Diversity array technology

SAAKRE MANJESH2014-11-105

OVER VIEW• Progress in development of genomic resources in the leading legume

crops of the semi-arid tropics (SAT) chickpea, pigeon pea and groundnut as compared to other crop species like cereals, has been very slow.

• SSR and SNP markers have become the markers of choice for genetic analysis and breeding applications in the SAT legume crops

• Next-generation sequencing (NGS) and high-throughput (HTP) genotyping methods.

• Diversity array technology (DArT) marker system became popular in many other crop species since no sequence information is needed

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• DArT arrays comprising 15,360 clones in each of the three species

• The parental genotypes of mapping populations including intra-specific mapping populations in chickpea and pigeonpea, when screened with the available DArT arrays, showed 35% and 9% polymorphism, respectively.

• DArT markers are not cost-effective or attractive marker system for detecting polymorphism in cultivated germplasm of the SAT legume crops.

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• DArT markers may prove useful for introgression of segments from alien species to the elite varieties of the legume crops.

• In pigeonpea, by using 1,225 DArT markers in the cross between C. platycarpus and C. cajan, 2–5% C. platycarpus genome carrying genes for disease and insect resistance was observed (Mallikarjuna et al. 2011).

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The development and application of genomics resources in accelerating genomics research and breeding applications in the SAT legume crops.

Conti…..

• SSR markers- 454/FLX sequencingChickpea- 2,000Pigeonpea- 3,200Groundnut- 2,500

Candidate Markers for :Drought-tolerance-related root traits in chickpea Resistance to foliar diseases in groundnutsterility mosaic disease (SMD) and fertility restoration in

pigeonpea

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• DArT Pty Ltd, ICRISAT has developed DArT arrays comprising 15,360 clones in each of the three species.

• SNP genotyping platforms including GoldenGate, VeraCode and Competitive Allele Specific PCR (KASPar) assays have been developed in chickpea and pigeonpea.

• 454/FLX sequencing for SSR markers

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Transcriptomic resources and molecular markers developed at ICRISAT using next generation sequencing and highthroughput genotyping technologies

Diversity array technology (DArT)

high-throughput marker system

No sequence information is needed

DArT is based on microarray hybridizations

Detect the presence v/s absence of individual fragments

Efficiently and economically scan from hundreds to thousands of polymorphic markers.

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DArT technology consists of several steps:

1. Complexity reduction of the DNA of interest

2.  Library creation Microarraying libraries onto glass slides 

3. Microarraying fragments onto glass slides

4. Hybridisation of fluoro-labelled DNA onto slides 

5. Scanning of slides for hybridisation signal 

6. Data analysis and extraction.

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1. Complexity reduction of the DNA of interest

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DArT operates on the principle that the genomic 'representation' contains two types of fragments:

• Constant fragments, found in any 'representation' prepared from a DNA sample from an individual belonging to a given species, and

• Variable (polymorphic) fragments called molecular markers, only found in some but not all of the 'representations'.

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Presence vs. absence in a genomic 'representation' is assayed by hybridizing the 'representation' to a DArT array consisting of a library of that species.

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 2. Library creation

DNA amplification

Cloning

Library in E coli

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Each colony contains one of the fragments from the

genomic 'representation'.

3. MicroarrayingSelection of clones

Arranged into a plate format (usually 384-well plates)

 Fragments within library

amplified

Spotted onto glass slides

14 Genotyping array

4. Hybridisation

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5. scanning The hybridised slides are Washed and processed to remove

unbound labelled DNA.

Then scanned using a scanner to detect fluorescent signal emitted from the hybridised fragments.

The result from each fluorescent channel is recorded

The resulting images are stored in 'tif' format.

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6. Data analysisThe data from the scanned images is extracted and analysed using the DArTsoft software and the information is managed by the DArTdb Laboratory Information Management System.

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DArTsoft- Software

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Advantages of DArT technology

Marker density relevant to application

Sequence information and platform independence

High throughput due to a high level of multiplexing

Matching most cost-effective technology with the application on modern platforms

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DArT Applications• Genome profiling and diversity analysis

• Genetic and physical mapping

• Identification of QTL

• Rapid introgression of genomic regions in accelerated backcrossing programs

• Simultaneous marker-assisted selection for several traits

• Genomic Selection

• Varietal identification of crops and genetic purity testing

• Monitoring the composition of complex DNA samples

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454/FLX sequencing

• 454/FLX sequencing

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Applications

Full genome sequencing

Amplicon sequencing

Transcriptome sequencing

Metagenomics

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References• Advances in genetics and molecular breeding of three legume

crops of semi-arid tropics using next-generation sequencing and high-throughput genotyping technologies, Rajeev k varshney et.,al, ICRISAT, CIMMYT.

• http://www.diversityarrays.com/dart-application-microarray

• http://ilmn-site2.azurewebsites.net/technology/beadarray-technology/goldengate-genotyping-assay.ilmn

• http://www.wellcome.ac.uk/Education-resources/Education-and-learning/Resources/Animation/WTX056046.htm

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Thank you