This document summarizes information from a student's assignment on plant genome sequencing techniques. It discusses early phenotypic selection methods and their limitations. It then summarizes different sequencing strategies used for important crop plants like rice, poplar, and Arabidopsis. These include BAC-by-BAC, whole genome shotgun, and various next-generation sequencing platforms. The document also summarizes applications of sequencing including identifying genes related to rice yield and flowering time and using sequencing to improve potato and maize varieties.

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

2. 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.

4. Frederick Sanger and his colleagues created the
di-deoxy sequencing method based on the
“elongation of DNA molecule during replication”
Shendure & Hanlee, 2008

5. 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

7. 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.

8. 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.

9. 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.

11. 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.

13. 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.

14. ssDNA template is immobilized, hybridized to a sequencing primer and
incubated with,
Enzymes: DNA polymerase, ATP sulfurylase, luciferase and apyrase
Substrates: 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.
The released ATP aids in luciferase-mediated conversion of luciferin to oxy-
luciferin that generates visible light

16. 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

17. 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

18. Shendure & Hanlee, 2008

20. 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.

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

22. 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.

23. 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.

25. 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.

26. 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.

27. 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.

28. 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.

29. 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.

30. 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.

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

33. 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.

34. Munroe & Harris, 2010
SMRT Ion-TorrentFRET Nanopore

35.  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.