background
Rice (Oryza
sativa L.)
Bacterial leaf blight
resistance
xa21, xa5, xa13 STS, SSR or
CAPS
RFLP, AFLP
Blast resistance Pil SSR ISSR
Quality Waxy (6) RFLP AFLP
Root traits and
aroma
QTL on
chromosomes 2,
7, 8, 9 and 11
RFLP and
SSR
RFLP and
SSR
Submergence
tolerance
QTL Sub1 SSR and
phenotyping
SSR
Sterility WA-CMS SSR SSR
Fertility restoration Rf1 STMS STMS
Collard and Mackill, 2008
Selected examples of MAS for gene transfer through Marker Assisted
Backcrossing programme
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9. Material and method
Yosen B line (maintainer line) Recipient parent
IR68897A (CMS) Donor parent
IR24 and IR36 fertility restorer lines
CASE STUDY -1
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10. Molecular breeding scheme for converting Yosen B fertile line to a novel CMS line via
marker-assisted backcrossing (from BC1F1 to BC4F1).
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11. 11
Ahmadikhah et. Al., 2015

12. Ahmadikhah et. Al., 2015
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13. INFERENCE
 Results showed that three backcrosses facilitated by SSR and
ISSR markers were sufficient to recover the RPG with high
efficiency.
 Thus MABC greatly accelerated the efficiency of CMS
development program and could save three to four
generations of backcrossing.
 Finally, developed a novel CMS line (Yosen A) comparable
to the original maintainer counterpart maintainer line, which
can be integrated in hybrid seed production program.
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14. What Is GENE
PYRaMIDING ?
 Process of combining two or more genes
from multiple parents to develop elite lines
and varieties.
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15. GENE PYRaMIDING
 Widely used for combining multiple disease resistance
genes for specific races of a pathogen.
 Important to develop ‘durable’ insect and pest resistance
against different races.
 It also used for to improve the qualitative characters into
those have absence that traits.
- e.g. amylose content, aromatic fragrance etc
 Gene pyramiding is extremely difficult to achieve using
conventional methods,
- Consider: phenotyping a single plant for multiple forms
of seedling resistance – almost impossible
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16. Resistant Rice Plant
GENE PYRaMIDING IN
RICE
BaCtERIal
REsIstaNCE
shEath
BlIGht
INsECt
REsIstaNC
Dattaet.Al.,2002
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17. sElECtED ExaMPlEs oF GENE PYRaMIDING IN
RICE.
Crop Character Gene transferred DNA markers
Parent 1 Parent 2
Rice (Oryza
sativa L.)
Bacterial leaf blight
resistance
xa5, xa13 Xa4, Xa21 RELP, STS
Blast resistance Pil, Piz-5 Pil, Pita RFLP, STS
Brown plant hopper
resistance (Bpt12, Bpt15)
Bph1 Bph2 STS
Grain quality, fertility
restoration
Rf1, Rf3 - STMS, AELP
Aromatic fragrance (badh
2.1)
Mianhui 725 WH6725 SSR, STS
Collard and Mackill, 2008
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18. CasE stUDY - 2
OBJECTIVE
 To transfer of Bacterial Blight Resistance Genes.
 To transfer of insect Resistance Genes.
 To transfer of Sheath Blight Resistance Genes.
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20. 1. Different lines carrying the Xa21 and RC7 genes showing a variable
response to their respective bioassays
Datta et. Al., 2002a : Selected for next generation
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21. INFERENCE
 Identified of F1 plants introgressed with RC7, Xa21 and Bt genes.
 All these PCR-positive F1 plants showed the HindIII/BamHI 1.6-
kb RC7 fragment received from the male parent TT-9 (carrying
the RC7 and Bt genes) was a female parent, PCR analysis of the
F1 plants identified the presence of the RC7 gene (Fig. 2a).
 All the PCR-positive F1 plants showed the expected 3.8-kb
EcoRV fragment inherited from the male parent TT-103. PCR-
negative lanes in Fig. 2a and b represent the plants from the selfed
seeds.
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22. TRANSGENIC
BREEDING / GENETIC
ENGINEERING /
TRANSFORMATION
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24. CASE STUDY -3
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25. In this study, to improve plant height and panicle
exsertion of the A line
two components
artificial micro-RNA (ami-RNA)
artificial target mimicry
A ami-RNA is a recently developed gene silencing
method with high specificity, while target mimicry
is a natural mechanism inhibiting the miRNA
function that was also recently characterized.
This approach provides a paradigm to tune the
expression of endogenous genes to achieve the
desired phenotype by combining ami-RNA and
artificial target mimicry technologies. 25

26. ExpERIMENTAl pROCEDURES
1. Construction of ami-RNA and MIM expression vectors
 The ami-RNA precursor was released from the T vector with the
digestion of BamHI and KpnI and cloned into the vector pC1300-
Ubi-nos, between the maize ubiquitin promoter and the nos
terminator, to form the final ami-RNA expression vector pEUI-ami-
RNA. PC1300-Ubinos is a previously modified vector derived from
the Ti binary vector pCAMBIA1300 by inserting the maize ubiquitin
promoter and the nos terminator into its multiple cloning site.
 The artificial MIM genes were generated by modifying the native
rice target mimic gene OsIPS1. The OsIPS1 had been isolated and
cloned into the plasmid MT375. The MIMa and MIMb were
finally cloned into pC1300-Ubi-nos between the maize ubiquitin
promoter and the nos terminator.
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27. Experimental procedures
Collection of Parent Materials
Construction of ami-RNA and MIM expression vectors
Rice transformation
Southern blot analysis
RT-PCR and qRT-PCR
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28. Chen et. Al., 2013
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29. Fig. (a) The design of the mimicry
sequences of MIMa and MIMb
mRNAs.
Fig. (b) flow diagram of all
transformations and cross
combinations in this study.
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30. Chen et. Al., 2013
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31. The phenoType of TeZS97A
Fig. (a) The teZS97A
has greater plant
height
Fig. (b) Panicle
exsertion
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32. The phenoType of Te-hybridS And The
relATive expreSSion level of eui1 in Te-
hybridS.
Fig. (a) The hybrid teZS97B/MH63 was significantly taller than wild-type ZS97B/MH63.
Fig. (b) The plant height of teZS97B/MH63-MIMa recovered to normal level after MIMa was
introduced.
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33. inference
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With the help of an ami-RNA and a target mimic (MIM) gene
improved the Panicle exsertion of newly developed line and
their hybrids also.
The hybrid derived from the crossing of the te-A line and the
MIM R line remains a semi-dwarf phenotype if the R line is
semi-dwarf because MIM genes from the R line suppress the
activity of amiRNA-eui1 from the te-A line.

34. concluSion
Developed a novel CMS line (Yosen A) comparable to the
original maintainer counterpart maintainer line, which can
be integrated in hybrid seed production program by MAB.
(Ahmadikhah et. Al., 2015)
Identified of F1 plants introgressed with RC7, Xa21 and Bt
genes by gene pyramiding. (Datta et. Al., 2002)
With the help of an ami-RNA and a target mimic (MIM)
gene i.e. Transgenic breeding improved the Panicle
exsertion and plant height of newly developed line and their
hybrids also. (Chen et. Al., 2013)
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35. Cooperation between breeders
and molecular biology scientists
can enhance the PLANT BREEDING
considerably.
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