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Integrated Physical and Genetic Mapping of Upland Cotton

Integrated Physical and Genetic Mapping of Upland Cotton. Lei E Presented by Mingxiong PANG. Introduction C otton is important in the USA. No. 1 natural fiber resource No. 2 seed oil resource No. 4 valuable crop in USA. There are four cultivated species.

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Integrated Physical and Genetic Mapping of Upland Cotton

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  1. Integrated Physical and Genetic Mapping of Upland Cotton Lei E Presented by Mingxiong PANG

  2. IntroductionCotton is important in the USA No. 1 natural fiber resource No. 2 seed oil resource No. 4 valuable crop in USA

  3. There are four cultivated species • Two new world tetraploid species, -G. hirsutum L. (upland cotton) -G. barbadense L. (Pima cotton) • Two old world diploid species, G. arboreum L. and G. herbaceum L.

  4. Upland Cotton • Allotetraploids • A + D subgenomes • 2n=4x=52 chromosomes • ~2,500 Mb of genome size

  5. Objectives • Localize existing SSR markers on BAC library to determine the physical BAC address of each locus. • Evaluate the utility of this BAC library. • Distribute PCR pools of the BACs as an additional resource to the BAC library.

  6. Material and MethodsSSRs • Brookhaven National Laboratory (BNL) • Fluorescent dye labeled. • HEX (yellow), NED (green), 6-FAM (blue) • 80 SSR marker primer pairs • Most assigned to genetic maps and chromosomes

  7. … … Missing … … … … Missing … … Physical mapping • Chromosomal assignment of DNA markers • Aneuploids stock • Monosomes • Monotelodisomes • Clone contig map • Overlapping clones of genome DNA w/o gaps • Vectors: YAC, BAC.

  8. BAC Library • Bacterial Artificial Chromosome (BAC) • Modified F factor plasmid in E. coli • Low chimeric and stable clones • Up to 500kb DNA insert • Cotton BAC libraries • Clemson University Genomics Institute • Upland cotton ‘Maxxa’

  9. BAC Library • “Maxxa” BAC library • 336 plates (384-well/ plate) • 129,024 clones • Mean insert: 137 kb • Genome size: 2,118 Mb • Coverage • W=NI/G =(129,024х137 Kb)/2,118Mb=~8.3X

  10. P2 P1 Row Pool 20 (RP 20) Single BAC Clone: P3D7 P3 P4 Column Pool 7 (CP 7) P5 P6 Pooling • Super pools (SP) • Every 6 BAC plates • 2,304 clones • Column pools (CP) & Row pools (RP) • 48 CPs/SP • 48 RPs/SP

  11. CPRP culture • Incubation • 37 C • 175 RPM • 16 hours • Subculture • 96-deep-well block • 96 individual 15 ml Falcon Tubes

  12. PCR CPRP & Detection • Positive SSR primers from PCR Super pool • One positive product each in CP&RP • Locate the positive BAC for the SSR marker

  13. Verification • Individual clones of positive BACs • Cultured, DNA extracted • PCR with corresponding SSR primers • Sequencing PCR products • Pairwise alignments

  14. Results • 10 super pools created & analyzed • Total chosen 23,040 BAC clones • Equivalent genome coverage (23,040 х 137 Kb)/2,118 Mb=1.5x

  15. Positive Loci

  16. Positive Loci • 80 SSR primers amplified 142 loci on “Maxxa” genome. • Expected: 142 х 1.5= ~210 loci • Observed: 184 loci • 94 unique loci amplified by 63 primers

  17. Target band on RP13 Target band on CP8 CPRP Gels Gel Images of CPRP#3-1606Y The green bands are positive controls, which were amplified by a primer pair that are designed to amply a 163-bp fragment on BAC conserved region.

  18. Positive BACs • 30 positive BACS • 8 sequences verified

  19. Discussion(1) • 1.5 genome equivalent covered • 94/142 loci, 63/80 primers • BAC library is adequate • SP creation is adequate

  20. Discussion (continue) • 700 existing SSRs • Need much more • High-resolution integrated map • BAC ending sequencing • Align contigs • Develop new markers

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