Genome sequencing in vegetable crops

Chairperson

Dr.P.Irene vethamoni,

Professor (Horticulture)

Dept. of Vegetable Crops

StudentBommesh J.C.II (M.Sc.) Vegetable scienceDept. of Vegetable Crops

Genome Sequencing in vegetable crops

Seminar on

INTRODUCTION

FIRST GENERATION SEQUENCING

SECOND GENERATION SEQUENCING

THIRD GENERATION SEQUENCING

LIST OF WHOLE SEQUENCED VEGETABLES

GENOME SEQUENCING IN POTATO MELON AND CABBAGE

SUMMARY & CONCLUSION

Introduction

(Lilian et al., 2002)

Sequencing

determines

Adenine

Thymine

Cytosine

Guanine

• DNA sequencing includes several methods and technologies that are

used for determining the order of the nucleotide bases—

adenine, guanine, cytosine, and thymine in a molecule of DNA.

Terminology Chromosome – It is a DNA-histone protein thread, occurring in the

nucleus of a cell - hereditary material – Waldeyer (1888)

DNA – Chemically linked chain of nucleotide which consists ofphosphate, sugar and nitrogenbase(ATGC) – Watson & Crick

Gene: The hereditary unit that occupies a fixed position on thechromosome.

Genome - The total complement of genetic material in thecell or an organism - H. Winkler (1920)

Genomics - The study of the structure and function ofgenomes - T.H.Roderick (1987)

History of Genome Sequencing

Discovery of DNA structure-

Watson and Crick (1953)

Development of Sanger

Sequencing - Frederick Sanger

(1977)

Polymerase Chain Reaction -Kary

Mullis (1983)

Development of Pyrosequencing -

Pal Nyren (2000)

Sequence of Human Genome

Completed (2003) – John craig

venter

U.S. Department of Energy Office of Science

2000

2009

2011

2011

2011

2012

2012

2012

2012

2013

2013

2014

Arabidopsis

The journey in sequencing world

Genome Sequencing - Important?

To understand how the genome as a whole works

To study gene expression in a specific tissue.

Understand how gene expression is regulated in a particular environment

To find correlations how genome information relates to susceptibility / resistance to diseases , physiology and metabolism etc.

To improve the quality and productivity of the crop plants

The whole genome sequence is an tool for make an organism.

Michael and Jackson (2013)

Steps in genomics

Functional genomics

Gene location in Sequences

Genome Sequencing

(Brown., 2010)

Sequencing Platforms

First Generation Sequencing Platforms (FGS)

Sanger’s method

Maxam and gilbert method

Second Generation Sequencing Platforms (SGS)

Roche/454 FLX Platform

Illumina/Solexa Genome Analyzer Platform

Third Generation Sequencing Platforms (TGS)

Pacific Biosciences SMRT

Ion Torrent

Oxford Nanopore (Hamilton et al., 2012)

Sanger’s Method

Chain termination method

DiDeoxyNucleoside TriPhosphate (ddNTP)

dNTP and ddNTP is added in (10:1 ratio)

F. SANGER,


Annealing

Polymerizationandlabeling

Termination

Polyacrylamide/ureagel

electrophoresis


Shot gun approach• Longer sequences must be subdivided into smaller fragments, and

subsequently re-assembled to give the overall sequence

• DNA is broken up randomly into numerous small segments, which

are sequenced using any method to obtain reads.

• Multiple overlapping reads for the target DNA are obtained by

performing several rounds of this fragmentation and sequencing.

• Computer programs then use the overlapping ends of different

reads to assemble them into a continuous sequence

Dye-terminator sequencing(Automated

Sanger method)

• DNA fragments are labeled

with a fluorescent tag on the

primer.

• In the new DNA strand with

a dNTP.

• Terminate with a labeled

ddNTP.


Capillary electrophoresis

Samples passing a detection window

are excited by laser and emitted

fluorescence is read by CCD camera.

Sequence ladder by fluorescent peaks


G : Dimethyl sulphate(DMS) A+G : PiperidineC+T : HydrazineC : Hydrazine + 1.5M NaCl

(Chemical cleavage method - 1977)


• Founded in 2000 by Jonathan Rothberg• First commercially available NGS platform• This platform combined the single-molecule

emulsion PCR with pyrosequencing

Roche/454’s GS FLX Titanium - Pyrosequencer

(Hamilton et al., 2012)

Pyrosequencing


StepsPreparation of the DNA

Adapters - A and B adapters are used as priming - since their composition is known.

Denatured using sodium hydroxide to release the ssDNA template library (sstDNA).

Roche/454’s GS FLX Titanium - Pyrosequencer

Each ssDNA in the library is hybridized onto a primer coated bead in micro reactor.

PCR takes place in each of these beads individually

The DNA in the beads are denatured , This ssDNA rich beads ready for sequencing.

The beads are loaded into individual wells created from finely packed and cut fiber-optics (PicoTiterPlate device).

Enzyme beads are added. These containing sulfurase and luciferase,

The nucleotide bases are added in a timed fashion . One by one

The intensity of the light emitted by luciferase is proportional to the number of nucleotides incorporated and each nucleotide different light.

Pyrosequencingreaction

sequencing-by synthesis.


Ion Torrent

The technology was licensed from DNA Electronics Ltd, developed by Ion Torrent Systems Inc. and was released in February 2010.

Also be referred to as Ion Torrent sequencing, pH-mediated sequencing,

It has microwells on a semiconductor chip Sequencing by synthesis

Schadt et al.(2010)

• In nature, the incorporation of a (dNTP) into a growing DNA strand involves the formation of a bond and the release of PPi and a positively charged H+.

• A dNTP will only be incorporated if it is complementary to the leading unpaired template nucleotide.

Schadt et al.(2010)

The hydrogen ion that is released in the reaction changes the pH of the solution, which is detected by an ISFET (ion-sensitive field-effect transistor used for measuring ion concentrations)

The unattached dNTP molecules are washed out before the next cycle when a different dNTP species is introduced.

Schadt et al.(2010)

Oxford Nanopore Technology

The translocation of nucleotides cleaved from a DNA molecule across a pore, driven by the force of differential friction across the membrane.

Schadt et al.(2010)

Up coming methods

• Tunnelling currents DNA sequencing

• Sequencing by hybridization

• Sequencing with mass spectrometry

• Microfluidic Sanger sequencing

• Microscopy-based techniques

Schadt et al.(2010)

Advancement of sequencing technology

Dijk et al.(2014)

Sl.

No

Crop Genome

size

Genes

predicted

Year of

completion

Sequencing

method

1 Cucumber 350 Mbp26,682 2009 Sa,I

2 Potato 844 Mbp 39,031 2011 Sa,4,I

3 Chinese

cabbage

485 Mbp 41,174 2011 I

4. Cabbage 650 Mbp 454,274 2011 I,4, Sa

4 Watermelon 425 Mbp 23,440 2012 I

5 Muskmelon 450Mbp 27,427 2012 Sa,4,I

6 Cassava 760Mb 30,666 2012 I,4

7 Tomato 900Mbp 34,727 2012 Sa,4,S,I

8 Solanum

pimpinellifolium

739Mbp 27,283 2012 Sa,4,S,I

9 Sugar beet 758 Mbp 27,421 2013 I,4, Sa

10 French beans 520Mbp 31,638 2013 I,4

11 Brinjal 833.1Mbp 85,446 2013 l

12 Chilli 3.48 Gbp 34,476 2014 l

Completely sequenced vegetables

Sa – Sanger

I – illumina

4- Roche-

FLX

Michel et al.

(2013)

Kim et al.

(2014)

• Under the MELONOMICS project, the double haploid line

DHL92 was used for sequencing.

• The homozygous DHL92 double-haploid line, derived from the

cross between PI 161375 (spp. agrestis) and the Piel de Sapo T111

line (ssp. inodorus) was chosen to get better assembly of the

genome sequence.

• DNA was prepared from nuclei extracted from leaves of the

doubled haploid line DHL92

• The whole genome sequence done by shot gun strategy with

Roche 454 GS FLX Titanium system.

The Genome of Melon (Cucumis melo L.)

(Mas et al., 2011)

• It representing the 83.3% of estimated melon genome. They

predicted 27,427 protein coding gene.

• The estimated genome Analyzed by reconstructing 22,218

phylogenetic trees, allowing mapping of the orthology and paralogy

relationships of sequenced plant genomes like cucumber.

• Musk melon genome size is 450 Mb.

• The genome sequencing completed in 2012

(Mas et al., 2011)

Assembly MeasureBases in contigs 335,385,220No. of contigs (>100 bases) 60,752No. of large contigs (>500 bases) 40,102Average large contig size (bases) 8,233No. of scaffolds 1,594No. of contigs in scaffolds 30,887No. of bases in contigs in scaffolds 321,933,769Average scaffold size (bases) 226,731

Metrics of the melon genome assembly

(Mas et al., 2011)

Melon

chromosomeCucumber

chromosome

Relationships between melon and cucumber chromosomes.

(Mas et al., 2011)

Comparative analysis of the melon and cucumber genomes.- Alignment of melon and

cucumber genomes

1

3

2

2

1

54

7

6

4

3

8 5

9

6

10

7

CUCUMBER

12

11

Melon

(Mas et al., 2011)

Dark green : widespread genes that are found in at least 23 species, yellow: widespread but plant-specific genes that are found in at least 20 of the 23 plant species.Gray: Species-specific genes with no (detectable) homologs in other species. Brown: genes without a clear pattern.

Phylogeny - Comparative genomics of 23 fully sequenced plant species

(Mas et al., 2011)

Gene prediction statistics of melonGenome size (bp)* 375,485,313

Genome GC content (%) 33.2

Number of genes 27,427

Disease resistance genes 411

Mean gene length 2,776

Total genic length 76,125,905

Gene density (kb/gene) 13.69

Number of exons 160,598

Mean exon length 271

Number of introns 125,750

Mean intron length 506 (Mas et al., 2011)

Disease resistance genes identified in melon

R-protein type No. of genes

Cytoplasmic class 81

Transmembrane 290

other 40

Total 411

(Mas et al., 2011)

• The melon assembly done by using software MEGABLAST.

• The availability of genome sequences in melon an important tool for understanding plant evolution and the genetic variability existing within cultivated species.

• Genome sequences are also becoming a strategic tool for the development of methods to accelerate plant breeding.

(Mas et al., 2011)

• Potato Genome Sequencing Consortium (PGSC)

• S. tuberosum group Phureja DM1-3 516 and S. tuberosum group Tuberosum RH89-039-16. these homozygous doubled- monoploidclones was used for sequencing.

• DM1-3 516 R44 (DM) resulted from chromosome doubling of a monoploid (1n = 1x = 12) derived by anther culture of a heterozygous diploid (2n = 2x =24) S. tuberosum group Phureja clone (PI 225669).

• RH89-039-16 (RH) is a diploid clone derived from a cross between a S. tuberosum ‘dihaploid’ (SUH2293) and a diploid clone (BC1034).

Genome sequence and analysis of the tuber

crop potato

Xu et al.(2011)

Sequencing Method - 454 GS FLX , illumina GA2 platforms andSanger sequencing .

The genome size is 844 Mbp.

Predicted genes are 39,031.

• Whole genome sequence completed in 2011.

• Central Potato Research Institute – Shimla. Member of Potato Genome

Sequencing Consortium (PGSC)

• They are involved in sequencing - S. K. Chakrabarti (PrincipalInvestigator), Virupaksh U. Patil.

Xu et al.(2011)

Ideogram of the potato genome

Xu et al.(2011)

Xu et al.(2011)

Sequence data of BACs, organized per chromosome. All BACs not assigned to any chromosome were combined into a virtual chromosome 0.

1 2 3 4 5 6 7 8 9 10 11 12 0 Total

Chromosomes

Venn diagram of orthologous gene families

Xu et al.(2011)

Syntenic blocks between genomes of Arabidopsis,

Potato and Grape

Xu et al.(2011)

• They identified 15,235 genes were expressed in the transition from stolons to tubers formation.

• Late blight resistance genes R1, RB, R2, R3a, Rpi-blb2 and Rpi-vnt1. were identfied.

Identified genes

Xu et al.(2011)

Outcome

• The potato genome sequence may elucidate the evolution of tuberization.

• Using a combination of data from the vigorous, heterozygous diploid RH and relatively weak, doubled-monoploid DM, we could directly address the form and extent of heterozygosity in potato.

• The potato genome provides a new resource for use in breeding.

• It Given the pivotal role of potato in world food production and security.

Xu et al.(2011)

The genome of Brassica oleracea

• The Brassica Genome Sequencing Project (BrGSP) consortium.

• B. oleracea sp. Capitata homozygous line 02 – 12 used for genome

sequencing.

• DNA was extracted from leaves.

• 454 GS FLX Titanium sequencing technology was used to achieve a

B. oleracea whole genome.

Liu et al.(2013)

This assembly represents 85% of the estimated Brassica genome.

The genome size is 650 Mbp

Predicted genes are 454,274 with mean transcript length of 1,761 bp,

• Whole genome sequence completed in 2011.

Liu et al.(2013)

B. oleracea genome assembly

Contig size (bp) 26828

Contig no. 5425

Scaffold size (bp) 1457055

Scaffold number 224

Liu et al.(2013)

Genomic landscape of Brassica oleracea

Liu et al.(2013)

Syntenic blocks between genomes of B. oleraceae,

Arabidopsis and B. rapa

Liu et al.(2013)

Identified genes for bio- chemical and metabolic pathways

Bio - chemical and metabolic pathways No.of genes

Glucosinolate biosynthesis and breakdown 127

Indole alkaloid biosynthesis 40

Tropane,piperidine and pyridine alkaloid biosynthesis 45

Flavone and flavonol biosynthesis 56

Carotenoid biosynthesis 151

Steroid biosynthesis 75

Biosynthesis of unsaturated fatty acids 79

Tryptophan metabolism 129

Liu et al.(2013)

Venn diagram of orthologous gene between

different species.

Liu et al.(2013)

Out come

• The brassica assembly done by using software TBLASTN.

• The B. oleracea genomic sequence helps in comparison with its

relatives with respect to genome evolution .

• It provide a fundamental resource for the genetic improvement of

important traits.

• The genome sequence is platform for investigation of the

morphological variation in B. oleracea and its relatives.

Liu et al.(2013)

Future thrust

Comparative analysis of other vegetable crops

Genetic engineering

Enhanced Crop improvement

Evolution and diversification

Genomics is novel technology

Genome sequencing is first step in genomics

Each and everyone is a unique creation!

DNA is a blue print of life!

Each cell contain - A, C, G, T

A hidden language/code determines which proteins should be made and when

Understand the mechanisms of genetic heredity and how different organisms

relate to each other.

Gene discovery for useful traits

Genome have wide regulatory networks to improve traits

Summary

The genome sequencing era is just starting. This erais to solve many of the major challenges we facetoday and many which are still unknown ,

Conclusion

Education

Genome sequencing in vegetable crops