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

2. Introduction Cotton 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) Two new world tetraploid species, -G. hirsutum L. (upland cotton) -G. barbadense L. (Pima cotton) -G. barbadense L. (Pima cotton) Two old world diploid species, G. arboreum L. and G. herbaceum L. Two old world diploid species, G. arboreum L. and G. herbaceum L.

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

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

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

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

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

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

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

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

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

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

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

15. Positive Loci Super PoolsPositive Loci Number SP#120 SP#228 SP#315 SP#424 SP#512 SP#617 SP#716 SP#8 13 SP#9 12 SP#10 27 Total BACs: 23,040 Total Positive BACs: 184

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

17. CPRP Gels Target band on RP13Target band on CP8 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 30 positive BACS 8 sequences verified 8 sequences verified

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

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