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Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Northern Red Oak and Black Walnut BAC-based Comparative Genomics
Chris Saski
Tatyana Zhebentyayev
Clemson University
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Objectives
• BAC libraries for Northern Red Oak (NRO) and Black Walnut (BW)
• Phythopthora response and other abiotic/biotic stress response elements
• Target and sequence homologous chestnut blight QTL region in NRO and BW
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
BAC Libraries
Northern Red Oak Black Walnut
QRNRBa QRNRBb JNC_Ba JNC_Bb
RE HindIII BstYI HindIII BstYI
# Clones 27,648 46,080 27,648 27,648
# Plates 72 120 72 72
# Filters 2 3 2 2
Average insert size 110 kb 120kb 130kb 120kkb
Coverage4.0X 7.3X 4.5X 4.0X
(760Mb genome) (760Mb genome) (800Mb genome) (800Mb genome)
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
BAC Recruitment
• Single Gene: 3D hybridization to ID flowering and stress response elements
– 144 probes
– Single BAC ID for NRO & BW
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
BAC Recruitment
• Pooled: Bulk hybridization and fingerprinting for blight QTL
– 24 probes
– Bulk BAC ID for NRO & BW
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Sequenced BAC Stats
Source of information Genes Targeted
Flowering pathway homologs (Arabidopsis) 21
Association study in apricot (A. Abbott) 31
Fagacea ESTs from GeneBank 5
Dormancy-related candidate genes (J. Romero-Severson) 10
Divergent EST-SSRs in two oak species (O. Gailing) 5
Cbr1, Cbr2, Cbr3 (M. Staton) 24
Total 96
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Sequenced BACs: Functional Groups
Functional groups Genes Targeted
Flowering pathway homologs (Arabidopsis) 12
Epigenetic control/chromatin modification 12
Hormone regulation 7
Biotic/abiotic stress 25
Cell division/chromosome segregation 4
Miscellaneous (mainly unknown function) 12
MTP: Chestnut blight QTLs (Cbr1, Cbr2, Cbr3) 24
Total 96
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Candidate genes from Flowering Pathways in Arabidopsis
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Single Gene BACs: Stats
Finished BACs(Single Contig)
Total Finished Bases G+C N50
Average BAC Length (bp) No. Genes
Northern Red Oak 77 7,320,181 36% 114,670 106,089 665
Black Walnut 69 8,610,043 35% 120,554 111,818 654
NRO % Genome Captured: 1%
BW % Genome Captured: 1%Long stretches of homopolymer runs....
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Dormancy CG SM1_HOS1Chromatin Modification
• RING finger motif near the amino terminus.
• Negative regulator of cold responses. • Functions as an E3 ligase required for the
ubiquitination of ICE1. • HOS1 physically interacts with ICE1 and
mediates the ubiquitination of ICE1 both in vitro and in vivo.
• Overexpression represses the expression of CBFs and their downstream genes and confers increased sensitivity to freezing stress.
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Dormancy CG SM1_HOS1
BW Clone: 130,189kb NRO Clone: 138,635kb Comparison
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
PPA000367 Putative SWI/SNF
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
PPA000367 Putative SWI/SNF
BW Clone: 124,830kb NRO Clone: 147,484kb Comparison
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
PPA000367 Putative SWI/SNFNRO GENE Sequence desc.
QRNRBa019C05_1 ap-2 complex subunit alpha-1-like
QRNRBa019C05_2 reticulata-related 1
QRNRBa019C05_3 gaba transporter 1-like
QRNRBa019C05_4 ribonuclease h protein at1g65750-like
QRNRBa019C05_5 orf120 gene product
QRNRBa019C05_6 uncharacterized loc101221444
QRNRBa019C05_7 retrotransposon unclassified
QRNRBa019C05_8 chromodomain-helicase-dna-binding protein mi- isoform 1
QRNRBa019C05_9 dna binding
QRNRBa019C05_10swi snf-related matrix-associated actin-dependent regulator of chromatin subfamily a member 3-like 2-like
QRNRBa019C05_11 ribonuclease h protein at1g65750-like
BW GENE
JN_CBa008B02_1 btb poz domain-containing protein at3g44820-like
JN_CBa008B02_2 ap-2 complex subunit alpha-1-like
JN_CBa008B02_3 reticulata-related 1
JN_CBa008B02_4swi snf-related matrix-associated actin-dependent regulator of chromatin subfamily a member 3-like 2-like
JN_CBa008B02_5 uncharacterized loc101221444
JN_CBa008B02_6 chromodomain-helicase-dna-binding protein mi- isoform 1
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Chestnut Blight QTL in NRO & BWNorthern Red Oak
• 135 BAC identified
• MTP = 24 BACs
• Length: 1,740,787bp
• Scaffolds: 11
• N50: 213,671bp
Black Walnut
• 256 BACs identified
• MTP = 31 BACs
• Length: 1,399,219
• Scaffolds: 8
• N50: 194,078bp
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Chestnut Blight QTL in NRO & BW
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Future Directions
• Complete BAC analysis and finalize manuscript
– Assemblies, functional annotations, QTL interval analysis
– Tatyana, Chris
• Phenology pilot?
• Draft genome(s)
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Comparative Genomics of
Hardwood Tree Species
http://www.hardwoodgenomics.org
Scientists
