Genomic In-Situ Hybridization (GISH)-Principles, Methods and Applications in Crop Plants.pptx

Banoth Madhu: Genomic In-Situ Hybridization (GISH)-Principles, Methods and Applications in Crop Plants. It is a cytogenetic technique that allows the detection and localization of specific nucleic acid sequences on morphologically preserved chromosomes using genomic DNA of donor specie as probe. It is a cytogenetic technique that allows the detection and localization of specific nucleic acid sequences on morphologically preserved chromosomes using genomic DNA of donor specie as probe

1. Genomic In-Situ Hybridization(GISH ); Principles , Methods and Applications In Crop Plants 1 Banoth Madhu Ph.D Research Scholar Genetics and Plant Breeding Tamil Nadu Agricultural University, Coimbatore

2. 2 It is a cytogenetic technique that allows the detection and localization of specific nucleic acid sequences on morphologically preserved chromosomes using genomic DNA of donor specie as probe. What is GISH(Genomic In-Situ Hybridization)?

3. 3 GISH is an advancement in the FISH and allows distinguishing the genomes in a cell . An unlabeled DNA of parental specie is used as competitor DNA and Total labeled genomic DNA is used as probe in hybridization. GISH is quick, accurate, sensitive, informative and a comparative approach . Detect specific nucleotide sequences within cells and tissues . U nique link among the studies of cell biology, cytogenetics, and molecular genetics. D etect single-copy sequences on chromosome with probes shorter than 0.8 kb . More precisely, it investigates the evolutionary relationships of crops & identification of inserted regions in parent from alien species . These advantage GISH has over FISH Introduction

4. 4 GISH was mainly developed for the animal hybrid cell in 1986. GISH for plants was developed in 1987 by M.D. Bennett and J.S. Heslop-harrison . 1 st used to discriminate genomes of intergeneric hybrid between Hordeum chilense & Secale africanum at Plant Breeding institute Cambridge in 1987. Schwarzacher (1989) modified the FISH technique to GISH. Background M.D. Bennett

5. 5 This technique involves the extraction of labeling DNA of one organism and to use as a probe to target the genome of another organism. The part of genome that are similar to the probe hybridize to from a probe target complex. Principle

6. 6 Case Study Characterization of chromosome types among accessions of a germplasm collection- FISH T o distinguish parental chromosomes (or chromosome segments) in an interspecific hybridization or the distinct genomes of an allopolyploid- GISH

7. 7 Main steps of GISH

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9. 9 DNA isolation (probe and blocking DNA) CTAB method from Embrapa Wheat Selective precipitation of polysaccharide, according to Michaels et al .

10. 10 DNA quantification Analysis of genomic DNA by gel electrophorese in 0.8% agarose

11. 11 Fragmentation of the blocking DNA and Nick translation Nick translation reaction

12. 12 Seed germination and collecting, pretreating and fixating root tips Washing and germinating seeds in (A) and (B); pretreatment and fixation of root tips in (C) and (D ) .

13. 13 Slide preparation Preparation of slides for genomic in situ hybridization

14. 14 S lide treatment for GISH Slide treatment

15. 15 Preparation of the hybridization mixture Preparation of hybridization mixture(Probe + Blocking DNA) for GISH

16. 16 Preparation of the hybridization mixture

17. 17 Post-hybridization baths and probe detection

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19. 19 Results

20. 20 Blocking DNA : probe should be high enough to prevent labeling of both the genomes at the same time. Blocking DNA is more important in intraspecific hybrids eg :- Lycopersicon esculentum and L. peruvianum – Need more(100X) blocking DNA ( Parokonny et al ., 1997). While, Dendranthema nankingense and Tanacetum vulgare – No Blocking DNA (Tang et al ., 2011) Precautions

21. 21 It is possible to distinguishing the genomes in a interspecific/intergeneric hybrid. To the study of hybrid lineages detecting the amount of introgressed chromatin during the production of new lineages. The GISH has direct applications on the understanding of the genome evolution of partial allopolyploids and recombinant inbred lines. GISH can be applied to the analysis of the meiotic behavior in hybrids and polyploids, providing information concerning the relationship between species. Karyotype analysis Detection of Chromosomal aberration Chromosome paintings & diagnostic cytogenetics Applications

22. 22 Cannot detect small mutations. Probes are not yet commercially available for all chromosomal regions Relatively expensive C annot discriminate closely related genomes in certain allopolyploids GISH can distinguish between genomes with 80-85% homogenousity . But if stringency is high & amount of blocking DNA is also high, almost identical genomes can also be identified ( Parokonny et a l,1997) Limitations