Diversity Array technology

1. Diversity array technology
SAAKRE MANJESH
2014-11-105

2. 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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3. • 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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4. • 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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5. 4
The development and application of genomics
resources in accelerating genomics research
and breeding applications in the SAT legume
crops.

6. Conti…..
• SSR markers- 454/FLX sequencing
Chickpea- 2,000
Pigeonpea- 3,200
Groundnut- 2,500
Candidate Markers for :
Drought-tolerance-related root traits in chickpea
Resistance to foliar diseases in groundnut
sterility mosaic disease (SMD) and fertility restoration in
pigeonpea
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7. • 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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8. 7
Transcriptomic resources and molecular markers developed at ICRISAT using
next generation sequencing and highthroughput genotyping technologies

9. 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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10. 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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11. 1. Complexity reduction of the DNA of interest
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12. 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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13. 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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14. 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'.

15. 3. Microarraying
Selection of clones
Arranged into a plate format
(usually 384-well plates)
Fragments within library
amplified
Spotted onto glass slides
14Genotyping array

16. 4. Hybridisation
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17. 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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18. 6. Data analysis
The 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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19. DArTsoft- Software
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20. 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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21. 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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22. 454/FLX sequencing
• 454/FLX sequencing
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23. Applications
 Full genome sequencing
 Amplicon sequencing
 Transcriptome sequencing
 Metagenomics
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24. 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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25. Thank you