Plant Genome sequencing and Crop improvement

Student : Ragavendran Abbai ID. No. : 2015315514Discipline : I MS Biotechnology

Crop Improvement In Pre-sequencing Era

Morphology based phenotypic selection was the only method adopted in regard with crop improvement in the pre-sequencing era.

Demerits of Phenotypic selection:Inconsistent due to GxE interaction.Time-consuming.Morphological markers are limited in number.

Merits of Phenotypic selection:Morphological markers are readily available.Usually require only simple equipments.

ORIGIN OF DNA SEQUENCING TECHNOLOGY

Conventional DNA Sequencing Strategies

Di-deoxy sequencing method

Frederick Sanger and his colleagues created the di-deoxy sequencing method based on the “elongation of DNA molecule during replication”

Shendure & Hanlee, 2008

Partial digestion of the entire genome

Construction of Physical map – either based on molecular marker data or based on restriction fingerprinting

Generate clones of large segments (~300 kb) of DNA from the organism of interest

Minimally overlapping clones are selected for sequencing based on physical mapping (Minimal Tiling Path)

Sanger sequencing after the fragmentation of cloned DNA into smaller parts

An insight into BAC by BAC sequencing strategy

Whole Genome Shotgun (WGS) strategy

The Arabidopsis thaliana genome

Arabidopsis thaliana was the first crop to be sequenced owing to its simple genome and also to its benefit as a model plant in genomic research. The technique adopted was BAC by BAC sequencing strategy.

Unraveling the Rice genome

Rice is the most highly consumed staple food crop in the world. With the notion of understanding the molecular mechanisms controlling yield, stress resistance, nutrient absorption and many more desired traits, rice was the first crop pant to be sequenced. The technique adopted was BAC by BAC sequencing strategy.

Understanding the Populus trichocarpa genome

Populus trichocarpa is the first woody tree to be sequenced and is indicated as the model forest species due its rapid growth and relative ease of experimental manipulation. The technique adopted was WGS sequencing strategy.

Elucidation of the Grapevine genome through WGS strategy

Demerits:Generation and maintenance of BAC libraries.WGS vs Assembly quality.

Merits:Genome sequencing was made possible for the first time.Draft genome of vital plant species were completed.

Conventional genome sequencing srategies

ADVANCES IN DNA SEQUENCING TECHNOLOGY

Next Generation DNA Sequencing platforms

Pyrosequencing approach

Pyrosequencing works on the principle of "sequencing by synthesis" principle. The technique was developed by Pål Nyrén and his student Mostafa Ronaghi at the Royal Institute of Technology in Stockholm in 1996.

ssDNA template is immobilized, hybridized to a sequencing primer and incubated with,

Enzymes: DNA polymerase, ATP sulfurylase, luciferase and apyraseSubstrates: adenosine 5´ phosphosulfate (APS) and luciferin.

PYROPOSPHATE (PPi) is released when correct nucleotide is incorporated

ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate.

Methodology of Pyrosequencing

The released ATP aids in luciferase-mediated conversion of luciferin to oxy-luciferin that generates visible light

Roche Applied Science’s 454 platform works based on Pyrosequencing

Illumina's Solexa platform works based on Bridge PCR

Shendure & Hanlee, 2008

In-vitro constructed adapter flanked shotgun library is PCR

amplified on a flow cell where the primers are attached

The 5` end of the primer is attached with a flexible linker

which leads to local tethering of the amplicons

Action of modified dNTPs with 3` Reverse terminating motif +Fluorescent labeling enables dissecting out the sequence

AB’s SOLiD platform works based on Emulsion PCR

Shendure & Hanlee, 2008

In-vitro constructed adapter flanked shotgun library is PCR

amplified on a bead

Selective recovery of beads harboring amplicons and

immobilization on a solid chip to produce dense, disordered array

Universal primer targeting the adapter is hybridized in the array

and fluorescently labeled octamer is used for sequencing.

Sequencing by synthesis is driven by DNA ligase

Comparison of leading NGS platforms:454, Solexa & SOLiD

Shendure & Hanlee, 2008

Whole genome sequencing through NGS platforms

Sequencing of the cucumber genome through hybrid approach utilizing Sanger’s and Illumina platforms

Cucumber is an economically important crop as well as a model system for sex determination studies and plant vascular biology.This hybrid approach resulted in 72.2-fold genome coverage.

Understanding of the Apple, Cocoa & Muskmelon genome

Combined approach of 454 pyrosequencing and Sanger’s sequencing was employed to dissect out the genome of Apple (2010), Cocoa (2010) and Muskmelon (2012).

Unraveling the Woodland Strawberry genome solely through NGS platforms

Woodland Strawberry genome was the first plant genome to be sequenced (2011) solely through NGS platforms. All 454, Illumina and SOLiD platforms were employed for this purpose.The strawberry genome was sequenced to ×39 coverage using NGS platforms, assembled de novo and then anchored to the genetic linkage map into seven pseudo-chromosomes.

Domination of Illumina sequencing platform

In recent years Illumina sequencing platform is increasingly being adapted for whole genome sequencing of plants.

Chinese cabbage (2011), Potato (2011), Banana (2012), chickpea (2013), orange (2013), pigeon pea (2012) and water-melon (2013) were sequenced through Illumina sequencing platforms.

IMPACT OF WHOLE GENOME SEQUENCING IN CROP IMPROVEMENT

CASE STUDIES

Enhancing Rice grain yield

The QTL Gn1a is reported to posses significant control in the spikelet production and hence yield. Gn1a co-localizes a gene, cytokinin oxidase (OsCKX2) which is associated with degradation of phytochrome cytokinin. Reduced expression of OsCKX2 leads to cytokinin accumulation in inflorescence -> increase in reproductive organs -> increase in grain yield.

The identification of the major effect QTL, qEMF3 associated with FOT (Flower Opening Time) which promotes Early Morning (EMF) Flowering has opened up avenues for generation of heat tolerant rice varieties.It has been reported that the flowering in the QTL introgressed lines gets completed before the temperature reaches the threshold limit of 35◦C, thus reducing sterility percentage.

QTL for FOT and Heat Escape

3000 Rice genomes project The Future of Rice Biotechnology

Deep sequencing (Avg.14x) of 3000 diverse rice genotypes was carried out under “3000 Rice genomes project” and it is a gold-mine for rice improvement.

Haplotype map of Maize

Several million sequence polymorphims among 27 diverse maize inbred line were brought into light and this would act as the platform for Genome Wide Association studies (GWAS) for dissecting out molecular basis of complex traits in maize.

Molecular basis of uniform ripening in Tomato

SlGLK2, the Golden 2-like transcription factor influences the chlorophyll accumulation and distribution in developing fruits.Appropriate manipulation of SlGLK2 will contribute to the uniform ripening uniform harvest.

Understanding the genetic link between photoperiodism and tuber formation in Potato

Potato requires “short-day” conditions for tuber formation. A major-effect QTL controlling plant maturity and tuber formation has been identified.This QTL paves way for the potato breeders to generate varieties specific to their geographical locations.

RNA-seq reveals molecular basis of salinity responsiveness in finger millet.

RNA-seq for unraveling salinity responsiveness in Finger-millet

FUTURE PROSPECTS

Problems with NGS platform and overview of high-throughput sequencing platforms in pipeline

Repetitive elements vs NGS platforms

Success of NGS technology in Triticeae genomes: Wheat, Barley and Rye is very limited due to the presence of ~80% of repetitive elements.

In-order to combat this issue, high throughput third generation “Real-time single molecule sequencing” technologies are in the pipeline.

Third generation sequencing Real-time single molecule sequencing

Munroe & Harris, 2010

SMRT Ion-TorrentFRET Nanopore

Selected References

Kloosterman et al. 2013. Naturally occurring allele diversity allows potato cultivation in northern latitudes. Nature, 495:246-250.

3K RGP: The 3,000 rice genomes project, 2014. GigaScience 3:7.

Bolger et al. 2014. Plant genome sequencing — applications for crop improvement. Current Opinion in Biotechnology, 26:31–37.

Shendure & Hanlee, 2008. Next-generation DNA sequencing. Nature biotechnology, 10.1038/nbt1486

Munroe & Harris, 2010. Third-generation sequencing fireworks at Marco Island. Nature, vol.28 no.5.

Feuillet et al. 2011. Crop genome sequencing: lessons and rationales. Trends in Plant Science, 10.1016/j.tplants.2010.10.005.