1

WELCOME

Ph. D. CREDIT SEMINAR2016-17

ON

Department of Plant Breeding and Genetics

JNKVV, JABALPURM.P -482004

Presented by :Kanak SaxenaRoll No.-234

Guided by:Dr (smt.) Rajani

Bisen

Advances in Cereal Genomics and its application in crop improvement

CONTENTS

Introduction

Genomics

Need of Genomic research in cereal crops

Laboratories for Cereal Genomic Research in India

Genomics based approaches and techniques

Role of genomics in crop improvement

Achievements in Cereal Genomics

Case Study

Challenges in using genomics for breedingConclusion

References

(Genomics word was coined by Thomas Roderick in 1986.)(Study of structure & function of entire genome of a living organism)

(Study of the structure of entire genome of an organism)

(Study of the function of entire genome of an organism)

GENOMICS

Structural Genomics Functional Genomics

Comparative Genomics

(Study of the relationship of genome structure and function across different biological species or strains)

1980 – DNA markers (RFLP) 1983 – Karry Mullis invented the PCR technique, and Several

PCR based markers developed i.e. RAPD, AFLP, SSR, SNP, CAPS, STS, SCAR, EST, etc..

1986 – Leroy Hood and Lloyd Smith developed the first semi-automatic DNA sequencer

1990 – Development of Pyrosequencing by Pal Nyren 1990 – The U.S. National Institutes of Health (NIH) begins large-

scale sequencing trials on Mycoplasma , Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae

1995 – Craig Venter, Hamilton Smith at The Institute for Genomic Research (TIGR) publish the first complete genome of a free-living organism, the bacterium Haemophilus influenzae

1996 – Sequence of saccharomyces cervisiae genome completed.

Nature Review, 2010 & Nature Biotechnology, 2001-2013

1998 – The genome of the 1st multi-cellular organism of the Round worm (Caenorhabditis elegans) was completed.

1999 – Sequence of first human chromosome (chromosome 22nd) 2000 – The first plant to be completely sequenced is that of the

Arabidopsis thaliana.

2001 – A draft sequence of the human genome is published.

2002 – Rice genome sequencing was completed

2003 – Human genome sequencing was completed

2004 – 454 Life Sciences markets a version of pyrosequencing machine developed. ‘454 Sequencing’ used in Barley this machine reduced sequencing costs 6-fold compared to automated Sanger sequencing methods.

Recently, 2012 – Wheat genome was sequenced – R. Brenchley et al. 2012 –Barley genome was sequenced – RKV et al.

Nature Review, 2010 & Nature Biotechnology, 2001-2013

Genome size, structure and genomic

resources of major cereal species

Rajeev K. Varsney et al (2013)

CROP BOTANICAL NAME GENOME SIZE(Mb)

RICE Oriza sativa 400

SORGHUM Sorghum bicolar 1000

MAIZE Zea mays 2500

BARLEY Hordeum vulgare 8000

BREAD WHEAT Ttriticum aestivum 16000

ESTIMATED GENOME SIZE OF MAJOR CEREAL CROPS

Rajeev K. Varsney et al (2013) Trends in biotechnology

Comparison of Rice with Cereal genomes

?WHY GENOMI

CS?

Through conventional breeding, selection for crop improvement is carried out on phenotypic character, which is the result of genotypic and environmental effects but by the use of molecular markers exact location of particular gene on chromosome can easily be identified.

Many potential genes that confer resistance have been mapped in most of the economically important cereal crops like Rice, Maize, Wheat, Sorghum and Barley.

QTLs identified and markers linked with the promising QTLs are useful resource for genomics assisted breeding. For ex. resistance to heat tolerance in wheat.

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Presence of all of the important genes and related markers will be very much helpful to identified and development of new cultivars as we desire - Varietal identification

Insertion and deletions are desirable or undesirable, easily be possible to identify with the sequencing of cereal crops.

Generated set of ESTs serves as a resource of high quality transcripts for gene discovery and development of markers associated with abiotic stress tolerance that will be helpful to facilitate cereal genomic breeding.

Laboratories for genomic research in India

National Research Centre for Plant Biotechnology, IARI, New Delhi

International Centre for Genetic Engineering & Biotechnology, New Delhi

Jawaharlal Nehru University , New Delhi

National Institute of Plant Genome Research, New Delhi

ICRISAT and DRR ,Hyderabad

International Institutes for Cereal Genomic Research

Genome Institutes/ Countries Involved

Rice GenomeJointly Mapped By IRRI, USA, China, Japan Scientist through International Rice Genome Sequence Project.

Wheat GenomeInternational Genome Research on Wheat by Mexico (CIMMYT), UK, USA & Japan

Arabidopsis Genome China, Japan, & USA

GENOMICS- BASED APPROACHES

Rajeev K. Varsney et al (2013)

Genomic technologies for high-throughput genome sequencing

Diversity Panel

1. ASS0CIATION MAPPING

Zhu et al., (2008)

A holistic approach, where genomics technologies including molecular markers, trasncriptomics, metabolomics, proteomics and bioinformatics are integrated with conventional breeding strategies for breeding crop plants resistant/ tolerant to biotic and abiotic stresses or improved for quality and yield.

3 Next generation sequencing (NGS) technologies Include various novel sequencing technologies for example

454/FLX (Roche Inc.), ABI SOLID (Applied Biosystems), Solexa (Illumina Inc.) etc. that have surpassed traditional Sanger sequencing in throughput and in cost-effectiveness for generating large-scale sequence data.

2 Genomic Assisted Breeding

Clone by clone sequencing also called as the directed sequencing of the BAC contigs.

The chromosomes were mapped

Then split up into sections

A rough map was drawn for each of these sections

Then the sections themselves were split into smaller bits.

Each of these smaller bits would be sequenced.

(BAC clones (80-100 kb long DNA fragments ) arranged in contigs.)

4. CLONE BY CLONE SEQUENCING

In this approach, genomic DNA is cut into pieces

Inserted into BAC vectors

Transformed into E. coli where they are replicated

The BAC inserts are isolated

Mapped to determine the order of each cloned fragment.

Each BAC fragment in the Golden Path is fragmented randomly into smaller pieces

Each piece is cloned into a plasmid

Sequenced on both strands.

These sequences are aligned so that identical sequences are overlapping.

5. SHOTGUN SEQUENCING METHOD

This is referred to as the Tiling Path.

Moto Ashikarc et al., (2009)

COMPARATIVE GENOMICS OF CEREALS

Several draft sequences of the rice genome are also available and are being extensively used for study of other cereal genomes

Analyzing & comparing genetic material from different species

Evolution, gene function, and inherited disease Understand the uniqueness between different species

HR- Highly repetitiveMR-Moderately repetitiveSL- Slow/ Low repetitiveGnSz – Genome sequenceSqCx- Sequence Complexity

Gupta, P. et al., (2009), PAU

ACHIEVEMENTS IN CEREAL GENOMICS

Major QTLs responsible for yield

Gupta, P. et al., (2009), PAU

Major QTLs responsible for grain protein and enzyme activity

Gupta, P. et al., (2009), PAU

Case study

GENOTYPE Station trials Multilocation evaluation

DBW 16, DBW 17, PBW 568 + Yr36/GpcB1 2009-10 2010-11

Advanced lines + Lr 34/Yr18 2009-10 2010-11

PBW 343 + Lr37/Yr17 2009-10 2010-11

HD 2733 + Lr37/Yr17 2009-10 2010-11

PBW 343+Yr+Lr gene(s) from Ae. umbellulata

2010-11 2011-12

PBW 343 + Lr24+Lr28+Yr10+Yr15 2011-12 2012-13

PBW 343+KB resistance+Yr40/Lr57

2011-12 2012-13

PBW 550, PBW 533 +Yr 10/Yr 15 2011-12 2012-13

Yield testing of Wheat MAS products(4 rust resistant genes pyramided)

Pyramiding of Lr 24, Lr 28, Yr 10 and Yr 15 in PBW 343 Gupta, P. et al., (2009), PAU

OryzaSNP Project <http://www.oryzasnp.org>

IR64 IA

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SNP scan >100 Mb/genome20 diverse genotypes

Zhu et al., (2009)

FUNCTIONAL SNP’S FOR TARGET TRAITS IN RICE

Bacterial blight xa5

Blast Pia, Pi9,Pita, Pizt

Tungro RTSV

Grain quality GBSS, Wx, SBE3, SSlla

Submergence tolerance

Sub1

Zhu et al., (2009)

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Golden RiceVariety :- Rice (Oriza sativa)Scientist:- Igno Potrycus & Peter Boyer.Produce :- β-varotene Precursor of Vit. A.

Golden rice was created by transforming rice with two beta-carotene biosynthesis genes:psy (phytoene synthase) from daffodil (Narcissus pseudonarcissus)

crtI (carotene desaturase) from the soil bacterium (Erwinia uredovora)

Ye, Xi et al., (2000)

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Original golden rice was called SGR1

Application of candidate gene strategy to identify CrtRB1 locus

Jiabing et al., (2014), Nature Genetics

GENETIC ENHANCEMENT OF NUTRITIONAL QUALITY OF GRAIN SORGHUM

Genetically enhancing the nutritional quality of grain sorghum by the introduction of genes encoding the methionine-rich maize beta-zein and the lysine-rich barley chymotrypsin inhibitor CI-2 proteins.

The goal to produce transgenic sorghum plants with elevated lysine and methionine contents.

A biolistic and Agrobacterium-mediated transformation protocol for selected grain sorghum lines was established. This would form the technological basis for nutritional quality improvement of grain sorghum.

A Grootboom and M M O’Kennedy (2013)

VARIETIES DEVELOPED THROUGH GENOMIC TECHNOLOGIES

Varshney et al.,(2006)

Challenges in using genomics for breeding

Precision phenotyping Density of genetic maps. Low heritability of traits. Contribution of regulatory variation Consideration of epistasis Technical skill Technical difficulties Cost investment issues

Genomics based technologies is producing enormous amounts of DNA sequence information that is opening up new experimental opportunities for functional genomics analysis.

The incorporation of metabolomic data and data from phenotype studies will close the loop and create the foundation for advanced knowledge.

The recent integration of advances in molecular biology, transgenic breeding and molecular marker applications with conventional plant breeding practices has created the foundation for genomic research.

Genomics will affect every aspect of cereal breeding and will modernize this old science, although genomic approaches will not succeed unless they are combined with traditional breeding programs.

CONCLUSION

Some of References

Jiang Y, Cai Z, Xie W, Long T, Yu H, Zhang Q. 2012. Rice functional genomics

research: Progress and implications for crop genetic improvement.

Biotechnology Advances (30) 1059–1070.

Sasaki T, Wu J, Mizuno H, Antonio BA, and Matsumoto T. 2008. The Rice Genome

Sequence as an Indispensable Tool for Crop Improvement. Chapter 1.1,

Springer-Verlag Berlin Heidelberg.

Tyagi AK, Mohanty A. 2000. Rice transformation for crop improvement and functional

genomics. Plant Science (158) 1–18.

Cai Q, Yuan Z, Chen M, Yin C, Luo Z, Zhao X, Liang W, Hu J & Zhang D.

2014. Jasmonic acid regulates spikelet development in rice. ISSN (online)

2041-1723.

Varshney R.K., Thiel T., Stein N., Langridge P., Graner A. Cereal Genomics. Cell Mol

Biol Lett 2004; 7: 537-546

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