integrated physical and genetic mapping of upland cotton lei e presented by mingxiong pang
Post on 21-Dec-2015
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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.
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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