2. LG1LG2LG3LG4LG5LG6LG7LG8LG9LG10LG11 LG14 WEYQTL WEYVEQ WEYVEB WEYPRE Fig. 5 Wood property QTLs and Candidate Genes in loblolly pine ewsg %lw *lwsg %lw *%lw lwsg *ewsg *emfa ewsg *emfa *ewsg ewsg *%lw *emfa lwsg *ewsg *lwsg lwsg *ewsg *lwsg lwsg %lw *emfa *lmfa %lw *lmfa * %lw lwsg *emfa lwsg *wsg *lwsg *swv *lmfa *%lw *swv *%lw lwsg *lwsg lwsg *%lw lwsg swv lmfa *swv *%lw emfa *%lw %lw *ewsg swv lmfa lwsg *ewsg *%lw swv lwsg *ewsg lwsg swv *ewsg swv *ewsg swv *ewsg swv *ewsg %lw *ewsg lmfa *ewsg *swv *lwsg swv *lwsg %lw swv *lwsg *lmfa *lwsg *%lw 4CL 4ch-2 SAM synthetase-1 SAM synthetase-2 diphenol oxidase AGP6-like/I4A9 CCoAOMT AGP6 UTP glucose gt PAL glycine hydroxy-mt cellulase (ce12) 4CH-1 CAD beta-1,3 glucanase AGP6-like/3H6 Candidate Genes

3. SNP PIPELINE DISCOVERY VALIDATION & HAPLOTYPE DETERMINATION VALIDATION & HAPLOTYPE DETERMINATION GENOTYPING

4. 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 SNP discovery in silico

5. Most abundant gene contigs in xylem

6. Location of SNPs within a sequence contig for the gene AGP6

7. 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

8. 2 GENE MEMBERS FOR TRANS-CINNAMATE 4-HYDROXYLASE

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

10. Haplotype determination

13. 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

14. AGP6 pyrograms Nucleotide Addition T A C A T C G A Sequence SNP Genotype TTACAT-GAAA TTACA--GAAA 3 2 1 3 2 1 A/T A/A Target: TTAC A / T GAAA

15. 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 Pyrosequencing Advantages & Cost PCR - 50 ul90 c Pyrosequencing reaction $1