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 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 BAC Libraries RE # Clones # Plates # Filters Average insert size Coverage Northern Red Oak QRNRBa QRNRBb HindIII BstYI 27,648 46,080 72 120 2 3 110 kb 120kb 4.0X 7.3X (760Mb genome) (760Mb genome) Black Walnut JNC_Ba HindIII 27,648 72 2 130kb 4.5X (800Mb genome) JNC_Bb BstYI 27,648 72 2 120kkb 4.0X (800Mb genome) 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 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 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 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 Candidate genes from Flowering Pathways in Arabidopsis Comparative Genomics of Hardwood Tree Species http://www.hardwoodgenomics.org Single Gene BACs: Stats Finished BACs Total Finished (Single Contig) 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 Dormancy CG SM1_HOS1 Chromatin 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 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 PPA000367 Putative SWI/SNF 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 PPA000367 Putative SWI/SNF NRO GENE QRNRBa019C05_1 QRNRBa019C05_2 QRNRBa019C05_3 QRNRBa019C05_4 QRNRBa019C05_5 QRNRBa019C05_6 QRNRBa019C05_7 QRNRBa019C05_8 QRNRBa019C05_9 QRNRBa019C05_10 QRNRBa019C05_11 BW GENE JN_CBa008B02_1 JN_CBa008B02_2 JN_CBa008B02_3 JN_CBa008B02_4 JN_CBa008B02_5 JN_CBa008B02_6 Sequence desc. ap-2 complex subunit alpha-1-like reticulata-related 1 gaba transporter 1-like ribonuclease h protein at1g65750-like orf120 gene product uncharacterized loc101221444 retrotransposon unclassified chromodomain-helicase-dna-binding protein mi- isoform 1 dna binding swi snf-related matrix-associated actin-dependent regulator of chromatin subfamily a member 3-like 2-like ribonuclease h protein at1g65750-like btb poz domain-containing protein at3g44820-like ap-2 complex subunit alpha-1-like reticulata-related 1 swi snf-related matrix-associated actin-dependent regulator of chromatin subfamily a member 3-like 2-like uncharacterized loc101221444 chromodomain-helicase-dna-binding protein mi- isoform 1 Comparative Genomics of Hardwood Tree Species http://www.hardwoodgenomics.org Chestnut Blight QTL in NRO & BW • • • • • Northern 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 Chestnut Blight QTL in NRO & BW 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 Scientists Comparative Genomics of Hardwood Tree Species http://www.hardwoodgenomics.org
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