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Welcome

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RICE FLOWER

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MARKER ASSISTED SELECTION OF

MALE STERILITY IN RICE

VIPIN MOHAN 2011-09-112

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Ric

e –

the

grai

n

Second in production & consumption

Staple food

90% of world’s rice is produced and

consumed in Asia

Wide adaptability

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but….

• Growth in rice production :• 2.5 – 3.0 % during 1970 – 80• 1.5 % during 90’s

• Population growth :• by 2025 - 8 billion

• Required rice production :• 40 % more

Nas et al., 2013

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What is Male Sterility? Failure of plants to produce functional anthers, pollen,

or male gametes. Agronomically important for the hybrid seed

production Occurs mainly in bisexual plants. Pollen sterility/structural sterility/functional sterility

Flower of male-fertile chilly Flower of male-sterile chilly

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Based on its inheritance or origin Chemically induced male sterility Transgenic male sterility Cytoplasmic male sterility (CMS) Nuclear male sterility (NMS) Cytoplasmic-genetic male sterility

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Biochemical means of producing male sterile plants

Feminizing hormones

Inhibitors of anther or pollen developmenta. acting on sporophytic tissueb. acting on gametophytic tissue (gametocides)

Inhibitors of pollen fertility

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Advantages of Chemical hybridization

High degree of efficacy and developmental selectivity Persistence during the development of flower or spikes Low cost Acceptable levels of toxicity Low general phytotoxicity Agronomic performance of hybrid seed

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Barnase/Barstar system for engineered male sterility (Mariani et al., 1992)

Male sterility through modification of biochemical pathways (Marc et al., 2000)

MALE-STERILITY THROUGH RECOMBINANT DNA

TECHNOLOGY

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Barnase/Barstar system Targeting the expression of a gene encoding a

cytotoxin by placing it under the control of an anther specific promoter (Promoter of TA29 gene)

Expression of gene encoding ribonuclease (chemical synthesized RNAse-T1 from Aspergillus oryzae and natural gene barnase from Bacillus amyloliquefaciens)

Success in oilseed rape, maize and several vegetative species

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Male sterility through modification of biochemical pathwaysCarbohydrates :- Play a critical role in the anther and pollen development by sustaining

growth as well as signal pathways. Their transportation from photo synthetically active source tissues to

developing sinks is regulated by extra cellular invertase The extracellular invertase Nin 88 of tobacco shows expression pattern

in developing anther The tissue specific antisense repression of nin88 under the control of

Nin88 promoter in plant caused male sterility . Exogenous supply of carbohydrates able to partially overcome blocking

of pollen development so it maintaining the male sterility. Restoration by crossing this GMS system with plants expressing

distantly relate invertase (Marc et al.,2000)

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Cytoplasmic male-sterililty Structural changes in the cytoplasmic organelle

genome Maternally inherited Three lines of plants must be maintained(A,B and R

lines)

It is divided into:a. Autoplasmic b. Alloplasmic

Eg.: Pusa6A and IR262829A

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Drawbacks: insufficient or unstable male sterile

Difficulties in restoration system

Difficulties with seed production

Undesirable pleiotropic effect

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Genetic male sterility

Genic/genetic/Mendelian Inheritable

Controlled by a number of nuclear genes (a

pair of recessive alleles “msms”)

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Temperature - Changing the optimal temperature can induce sterility

eg.: EC720903, C815S

Photoperiod - It has a strong influence (Photoperiod sensitive)

Changing the growth habit can stimulate the sterility eg.: Nongken58S

Determining factor

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Advantages2 line systemStability100% male sterile progeniesInheritableRestoration

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Cytoplasmic - genetic male sterility

Controlled by - nuclear (with Mendelian inheritance) and cytoplasmic (maternally inherited) genes

Restorers of fertility (Rf) genes present Rf genes - no expression of their own unless the sterile

cytoplasm is present Plants with:

• N cytoplasm are fertile • S cytoplasm with genotype Rf- leads to fertile • S cytoplasm with rfrf produces only male steriles

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Limitations of CGMS systemUndesirable effects of the cytoplasmUnsatisfactory fertility restoration Unsatisfactory pollination Spontaneous reversionModifying genesContribution of cytoplasm by spermEnvironmental effectsNon- availability of suitable restorer line

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MARKER ASSISTED SELECTION

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MARKERA marker (morphological, biochemical or one based on DNA/RNA variation) is used for indirect selection of a genetic determinant or determinants of a trait of interest (e.g. productivity, disease resistance, abiotic stress tolerance, and quality). This process is used in plant and animal breeding.

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MARKERS

Morphological Biochemical DNA based

Hybridization based e.g. RFLP PCR based Chip based

Arbitrary primers e.g. RAPD,ISSR,AFLP Specific primers SNP based

Repeat based e.g. SSRs

Sequence based e.g. SCAR, CAPs, SNP

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F2

P2

F1

P1 x

large populations consisting of thousands of plants

PHENOTYPIC SELECTION

Field trialsGlasshouse trials

DonorRecipient

CONVENTIONAL PLANT BREEDING

Salinity screening in phytotron Bacterial blight screening Phosphorus deficiency plot

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Molecular Marker Assisted Breeding

Conventional screening difficulties

Molecular marker indicates the presence or absence of gene at an

early stage

A molecular marker very closely linked to the target gene can act

as a “tag” which can be used for indirect selection of target gene

(Jena et al., 2003)

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F2

P2

F1

P1 x

large populations consisting of thousands of plants

ResistantSusceptible

MARKER-ASSISTED SELECTION (MAS)

MARKER-ASSISTED BREEDING

Method whereby phenotypic selection is based on DNA markers

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Markers must be polymorphic

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8Primer A Primer B

P1 P2

P1 P2

Not polymorphic Polymorphic!

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Markers must be tightly-linked to target loci!

• Ideally markers should be <5 cM from a gene or QTL

• Using a pair of flanking markers can greatly improve reliability but increases time and cost

Marker A

QTL5 cM

RELIABILITY FOR SELECTION

Using marker A only:

1 – rA = ~95%

Marker A

QTL

Marker B

5 cM 5 cM

Using markers A and B:

1 - 2 rArB = ~99.5%

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Sl. No

TGMS gene

Linked Marker Chromosome location

References

1 tms1 OPB-19(RAPD) 8 wang et al., 1995

2 tms2 RM11,RM2 9Lopez et al., 2003,

Pitnjam et al., 2008,Yamaguchi et al., 1997

3 tms3 OPF-18,OPAC-3(RAPD) 6 Lang et al.,1999, Subudhi et al. 1997

4 tms4 RM27 2 Dong et al., 2000

5 tms5 RM174,RM5862,RM5897 2 Nas et al., 2005

6 tms6 RM3351 5 Lee et al., 2005

7 tms7 RM224,RM21 11 Hussain et al.,2012

8 tms9 Indel37,Indel57 2 Sheng et al. 2013

TGMS genes and their linked markers in rice

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(1) LEAF TISSUE SAMPLING

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

Overview of ‘marker

genotyping’

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Advantages of MAS

Simpler method compared to phenotypic screening

Selection at seedling stageIncreased reliability More accurate and efficient selection of

specific genotypesMore efficient use of resources

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Marker-assisted backcrossing (MAB)

Method to introgress a single locus controlling a trait of interest while retaining the essential characteristics of recurrent parent.

Marker-assisted backcrossing (MAB)

MAB has several advantages over conventional backcrossing:

– Effective selection of target loci

– Minimize linkage drag

– Accelerated recovery of recurrent parent

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P1 x F1

P1 x P2

CONVENTIONAL BACKCROSSING

BC1

VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY RESEMBLE RECURRENT

PARENT

BC2

MARKER-ASSISTED BACKCROSSING

P1 x F1

P1 x P2

BC1

USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS THAT HAVE MOST RP MARKERS AND SMALLEST %

OF DONOR GENOME

BC2

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Gene pyramiding

• MAS helps to identify the desired

resistant genes

• Resistance GENES can be

pyramided to make a line having

multi – race resistance

Samis et al. (2002)

• MAS has provided an approach to

pyramid beneficial alleles

F2

F1Gene A + B

P1Gene A

x P1Gene B

MAS

Select F2 plants that have Gene A and Gene B

Genotypes

P1: AAbb P2: aaBB

F1: AaBb

F2AB Ab aB ab

AB AABB AABb AaBB AaBb

Ab AABb AAbb AaBb Aabb

aB AaBB AaBb aaBB aaBb

ab AaBb Aabb aaBb aabb

Process of combining several genes, usually from 2 different parents, together into a single genotype

x

Breeding plan

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Studies…

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CONCLUSION

With the assistance of Biotechnology(marker assisted breeding), we can produce rice hybrids very effectively with in a short time. It will be a big solution for meeting demands of growing population.

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References:

1. Dong NV, Subudhi PK, Luong PN, Quang VD, Quy TD, Zheng HG, Wang B, Nguyen HT (2000). Molecular mapping of a rice gene conditioning thermosensitive genic male sterility using AFLP, RFLP and SSR techniques. Theor. Appl. Genet. 100: 727-734.

2. Jing J., Mou T., Yu H., and Zhoru F. 2015. Molecular breeding of TGMS lines of rice for blast resistance using Pi2 gene. Rice 8:11.

3. Nas, T. M. S., Sanchez, D. L., Diaz Ma. G. Q., Mendioro M. S. and Sant S. Virmani S. S. 2005. Pyramiding of thermosensitive genetic male sterility (TGMS) genes and identification of a candidate tms5 gene in rice. Euphytica 145:67-7.

4. Khora P., Priyadarshi R., Singh A., Mohan R., Gangashetti M. G., Singh B. N., Kole C. and Shenoy V. 2012. Molecular characterization of different cytoplasmic male sterility lines using mitochondrial DNA specific marker in rice. J. biosci. 98: 56-78.

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5. Lang, N. T., Subudhi, P. K., Virmani, S. S.,Brar, D. S., Khush, G. S. andli, Z. 1999. Development of PCR-based markers for thermosensitive genetic male sterility gene tms3(t) in rice (Oryzasatiua L.) heredital 131:121-127.

6. Ngangkham, U., Parida, S. K., De, S., Kumar, A. R., Singh, A. K., Singh, N. K., and Mohapatra, T. 2010. Genic markers for wild abortive (WA) cytoplasm based male sterility and its fertility restoration in rice. Mol. Breed. 26:275-292

7. Niya Celine V. J., Roy Stephen, Manju R.V. and Shabana R. 2014. Evaluation of thermosensitive genic male sterile lines in rice suitable to Kerala through marker assisted selection J. Trop. Agric. 52 (1) : 74-78.

8. Wang, B., Wang, J. Z., Wu, W., Zheng, H. G., Yang, Z. Y., Xu, W. W., Ray, J. P. and Nguyen, H. T. 1995. Tagging and mapping the thermo-sensitive genic male-sterile gene in rice (Oryzasativa L.) with molecular markers. Theor. Appl. Genet. 91:1111-1114.

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9. Wang YG, Xing QH, Deng QY, Liang, FS, Yuan LP, Weng ML, Wang B(2003). Fine mapping of the rice thermo sensitive genic male sterile gene tms5. Theor. Appl. Genet. 107: 917-921.

10. Yamaguchi, Y., Ikeda, R., Hirasawa, H., Minami, M. andUjihara, P. 1997. Linkage analysis of thermosensitive genic male sterility gene, tms-2 in rice (Oryzasativa L.). Breed Sci. 47:371-373.

11. Yang, R. C. and Wang, N. Y. 1988. 5460S Indica photosensitive genic male-sterile rice. Int. Rice Res. Newsl. 13:6-7.

12. Zhonghua Sheng, Xiangjin Wei, Gaoneng Shao, Mingliang Chen, Jian Song, Shaoqing Tang, Ju Luo, Yichao Hu, Peisong Hu, Liyun Chen.2013. Plant.Breed,http://www.bionity.com/en/publications/527796/genetic-analysis-and-fine-mapping-of-tms9-a-novel-thermosensitive4-genic-male-sterile-gene-in-rice-oryza-sativa-l.html(30/9/2014)

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Thank you….!