Integrated Physical and Genetic Mapping

of Upland Cotton

Lei E

Presented by

Mingxiong PANG

IntroductionIntroductionCCottonotton is important in the is important in the USAUSA

No. 1 natural fiber resource No. 1 natural fiber resource

No. 2 seed oil resourceNo. 2 seed oil resource

No. 4 valuable crop in USANo. 4 valuable crop in USA

There are four There are four cultivated speciescultivated species

Two new world tetraploid Two new world tetraploid species, -species, -G. hirsutumG. hirsutum L. (upland L. (upland cotton) cotton)

--G. barbadenseG. barbadense L. (Pima cotton) L. (Pima cotton)

Two old world diploid species, Two old world diploid species, G. arboreumG. arboreum L. and L. and G. G. herbaceumherbaceum L. L.

Upland CottonUpland Cotton

AllotetraploidsAllotetraploids

A + D subgenomesA + D subgenomes

2n=4x=52 chromosomes2n=4x=52 chromosomes

~2,500 Mb of genome size~2,500 Mb of genome size

ObjectivesObjectives

Localize existing SSR markers on BAC Localize existing SSR markers on BAC

library to determine the physical BAC library to determine the physical BAC

address of each locus.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 Distribute PCR pools of the BACs as an

additional resource to the BAC library.additional resource to the BAC library.

Material and MethodsMaterial and Methods

SSRsSSRs Brookhaven National Laboratory (BNL)Brookhaven National Laboratory (BNL)

Fluorescent dye labeled. Fluorescent dye labeled.

– HEX (yellow), NED (green), 6-FAM (blue)HEX (yellow), NED (green), 6-FAM (blue)

80 SSR marker primer pairs80 SSR marker primer pairs

Most assigned to genetic maps and Most assigned to genetic maps and

chromosomeschromosomes

Physical mappingPhysical mapping

Chromosomal assignment of DNA markersChromosomal assignment of DNA markers

– Aneuploids stockAneuploids stock

MonosomesMonosomes

MonotelodisomesMonotelodisomes

Clone contig mapClone contig map

– Overlapping clones of Overlapping clones of

genome DNA w/o gapsgenome DNA w/o gaps

– Vectors: YAC, BAC.Vectors: YAC, BAC.

…

…

…

…

…

…

…

…

Missing

Missing

BAC LibraryBAC Library

Bacterial Artificial Chromosome (BAC)Bacterial Artificial Chromosome (BAC)

– Modified F factor plasmid in Modified F factor plasmid in E. coliE. coli

– Low chimeric and stable clonesLow chimeric and stable clones

– Up to 500kb DNA insertUp to 500kb DNA insert

Cotton BAC librariesCotton BAC libraries

– Clemson University Clemson University

Genomics InstituteGenomics Institute

– Upland cotton Upland cotton ‘‘MMaaxxaxxa’’

BAC LibraryBAC Library

““Maxxa” BAC libraryMaxxa” BAC library

– 336 336 plates (plates (384-well384-well// plate plate))

– 129,024 clones129,024 clones

– Mean insert: 137 kbMean insert: 137 kb

– Genome size: 2,118 MbGenome size: 2,118 Mb

CoverageCoverage

– W=NI/GW=NI/G

=(129,024=(129,024хх137 Kb)/2,118Mb=~8.3137 Kb)/2,118Mb=~8.3XX

PoolingPooling

Super pools (SP)Super pools (SP)

– Every 6 BAC Every 6 BAC

platesplates

– 2,304 clones2,304 clones

Column pools (CP) Column pools (CP)

& Row pools (RP)& Row pools (RP)

– 48 CPs/SP48 CPs/SP

– 48 RPs/SP48 RPs/SP

P1

P6

P4

P2

P3

P5

Row Pool 20(RP 20)

Single BAC Clone: P3D7

Column Pool 7 (CP 7)

CPRP cultureCPRP culture

IncubationIncubation– 37 37 CC

– 175 RPM175 RPM

– 16 hours16 hours

SubcultureSubculture– 96-deep-well block96-deep-well block

– 96 individual 15 ml Falcon Tubes96 individual 15 ml Falcon Tubes

PCR CPRP & DetectionPCR CPRP & Detection

Positive SSR primers from PCR Super Positive SSR primers from PCR Super

poolpool

One positive product each in CP&RPOne positive product each in CP&RP

Locate the positive BAC for the SSR Locate the positive BAC for the SSR

markermarker

VerificationVerification

Individual clones of positive BACsIndividual clones of positive BACs

Cultured, DNA extractedCultured, DNA extracted

PCR with corresponding SSR PCR with corresponding SSR

primersprimers

Sequencing PCR productsSequencing PCR products

Pairwise alignmentsPairwise alignments

ResultsResults

10 super pools created & analyzed10 super pools created & analyzed

Total chosen Total chosen 23,040 BAC clones23,040 BAC clones

Equivalent genome coverage Equivalent genome coverage

(23,040 (23,040 хх 137 Kb)/2,118 Mb=1.5 137 Kb)/2,118 Mb=1.5xx

Positive LociPositive Loci

Super Pools Positive Loci Number

SP#1 20

SP#2 28

SP#3 15

SP#4 24

SP#5 12

SP#6 17

SP#7 16

SP#8 13

SP#9 12

SP#10 27

Total BACs:23,040

Total Positive BACs:184

Positive LociPositive Loci

80 SSR primers amplif80 SSR primers amplifiedied 142 loci 142 loci

on “Maxxa” genomeon “Maxxa” genome..

Expected: 142 Expected: 142 хх 1.5= ~210 loci 1.5= ~210 loci

Observed: 184 lociObserved: 184 loci

94 unique loci amplified by 63 94 unique loci amplified by 63

primersprimers

CPRP GelsCPRP Gels

Target band on RP13 Target band on CP8

Gel Images of CPRP#3-1606YThe 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.

Positive BACsPositive BACs

30 positive BACS 30 positive BACS

8 sequenc8 sequenceses verified verified

DiscussionDiscussion(1)(1)

1.5 genome equivalent covered1.5 genome equivalent covered

94/142 loci, 63/80 primers94/142 loci, 63/80 primers

BAC library is adequateBAC library is adequate

SP creation is adequateSP creation is adequate

Discussion Discussion (continue)(continue)

700 existing SSRs700 existing SSRs

Need much moreNeed much more

High-resolution integrated mapHigh-resolution integrated map

BAC ending sequencingBAC ending sequencing

– Align contigsAlign contigs

– Develop new markersDevelop new markers