1. Identification of Laser Dissected Cotton Chromosomes using PCR-based markers TaShundra J. Bryant

2. Outline of talk:  Introduction - Cotton - Markers  Materials & Methods - Laser Capture Micro-dissection (LCM) microscope. - Whole Genome Amplification (WGA) - Polymerase Chain Reaction (PCR) - Poly-Acrylamide Gel Electrophoresis (PAGE)  Results  Discussion  Conclusion

3. Introduction:  Cotton (Gossypium spp.)  Fiber & oil.  Genus cotton (50 sps) - Diploid species (2n = 2x = 26) ~ 45 species. {differentiated cytogenetically into A-G & K groups}. - Allotetraploid (2n = 4x = 52) ~ 5 species.  G. hirsutum ~ 95% world cotton production. (upland cotton) ~ Genome size 2,500Mb (30k genes).

4. Introduction: (continued…)  Molecular Marker  “- a readily detectable sequence of DNA or protein whose inheritance can be monitored”.  Key characteristics: - polymorphic, - reproducible, - co-dominant and - fast and inexpensive to detect.  Types: Bio-chemicalNon-PCR basedPCR based

5.  to dissect the cotton chromosomes using LCM.  to isolate the DNA and to amplify the product using Whole Genome Amplification method.  to identify the chromosomes using chromosome specific PCR based markers. Objectives:

6. Isolating the protoplasts from germinated seedlings Spreading the protoplast on PET membrane slides Picking the individual chromosomes using LCM Germinating the cotton seeds Isolation of chromosomes Amplification of chromosomes using WGA Outline of work:

7. Selecting the SSR makers for screening PCR amplification using selected markers Running the PCR product on PAGE gel Amplified WGA3 product PAGE gel Analysis Identification of chromosome

8. Materials & Methods:  The genetic standard, G. hirsutum TM-1 (Texas Marker- 1) was used for the preparation of protoplasts.  Protoplasts were isolated and spread on polyethylene tetraphthalate (PET) membrane slide.  Individual chromosomes were isolated using Laser Capture Micro-dissection microscope (Veritas).  25 chromosome samples, B2, B3, B4, B8, B9, B11, B12, B13, A2, A4, A5, A8, A9, A10, A13, A14, A17, A18, A19, A20, A21, A27, A28, A32, A33, TM-1 and -Ve were used for further analysis.

9. Chromosome specific markers: Fig.1a. Chromosome 4 map Fig.1a. Chromosome 11 map BNL3279 BNL1066 BNL836 BNL1231 BNL625 BNL3411 BNL3431 MUSS123 BNL2572 BNL4049 BNL530 BNL4047 NAU1267 CAPS & EST-SSRs

10. LCM: ( LCM: ( UV laser microdissection) PET membrane slide UV laser photovolatilizes cells Laser ablation of unwanted cells Cells and membrane are cut from tissue

11. PCR: Denaturation (94 0 C) Annealing (57 0 C) Extension (72 0 C)

12. PAGE:  6% PAGE gels were used  acrylamide:bisacrylamide (29:1)  PCR product was runned at 250V for 2:30 min’s  Visualized under UV light after ethidium bromide staining.  Gel images were saved and scoring was carried out manually. Vertical gel electrophoresis systems (C.B.S. Scientific Company, inc.)

13. Results: Fig.1a. Locating the chromosome using LCM Fig.1b. Dissecting the chromosome using LCM

14. Results: (continued…) TM-1 B2 B3 B4 B8 B9 B11 B12 B13 A2 A4 A5 A8 A9 A10 A13 A14 A17 A18 A19 A20 A21 A27 A28 A32 A33 -VE Marker 100 bp 200 bp 300 bp 100 bp 200 bp 300 bp Fig.2. PAGE gel Analysis for BNL-530 marker

15. Results: (continued…) MarkerChrMapped Locus B2B3B4B8B9B11B12B13A2A4A5A8A9 TM-1 BNL327911114 ------------ 85/ 95/ 114/ 120 BNL106611156------------- 135/ 155/ 180 BNL83611215 ------------ 210 BNL123111185------------- BNL62511205/ 185 -185/ 520 185185/ 200/ 100/ 125/ 135 110/ 125/ 135 140--205/ 490 100/ 110/ 700 100/ 490/ 520/ 800 100/ 230 - 265 BNL341111200------------- 205 BNL343111200100/ 180 --100--------- 250/ 270/ 280 MUSS12311 ------------- 120/ 130/ 150/ 225/ 260 BNL25724290------------- 160/ 190/ 290 BNL40494175-------160----- 80/ 160 BNL5304170100/ 150 85 50----150---- 50/ 60/ 70/ 110 BNL40474170------------- 195/ 85/ 70 NAU12674200------------- -

16. Results: (continued…) MarkerChromosomeMapped Locus A10A13A14A17A18A19A20A21A27A28A32A33A34TM-1 BNL327911114 --------85-85/ 95/ 114/ 120 -85/ 95/ 114/ 120 BNL106611156-----------135-135/ 155/ 180 BNL83611215-----------210- BNL123111185------------- BNL62511205/ 185 140-----100 - 265/ 270 -265 BNL341111200-------------205 BNL343111200--270-------- -250/ 270/ 280 MUSS12311 -------------120/ 130/ 150/ 225/ 260 BNL25724290-------------160/ 190/ 290 BNL40494175--90--80-110---90-80/ 160 BNL5304170----23050190100170--110-50/ 60/ 70/ 110 BNL40474170-------------195/ 85/ 70 NAU12674200--------------

17. Discussion: MarkerChromosomesAmplification BNL327911yes BNL106611yes BNL83611yes BNL343111yes BNL62511yes BNL123111no BNL341111no MUSS12311no BNL40494yes BNL5304yes BNL25724no BNL40474no NAU 12674no  Of 13 markers used, 7 markers were positive with selected samples while 6 markers were negative to all samples

18. Discussion: (Continued…)  Using chromosome specific markers, the samples A33 and A27 were potentially identified as a chromosome 11 and chromosome 4 respectively.  Samples B12, A9, A13 and A17 were neutral with selected markers, indicating that they were potential candidates for other chromosome specific markers.

19. Discussion: (Continued…)  Possible reasons for no amplification: - due to genotypic differences of the parents or - due to lack of proper PCR conditions - due to error in assigning the marker to mapped loci.

20. Conclusions:  Use of chromosome-specific markers will aid in sequencing of individual chromosomes.  It also aids in analyzing the genetic diversity among cultivated and wild cotton species.  Integrating the available polymorphic markers such as CAPS, EST-SSRs etc and linkage groups help in developing comprehensive cotton genetic map to assist the cotton breeders.

21. Acknowledgements:  Dr. Govind Sharma  Dr. Ramesh Kantety  Dr. Ramesh Buyyarapu  Sripathi Venkat &  All members of CMB group