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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,
(Lilian et al., 2002)
Annealing
Polymerizationandlabeling
Termination
Polyacrylamide/ureagel
electrophoresis
(Lilian et al., 2002)
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.
(Lilian et al., 2002)
Capillary electrophoresis
Samples passing a detection window
are excited by laser and emitted
fluorescence is read by CCD camera.
Sequence ladder by fluorescent peaks
(Lilian et al., 2002)
G : Dimethyl sulphate(DMS) A+G : PiperidineC+T : HydrazineC : Hydrazine + 1.5M NaCl
(Chemical cleavage method - 1977)
(Lilian et al., 2002)
• 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
(Hamilton et al., 2012)
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.
(Hamilton et al., 2012)
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