LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9 LG10 LG11 LG14 WEYQTL WEYVEQ WEYVEB WEYPRE Fig. 5 Wood property QTLs and Candidate Genes in loblolly pine ewsg %lw *lwsg %lw *%lw lwsg *ewsg *emf a ewsg *emfa *ewsg ewsg *%lw *emfa lwsg *ewsg *lws g lws g *ews g *lws g lws g %lw *emfa *lmfa %lw *lmfa * %lw lwsg *emfa lwsg *wsg *lwsg *sw v *lmf a * %lw *sw v * %lw lwsg *lws g lws g *%lw lwsg swv lmf a *swv * %lw emfa *%lw %lw *ewsg swv lmf a lwsg *ewsg *%lw swv lwsg *ewsg lwsg swv swv *ewsg swv *ewsg swv swv *ewsg swv *ewsg %lw *ewsg lmfa *ewsg *ewsg *swv *lwsg swv *lwsg %lw swv *lwsg *lwsg *lmfa *lwsg *lwsg *%lw *%lw *QxE *cw c lmfa *mfa *lwsg cwc *mfa *ewsg *%lw *%lw QxE *ewsg *wsg %lw wsg *ewsg *ewsg *lwsg lwsg *%lw *ewsg *%lw ewsg *%lw %lw *%lw ewsg *mfa lwsg *%lw *ewsg %lw *lmfa ewsg *mfa lwsg *%lw lwsg *%lw lwsg ewsg ewsg *lwsg *%lw *mfa *%lw cwc cwc *ewsg %lw *%lw *cwc cwc lwsg QxE *cwc *QxE *cw c *QxE *QxE *QxE *QxE *cwc *cwc cwc *cw c *QxE *cw c cwc *cw c *QxE cwc 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
LG1LG2LG3LG4LG5LG6LG7LG8LG9LG10LG11 LG14 WEYQTL WEYVEQ WEYVEB WEYPRE Fig. 5 Wood property QTLs and Candidate Genes in loblolly pine ewsg %lw *lwsg %lw.
Dec 13, 2015
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LG1 LG2 LG3 LG4 LG5 LG6 LG7 LG8 LG9 LG10 LG11
LG14
WEYQTL WEYVEQWEYVEBWEYPRE
Fig. 5 Wood property QTLs and Candidate Genes in loblolly pine
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QxE
*cwc
*QxE
*cwc
*QxE
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*QxE
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*QxE
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cwc
4CL
4ch-2
SAM synthetase-1
SAM synthetase-2
diphenol oxidase
AGP6-like/I4A9CCoAOMT
AGP6
UTP glucose gt
PAL
glycine hydroxy-mt
cellulase (ce12)
4CH-1
CAD
beta-1,3 glucanase
AGP6-like/3H6
Candidate Genes
SNP PIPELINE
DISCOVERYDISCOVERYVALIDATION &
HAPLOTYPE DETERMINATION
VALIDATION &HAPLOTYPE
DETERMINATIONGENOTYPINGGENOTYPING
NSF Loblolly Pine xylem EST databasehttp://web.ahc.umn.edu/biodata/nsfpine/
50 000 xylem EST’s
4 Libraries
Up to 12 possible alleles present
SNP discovery in silico
Gene Contig
Alpha tubulin 5678elongation factor 1 alpha 5677S-Adenosylmethionine synthetase 2 5676glycine-rich RNA binding protein 5675tonoplast aquaporin (TIP)/Delta tonoplast intrinsic protein 5674peptidylprolyl isomerase (EC 5.2.1.8) (cyclophilin1), cytosolic 5673allergen-like 5672S-adenosyl-L-homocysteine hydrolase (SAHH) 5671peptidylprolyl isomerase (EC 5.2.1.8) (cyclophilin1), cytosolic 5670TCTP 5669S-Adenosylmethionine synthetase (SAMS2) 5668metallothionein-like protein 5667PtaAGP6 putative arabinogalactan protein-alllele 5666chitinase homolog LP6 5665glycine-rich RNA-binding protein (RNP) 5664MJK13.14 protein (unknown function) 5663PtaAGP6 putative arabinogalactan protein-allele 5662polyubiquitin 56614-coumarate:CoA ligase (4CL) 5660PtaPHY2 phytocyanin homolog 5659phenylcoumaran benzylic ether reductase 5658trans-cinnamate 4-hydroxylase (C4H1) 5657alpha-tubulin 3 5656alpha-tubulin 5655 methionine synthase 5654S-adenosyl-L-homocysteine hydrolase (SAHH) 5653ADP, ATP carrier protein 5652p14A9 arabinogalactan-like protein 5651heat shock protein hsp70 5650glycine hydroxymethyltransferase 5649
unknown protein arabidopsis MYA6.1 5648porin Mip1 5647
Most abundant gene contigs in xylem
Location of SNPs within a sequence contig for the gene AGP6
11 1,000000641100 176 264 355 443 517 729 812 926
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
2 GENE MEMBERS FOR TRANS-CINNAMATE 4-HYDROXYLASE
SNP discovery by sequencing
4-Coumarate CoA Ligase (4CL)
0 500 1000 1500 2000 2500
992
1416
1615
1702
1850
1939
2009
2357
2563
F4 R4 F3 R3 F2 R1A59 584 945 1462 1492 2008
• 32 gametophytes from diverse genetic backgrounds
• Primers designed for SNP-rich regions detected in silico
• SNP discovery within introns
4-Coumarate CoA Ligase (4CL)
0 500 1000 1500 2000 2500
992
1416
1615
1702
1850
1939
2009
2357
2563
F4 R4 F3 R3 F2 R1A59 584 945 1462 1492 2008
Haplotype determination
DNA179
368
513
983
1059
1092
1128
1135
1174
1213
1214
1215
1219
1320
1322
1402
1558
1644
1675
1692
1734
1761
1779
1888
1917
1989
HAP InsLSG 370xpoly A 1 A A C T T - C G A G G G T A G G G T A G T C G G G CLSG 373xpoly A 1 A A C T T - C G A G G G T A G G G T A G T C G G G CLSG 374x Poly A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C
LSG 363 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G CDF 3364 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G CS4PT-6 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C
SH7 A 1 T T - C G A G G G T A G G G T A G T C G G G CUSFS-12 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C1-1120 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C7-1102 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C
8-61 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C8-1020 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C8-1564 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C
12-1020 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C12-1139 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C12-1060 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C17-1024 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C
2-08 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C6-13 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C
8-103 A 1 A A C T T - C G A G G G T A G G G T A G T C G G G C
LSG 413 A 2 G G C C T T A C A T G A T G C T A C T A C T G G A T8-76 A 2 G G C C T T A C A T G A T G C T A C T A C T G G A T8-33 A 2 G G C C T T A C A T G A T G C T A C T A C T G G A T
7-56 A 5 G G C C T T A C A T G A T G C T A C T A C T G A A T
18-1210 A 3 G G C C T T A C A T G A T G C T A C T A C C G G A T11-503 A 3 G G C C T T A C A T G A T G C T A C T A C C G G A T
6-1112 A 4 A A G T C T C C C T G A C G C T A C T A C C A G A C7-1502 A 4 A A G T C T C C C T G A C G C T A C T A C C A G A C
8-68 A 6 A A C T C T C C C T G A C G C T A C T A C C A G A C
15-1079 A 7 A A C T C T C C C T A A C G C T G T A G T C G G G C
7-1505 A 8 A A C T C T C C C T A A C G C T A C T A C C G G A C
8-203 A 9 G G C T C T C C C T A A C G C T A C T A C C G G A C
Arabinogalactan-like Protein AGP6
0 100 200 300 400 500 600 700 800 900 1000 1100 1200
-41 621 724 812 1079
F1 560 R1A F2 R2-12 547 574 988
DNA 79
98
116
124
148
164
166
176
187
210
247
481
496
514
550
684
687
691
707
736
743
745
771
796
832
868
875
882
887
901
910
918
930
957
966
989
1003
HAP INS
12-1139 A 1 G T G T G C A G G G G G G C C T T G T T T 1 A A A G A T C C A C G G C G CLSG 373xpoly A 1 G T G T G C A G G G G G G C C T T G T T T 1 A A A G A T C C A C G G C G C
LSG 363 A 1 G T G T G C A G G G G G G C C T T G T T T 1 A A A G A T C C A C G G C G CLSG 413 A 1 G T G T G C A G G G G G G C C T T G T T T 1 A A A G A T C C A C G G C G CDF 3364 A 1 G T G T G C A G G G G G G C C T T G T T T 1 A A A G A T C C A C G G C G C18-1210 A 1 G T G T G C A G G G G G G C C T T G T T T 1 A A A G A T C C A C G G C G C7-1505 A 2 G T G T C C A G G G G G G C C T T G T T T 1 A A A G A T C C A C G G C G C
8-68 A 2 G T G T C C A G G G G G G C C T T G T T T 1 A A A G A T C C A C G G C G CLSG 374x Poly A 2 G T G T C C A G G G G G G C C T T G T T T 1 A A A G A T C C A C G G C G C
1-1120 A 3 G T G T G C A G G A G G G C C T T G T T T 1 A A A G A T C C A C G G C G C
8-1564 A 4 G T G T C C A G G G A A G C C T T G T T T 1 A A C G A T C C A C G G C G C7-56 A 4 G T G T C C A G G G A A G C C T T G T T T 1 A A C G A T C C A C G G C G CSH7 A 4 G T G T C C A G G G A A G C C T T G T T T 1 A A C G A T C C A C G G C G C
12-1060 A 5 C T G T G C A G G G G A T C C C G G T G T 1 A A C G A - C T T T G G C G C11-503 A 5 C T G T G C A G G G G A T C C C G G T G T 1 A A C G A - C T T T G G C G C7-1502 A 6 C T G T G C A G G G G A T C C C G G T G T 1 A G C G A - C T T T G G C G C
8-203 A 7 G T G T G C A G G G G A T C C C G G T G T 1 A A C G A - C T T T G G C G C8-61 A 7 G T G T G C A G G G G A T C C C G G T G T 1 A A C G A - C T T T G G C G C
12-1020 A 7 G T G T G C A G G G G A T C C C G G T G T 1 A A C G A - C T T T G G C G C
2-08 A 8 G A G T G C A G G G G G G C T T T C T T G 1 A G C G G T T T A C T G A GTT17-1024 A 8 G A G T G C A G G G G G G C T T T C T T G 1 A G C G G T T T A C T G A G T
8-103 A 8? T T C T T G 1 A G C G G T T T A C T G A G TLSG 370xpoly A 8 G A G T G C A G G G G G G C T T T C T T G 1 A G C G G T T T A C T G A G T
8-76 A 9 G A G T G C A G G G G G G C T T T C T T G 1 T G C G G T T T A C T G A G T
6-13 A 10 G A G T G C A G G G G A G C T T T C T T G 3 A G C G G T T T A C T G A G T7-1102 A 11 G A G T G C A G G G G G G C T T T C T T G 1 A A C G G T T T A C T G A G T8-1020 A 12 G A G T G C A G G G G G G T T T T C C T G 2 A G C G G T T T A C T G A G T6-1112 A 12 G A G T G C A G G G G G G T T T T C C T G 2 A G C G G T T T A C T G A G T
S4PT-6 A 13 G A G G A C A G G G G G G C C T T G T T T 1 A A C G A T C C A C G G T G C
USFS-12 A 14 G A G T G G A G A G G G G C C T T G T T T 4 A G C T G T T T A C G C C C T15-1079 A 15 G A G T G C G A G G G G G C C T T G T T T 1 A G C T G T T T A C G C C C T
8-33 A 16 C T C T G C A G G G G A T C C C G G T G T 1 A A C G A - C T A T G G C G C
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
AGP6 pyrograms
NucleotideAddition
T A C A T C G A
Sequence
SequenceSNP Genotype
TT A C A T - G AAA
TT A C A - - G AAA
3
2
1
3
2
1
A/T
A/A
Target: TTACA/TGAAA
• 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 ul 90 c •Pyrosequencing reaction $1
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