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Fig. 5 Wood property QTLs and Candidate Genes in loblolly pine

WEYPRE. WEYVEB. WEYQTL. WEYVEQ. *ewsg. ewsg. *cwc. *cwc. *lmfa. *swv. QxE. * QxE. AGP6. *%lw. lmfa. %lw. *%lw. lwsg. PAL. lwsg. *swv. *ewsg. swv. *lwsg. *ewsg. %lw. *%lw. ewsg. *lwsg. %lw. *mfa. *cwc. wsg. cellulase (ce12). *lwsg. lwsg. *cwc. lwsg. 4ch-2. swv.

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Fig. 5 Wood property QTLs and Candidate Genes in loblolly pine

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  1. WEYPRE WEYVEB WEYQTL WEYVEQ *ewsg ewsg *cwc *cwc *lmfa *swv QxE *QxE AGP6 *%lw lmfa %lw *%lw lwsg PAL lwsg *swv *ewsg swv *lwsg *ewsg %lw *%lw ewsg *lwsg %lw *mfa *cwc wsg cellulase (ce12) *lwsg lwsg *cwc lwsg 4ch-2 swv * %lw *ewsg *%lw *cwc *ewsg *lmfa %lw lwsg *%lw *ewsg lwsg lwsg swv 4CL lwsg %lw *emfa *emfa *QxE lwsg *ewsg cwc %lw swv lwsg *lwsg swv cwc *emfa *QxE lwsg *%lw *%lw *cwc *QxE *QxE *mfa *ewsg swv *swv lmfa *ewsg %lw lwsg *lwsg *%lw *mfa lwsg glycine hydroxy-mt beta-1,3 glucanase lwsg *%lw *%lw *%lw *lwsg *wsg swv *swv *lwsg lmfa *%lw *%lw ewsg swv *%lw lwsg *%lw %lw *ewsg *%lw *lwsg lmfa ewsg *cwc *ewsg *ewsg *lmfa 4CH-1 emfa *QxE *%lw %lw ewsg CCoAOMT *%lw *ewsg AGP6-like/I4A9 *wsg SAM synthetase-2 cwc *cwc *ewsg swv CAD *lwsg *emfa *QxE *lwsg ewsg cwc *lmfa *ewsg *ewsg cwc *lwsg *lwsg *lwsg *cwc *ewsg *lmfa AGP6-like/3H6 lwsg lwsg diphenol oxidase *%lw *ewsg *ewsg *ewsg ewsg *QxE ewsg SAM synthetase-1 *%lw QxE *ewsg cwc lwsg swv *mfa %lw swv *%lw *mfa *emfa %lw *lwsg cwc UTP glucose gt *%lw Fig. 5 Wood property QTLs and Candidate Genes in loblolly pine LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9 LG10 LG11 LG14 Candidate Genes

  2. SNP PIPELINE DISCOVERY VALIDATION & HAPLOTYPE DETERMINATION GENOTYPING

  3. SNP discovery in silico NSF Loblolly Pine xylem EST database http://web.ahc.umn.edu/biodata/nsfpine/ 50 000 xylem EST’s 4 Libraries Up to 12 possible alleles present

  4. Most abundant gene contigs in xylem

  5. 11 176 264 355 443 517 641 729 812 926 100 1,000000 Location of SNPs within a sequence contig for the gene AGP6

  6. Allele or gene member ? Most divergent allele detected so far shares 97% homology. Secondary peaks in gametophyte sequencing indicate multiple gene members. Compare map position’s

  7. 2 GENE MEMBERS FOR TRANS-CINNAMATE 4-HYDROXYLASE

  8. SNP discovery by sequencing • 32 gametophytes from diverse genetic backgrounds • Primers designed for SNP-rich regions detected in silico • SNP discovery within introns

  9. Haplotype determination

  10. Genotyping by Pyrosequencing 1. Sequencing primer annealed 2. Addition of dNTPs sequentially 3. If dNTP incorporated PPi is released 4. Sulfurylase converts Ppi ATP 5. ATP drives luciferase reaction 6. Apyrase degrades unincorporated dNTP and ATP

  11. TT A C A T - G AAA TT A C A - - G AAA AGP6 pyrograms Target: TTACA/TGAAA SNP Genotype Sequence 3 2 A/T 1 Sequence 3 A/A 2 1 Nucleotide Addition T A C A T C G A

  12. Pyrosequencing Advantages & Cost • 96 SNP genotypes in ~15 minutes • primer placement not restricted to position immediately adjacent to SNP • > 99 % reproducibility & accuracy • Analyze multiple SNPs if within 20 bp • PCR - 50 ul 90 c • Pyrosequencing reaction $1

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