Genome Sequencing in Finger Millet Genome size estimation SOLiD Sequencing Technology Illumina Sequencing Technology Gene prediction and functional annotation of genes Mining of plant transcription factors and other genes

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CLINICAL
AND
GENOMIC
DIAGNOSTICS
GENOME SEQUENSING
IN FINGER MILLET

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CONTENTS
1. Introduction
2. Genome Sequencing in Finger Millet
3. Genome size estimation
4. SOLiD Sequencing Technology
5. Illumina Sequencing Technology
6. Gene prediction and functional annotation of genes
7. Mining of plant transcription factors and other genes
8. Future Perspectives
9. Case study
10. Conclusion
11. Reference

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GENOME
One complete set of genetic information (total amount of
DNA) from a haploid set of chromosomes of a single cell
in eukaryotes, in a single chromosome in bacteria, or in
the DNA or RNA of viruses.
Basic set of chromosome in a organism.
The whole hereditary information of an organism that is
encoded in the DNA.
In cytogenetics, genome means a single set of
chromosomes.
Denoted by X.
The genome is found inside every cell, and in those that
have nucleus, the genome is situated inside the nucleus.
 In Drosophila melanogaster (2n = 2x = 8); genome x = 4.

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GENOME SEQUENCING
Genome sequencing is the technique that allows
researchers to read the genetic information found in
the DNA of anything from bacteria to plants to
animals.
 Sequencing involves determining the order of bases,
the nucleotide subunits found in DNA :-
adenine(A), guanine(G),
cytosine(C), thymine(T).
Genome sequencing is figuring out the order of DNA
nucleotides.

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WHY GENOME SEQUENCING ?
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.

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FINGER MILLET
Classification :
Kingdom : Plantae
Subkingdom : Tracheobionta
Super division: Spermatophyta
Division : Magnoliophyta
Class : Liliopsida
Subclass : Commelinidae
Order : Cyperales
Family : Poaceae
Genus : Eleusine
Species : Eleusine coracana
DIFFERENT SPECIES :
Diploid species :
1. Eleusine indica
2. E. oligostachya
3. E.tristachya
4. E. poranansis
5. E. jaegeri
6. E. flacifolia
Tetraploid species :
1.Eleusine coracana
2. E. longipoides
3. E. verticillata
4. E. cagopoides

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FINGER MILLET
Two distinct subspecies :
Cultivated finger millet/ Ragi :
 2n = 4x = 36 (Tetraploid, AABB)
E. coracana ssp. coracana
 Short fingers, small grains, photo
insensitive.
 Wild finger millet :
 2n = 4x = 36 (Tetraploid, AABB)
 E. coracana ssp. africana
It has long fingers, stiff straw,
photosensitive and uneven grain
maturity phase.

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FINGER MILLET
It is the fourth most important millet after sorghum, pearl millet and foxtail
millet.
It occupies 12% of global millet’s area and is cultivated in more than 25
countries in African and Asian continents .
High nutraceutical and
antioxidant properties
Gluten-free
 Rich in calcium, fiber and
iron
Excellent malting qualities
Low glycemic index
It produces reasonable grain and
fodder yields under low input .
Survives on soils of low fertility.
It has an efficient carbon
concentrating mechanism
through C4 pathway.
In spite of its immense importance in human diet, development of
genomic resources and high throughput breeding efforts have been
limited.
Considering these, the whole genome sequence data was developed.

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GENOME SEQUENCING IN FINGER MILLET
Genotype used for genome sequencing : ML-365
ML-365 variety was developed and released in 2008.
 Features of ML-365 genotype :
Short duration (110– 115 days)
High yielding (5.0–5.5 tonnes/hectare)
Drought tolerant
Blast disease resistant
Good cooking qualities
The genome was sequenced by
SOLiD sequencing technology (Sequencing by
Oligonucleotide Ligation and Detection)
Illumina sequencing technology.

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Genome size estimation of finger millet and wild species
Seeds of ML-365 (Eleusine coracana subsp. coracana), EC516253 (Eleusine
jaegeri Pilg.), EC516251 (Eleusine multiflora Hochst.), EC516248 (Eleusine tristachya
Lam.), EC516245, EC516243 (Eleusine indica), EC541533, and EC541538 (Eleusine
coracana subsp. africana) were grown in pots containing red soil and fertilizer mix.
Young leaves were collected and chopped into pieces using sterilized blade and
stained using nuclear isolation buffer (NIB).
The liquid was filtered through 30 μm nylon mesh and samples were processed for
ploidy estimation as per the protocol suggested by Krishan .
Stained nuclei were analyzed using BD FACS cell sorter.
Genome sizes of all Eleusine species were estimated by comparing with Pisum
sativum as an internal standard.
The genome size was derived by multiplying the 1C value (pg) with 965 Mb (1pg
equivalent value).

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Shailaja et al. (2017)

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Data analysis
SOLiD SEQUENCING TECHNOLOGY
DNA Fragmentation
Adapter Ligation
Hybridization of target DNA with PCR beads
Amplification via PCR
Attaching beads to glass slide
Ligation reaction and imaging

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CONTENTS

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Data analysis
Adapter Ligation
Illumina Sequencing Technology
DNA Fragmentation
Adapter Ligation
Hybridization of target DNA with oligos on floor shell
Bridge amplification of DNA
Attaching beads to glass slide
Sequencing reaction and imaging

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Genome assembly statistics of ML-365
Details Value
Total length of sequence (Mb) 1196.06
No. contigs/scaffolds 525,759
Minimum length of contigs/scaffolds (bp) 200
Maximum length of contigs/scaffolds (bp) 454,778
Average length of contigs/scaffolds (bp) 2274.92
N50 (bp) 23,732
GC content (%) 44.76
No. of genes predicted
a. Non-TE related genes
b. TE related genes
85,243
78,647
6596
Mb - Million bases
Bp - base pairs
Genome sequencing and genome assembly
Shailaja et al. (2017)

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Gene prediction and functional annotation
The assembled scaffolds were used to predict the genes using
AUGUSTUS by Zea mays as a reference gene model.
In addition, RNA-seq data was incorporated to AUGUSTUS to assist
the gene prediction.
The gene ontology annotation was carried out by performing a BLAST
(Basic Local Alignment Search Tool) with the protein sequences of
Viridiplantae retrieved from UniProt database.
The pathway analysis of genes was carried out by KAAS (KEGG
Automatic Annotation Server) server using Arabidopsis thaliana
(ath), Glycine max (gmx), Oryza sativa japonica (osa), and Vitis
vinifera (vvi) as reference organisms.
The protein domain structures of all protein coding genes were
identified using InterProScan5 software .

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Gene prediction and functional annotation
Functional annotation confirmed that majority of the genes had
homologs with known functions in UniProt protein database.
The gene ontology (GO) annotation of genes revealed that
more number of genes were involved in molecular function
followed by cellular and biological processes. Majority of the
genes were involved in nucleic acid binding, zinc ion binding,
and ATP binding activities under molecular function.
Similarly, DNA integration and integral components of
membrane were major functions related to biological and
cellular processes, respectively.
The KAAS server based pathway prediction showed that
majority of genes were involved in carbohydrate metabolism,
amino acid metabolism, translation, lipid metabolism and
folding, sorting, and degradation pathways.

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Shailaja et al. (2017)

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Mining of plant transcription factors (TFs) in finger millet
The protein-protein homology analysis of genes of ML- 365 with
plant TFs protein database revealed 56 various families of TFs
distributed across 11,125 genes in finger millet.
Among them, bHLH, MYB, FAR1, WRKY, NAC, MYB related, B3, ERF,
bZIP, HD-ZIP, C2H2, C3H, G2- like, TALE, GRAS, ARF, M-type,
Trihelix, GATA, WOX, LBD, HSF, MIKC, S1Fa-like, HB other, CPP, and
YABBY were majorly distributed TFs in the finger millet genome.
These 11,125 genes were found to have homology with 75 plant
species and foxtail millet, rice, wild species of rice, apple,
Brachypodium, maize, sorghum, wheat and wild species of wheat
were major among other plant species.

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Shailaja et al. (2017)

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Repeat content in finger millet genome
Simple sequence repeats (SSRs) play an evolutionary relationship
in genome evolution.
 The scaffolds of ML- 365 genome were subjected for identification
of SSRs using MISA.
 A total of 114,083 SSRs were distributed across ML-365 genome
signifying the abundance of SSRs, of which 66,805 (58.56%),
40,578 (35.57%), 2179 (1.91%), 3010 (2.64%) and 1511 (1.32%) were
di-, tri-, tetra-, penta-, and hexa- types, respectively.
 SSRs identified in this study can be further effectively used in
diversity studies, linkage map construction, association mapping,
QTL mapping of agronomically important traits, and marker
assisted breeding programmes.

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Shailaja et al. (2017)

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Phylogenetic relationship of six Poaceae species
revealed based on single copy ortholog genes :
Shailaja et al. (2017)

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Mining of drought responsive genes
Finger millet is a drought tolerant cereal crop and mining for
drought responsive genes will hasten future breeding activities to
develop varieties for drought prone areas.
The Pfam based identification of drought responsive genes revealed
that 2866 genes were distributed across 19 Pfam domains.
Most of these genes were associated with WRKY, MYB, MYC, ZFHD,
NAC, ABF, AREB, GRF, and NF-Y transcription factors, which are
responsible for drought tolerance .
Utilization of these TFs to study the binding sites of TFs and
analyzing cis-acting elements will enhance further understanding of
drought tolerance in finger millet.

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Pfam ID Pfam domain name Gene count
PF00069 Protein kinase domain 1386
PF07714 Protein tyrosine kinase 546
PF00651 BTB/POZ domain 351
PF01370 NAD dependant epimerase/dehydratase family 175
PF04564 U-box domain 86
PF00582 Universal stress protein family 82
PF00571 CBS domain containing proteins (DCPS) 77
PF05699 hAT family dimerization domain 36
PF08879 WRC 32
PF00999 Sodium/hydrogen exchanger family 20
PF03061 Thioesterase superfamily 19
PF08880 QLQ 16
PF04185 Phosphoesterase family 12
PF07649 C1-like domain 10
PF00257 Dehydrin 10
PF03107 C1-domain 3
PF02637 GatB domain 3
PF04147 Nop 14-like family 2
Shailaja et al. (2017)

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Calcium transport and accumulation genes :
Finger millet grain contains rich sources of nutrients, specifically it possess 5–10
times higher calcium in grains as compared to other cereals.
Homology based analysis identified 330 calcium transport and accumulation related
genes.
Among 330, 28 CaM ATPase, 145 CaMK1, 125 CaMK2, 29 CAX1 and 3 TPC1 genes
were identified.
Out of 330, six genes (g5694.t1, g73960.t1, g89161.t1, g107035.t1, g135510.t1, and
g146823.t1) were found to be homologs to Calcium transport and regulation genes
identified previously.
Genes identified in this study will help in exploring finger millet germplasm for
calcium uptake, translocation and accumulation in various tissues in near future.

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Finger millet remains an important cereal crop in the semi-arid tropics and is likely to
gain more importance as more genomic resources become available and more people
show interest in healthy eating.
The high demand for finger millet in dry areas will require the release of better
yielding varieties coupled with unique resistance to biotic and abiotic stresses.
The current largely conventional breeding approaches in finger millet will not be
sufficient if the full potential of the crop is to be realized.
With the availability of a draft whole genome sequence, research will need to focus
on characterizing important traits and utilizing genomics-assisted breeding for more
efficient release of superior varieties.
Future Perspectives

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Case Study - 1
Continued….
Seeds of finger millet core collection for ML-365 variety were obtained from All India
Coordinated Research Project on Small Millets, University of Agricultural Sciences (UAS),
Bengaluru.
This project was a competitive grant from World Bank under the Sujala-II funded by
Watershed Development Department, Government of Karnataka, India to Shailaja
Hittalmani as a Principal Investigator.
The funding agency had no role in study design, data collection, analysis and
interpretation, decision to publish, or preparation of the manuscript.

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Results:
Experimental results from whole genome sequencing and assembling process of
ML-365 finger millet cultivar yielded 1196 Mb covering approximately 82% of total
estimated genome size.
Genome analysis showed the presence of 85,243 genes and one half of the
genome is repetitive in nature.
The finger millet genome was found to have higher colinearity with foxtail millet
and rice as compared to other Poaceae species.
Mining of simple sequence repeats (SSRs) yielded abundance of SSRs within the
finger millet genome.
Functional annotation and mining of transcription factors revealed finger millet
genome harbors large number of drought tolerance related genes.
Transcriptome analysis of low moisture stress and non-stress samples revealed
the identification of several drought-induced candidate genes, which could be
used in drought tolerance breeding.
Conclusions:
This genome sequencing effort will strengthen plant breeders for allele
discovery, genetic mapping, and identification of candidate genes for
agronomically important traits.
Availability of genomic resources of finger millet will enhance the novel breeding
possibilities to address potential challenges of finger millet improvement.

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CONCLUSION

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References
1. Shailaja Hittalmani, H. B. Mahesh, Meghana Deepak Shirke, Hanamareddy Biradar,
Govindareddy Uday, Y. R. Aruna, H. C. Lohithaswa and A. Mohanrao (2017), Genome
and Transcriptome sequence of Finger millet (Eleusine coracana (L.) Gaertn.) provides
insights into drought tolerance and nutraceutical properties, Hittalmani et al. BMC
Genomics , 18:465
2. S. Antony Ceasar, T. Maharajan, T. P. Ajeesh Krishna, M. Ramakrishnan, G. Victor
Roch, Lakkakula Satish and Savarimuthu Ignacimuthu (2018), Finger Millet [Eleusine
coracana (L.) Gaertn.] Improvement: Current Status and Future Interventions of
Whole Genome Sequence, Front. Plant Sci.
3. Damaris Achieng Odeny, Adnan Niazi, Kassahun Tesfaye, Dagnachew Lule, Solomon
Wanyonyi and Josephine Sarah Kungun (2020), Genomic Designing for Climate Smart
Finger Millet, DOI: 10.1007/978-3-319-93381-8_7, In book: Genomic Designing of
Climate-Smart Cereal Crops (pp.287-307)

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