PCR primers were designed according to Harry et al. (1998) and Temesgen et al.(2000). Briefly, all primer sets were chosen to operate under a single thermal cycling regime which included a hot start and touchdown stages. In many cases, the “melting profile” of the amplification product predicted by the EST sequence was observed using MacMelt software (BioRad). This step allowed the effects of both primer position and GC clamping on melting profile to be examined and was used to optimize amplfication products for denaturing gradient gel electrophoresis.

 

Sequences of loblolly pine primer pairs from xylem ESTs and seedling ESTs suitable as anchor loci for comparative mapping.

 

Table 1. PCR primers derived from cDNA clones of loblolly pine.
FROM:
Temesgen, B., Brown, G.R., Harry, D.E., Kinlaw C.S., Sewell, M.M. and Neale, D.B. 2001.
Genetic mapping of expressed sequence tag polymorphism (ESTP) markers in loblolly pine
(Pinus taeda). Theor. Appl. Genet. 102:664-675.

 

Clone
Namea		 Primer pairs (forward and reverse, 5′–>3′)
Product size
 Population, Detection methodb LG/CMc Best possible protein similarityd Scored E valued
cDNAs from seedling needle tissuese cDNA Genomic
Forward Reverse
0048f GAC ATT TGG ACT CTT CCT GGC AAC ATG AGA ATC ACG GGC A 270 270 Q, DGGE 2/72 Aquaporin 107 1e-26
0066f,g GCC CGC CCC CGC CGC CCG GCC CGC GCC CCG CGC CGC CCG CTG GCT GTT GGA ACC CAA G GGT ACC AGG GTA GGG TTT GC 385 385 B, DGGE 2/88.3 P65 protein Ectomelia virus 32 .91
0107 GCA GGA CCT TCT GGA CAA TC AGG TGG AGA AAG CCA AGC TC 450 450 B, DGGE 1/112.3 ATP synthase B’ chain 83 2e-95
0136f TGG CCA TCT ATT TCT GGT GG CAA TTG TCC CAT CCC CAT C 400 800 NPh — protein kinase-calcium dependent 290 6e-78
0149 GGT GAC GCA ATC ACC TTT G CCA ATC CAA CCG ATA CTT GG 410 550 Q, RE
(Dde I)		 21/0 apospory associated protein 96 3e-20
0459 TTA AAC GCT GCT GGT CAG G TGG AAA GAG CTG GAA GAT GG 450 550 Complexh — ubiquitin 74 2e-13
0464 TGT CAC TGC CCA GAG CTA TTC ATC ACA GCC GCT CCA AAA C 450 730 B, LV 2/58 aquaporin 65 7e-11
0500 GGC GAG TTG GCT TTC ATT C CAG CGA GGT ACC AGA TTT GC 400 500 B, DGGE 9/73.8 plastocyanin 61 1e-12
0606f TCA TGA GGG GAG AAA GTT GG GGG CAG TCA CTT GAA CTT CG 360 550 Q, DGGE 6/98.6 repJ 32 3.9
0624 CAC AAT TGC CAG ATG GGT C CTT CTC TAG CAA CGA TCC GG 620 940 B, RE
(HinP1 I)		 9/55.2 protein kinase 120 2e-27
0669f GTA TTC CTG CTT TCG TTC GG CGA GTC CCT GAT AGA CCC TG 370 600 NPh — photosystem II 22 KD protein precursor/psbS gene 94 7e-19
0670 GCC CGC CGC GCC CCG CGC CGG CCC GGC GCC CCC GGG CGG CAG AAG ACC AGA GCC AGG AC GAC AAA CGC CAA GGG AAT C 400 450 NPh — Pectinesterase 3 49 6e-06
0674f ATA ATT GGG GAG ATG CGG AG GCA GGC GGC GCG GGG CGC GGG CCG GGC GGC GGG GGC GGA GCA GAA GGA AGA CAG AGT GG 270 270 Q, DGGE 14/0 cyclophilin 99 2e-20
0708f TGT TGA CGA TCA AGC TGC C TCA AGC CAT CAG CGA AGT AC 310 350 Smear/ complexh — translationally controlled tumor protein 202 2e-51
0739 TAC AAA GGC TCG GGT ATT GG GTC ATG CCT AAC AAG CCC TG 350 350 Q, DGGE 6/67.6 phospholipase D 29 4.0
0893 GGA CTG AAG GGA TCT AGC TGG CAG CCC AAA TTC CAT CGT C 450 620 Q, DGGE 5/41.3 nonspecific lipid transfer protein 157 2e-38
0967 AAG GGG AGG AGC AAG TGA AG CTT GTT CAG GAA GTC TGC CC 350 350 NPh — glutathione
S-transferase		 36 0.039
0975 GAC GAA GAT GCG AAG AAA GG ATA CGG AAG CGT ACC TGG G 400 400 NPh — ribosomal B2 protein 80 4e-15
1165 CTG AGG GGA TGT GCA TGA C AAA CCA GCA GCC ACA TGA G 250 380 B, DGGE 6/50.8 metallothionein-like protein 55 8e-08
1454 ACA TCA ATC AAG TTG GCC TTG ACG ACC ATC TCC AAC CAC TC 350 350 B, DGGE 5/75.9 — — —
1576 TGG TAT GTG GAG GGA AGG C TAC AGC GTT TGC TCC TCC TG 480 480 Q, RE
(Dde I)		 1/59.7 regulatory factor RFX1 33 .55
1584 CGA AGC AAA GGA TGT CAC G TGT TGA GGT GGG GAT TGG 340 340 NPh — chalcone reductase 57 9e-11
1599 CAG GAT CAT ATG CTG AAG CG ACT CGC CAA TTT GCT CTA GC 520 520 NPh — ATP synthase delta chain chloroplast . 40 0.001
1623 TTT CTC AGG TGG GAG AGG TG TCG CAT CCA TGT GCG TAG 350 950 P, DGGEI — NS1-associated protein 102 2e-21
1626 TAA GAA GGC GGC GGT ACA G AAA CCA GCA GCC ACA TGA G 550 550 B, RE
(Hinf I)		 6/51 Metallothionein-like protein 62 8e-10
1635-3′ AAG AAG ACA ACG AGC AAC GG GCC CAC TCG AAT CAC AAA A 650 870 Q, RE
(HinP1 I)		 10/48.8 ribulose bisphosphate carboxylase, small subunit 145 3e-36
1643 AAT GGA GGA TGC CGT TAC AG AAC CAC TCT CGA ATC CCC AC 490 650 B, DGGE 10/56 ABI1 gene product (protein phosphatase) 59 5e-09
1750 TGT TTA CGT TCT TGA CGC GG TAG CAA GCA CTC TGA CTG TGG 300 300 B, DGGE 16/24.7 ferredoxin I 54 7e-17
1917 GCC CGC CCC CGC CGC CCG GCC CGC GCC CCG CGC CGC CCG ATA TCC GTC GCC TGG TTA AG GAT TCT CAA AGC AGC CCA AG 320 450 Smear/ complexh — chloroplast transit peptide 108 1e-23
1934 GAC GAA GTT GGT GGC GTA G TTC TGT TTG TGC GCC TAC TG 850 850 B, RE
(BsaJ I)		 3/2.5 light harvesting complex chlorophyll a/b binding protein 97 5e-21
1950 AAA CCA GCA GCC ACA TGA G TAT TAA GAA GGC GGC GGT AC 350 450 Q, DGGE 6/54.7 metallothionein-like protein 62 3e-09
1955 AGC CAA TGC ACC AAG AAG G ATC CAA CAA CAG AAC CCC TC 290 290 B, RE
(BsaJ I)		 5/82.3 metallothionein-like protein 29 0.017
1956 GAA GCT AGC GAA GGC TTT GG GGG TGT GAC CAT ATA ACA CCG 410 500 Q, RE
(Alu I)		 9/7.1 ubiquitin protein ligase 36 3e-16
2009 CAC AGT TCC CCA CAG CAA C ACA AGC GGT TCA GTG GCT C 400 600 B, DGGE 6/107.7 60S ribosomal protein L10A 126 2e-29
2053f TGA AAC TGC GGA TGG CTC CCG GAA TCG AAC CCT AAT TC 310 360 Q, DGGE Not mappedh folate binding protein 85 2e-21
2111 AGT CTT GGC CTT TTC TTG GG CAC CAA ACC TAA CCG AGA CG 350 350 GUh — — 40 0.002
2166 CTG CTG TTG AGC TTG TGT ACG TGC CCG TGT AAA GAT GAC AG 400 400 Q, RE
(Hinf I)		 4/56.5 pyruvate dehydrogenase 40 0.002
2253 CCA ATT TGC ACT TTG CCC CCA AAG CCC AAA TCC ATG 370 370 Q, DGGE 1/59.9 Fructose-bisphosphate aldolase 1, chloroplast 58 1e-08
2274 TGA TCA GAG AGC TGG TGC AG AGA TGA GCA TCA GGT CAG CC 400 600 B, DGGE 4/86 adenylyl cyclase 30 3.0
2290 AGC TTG CAG CAT CAA CCG GAA CCA AAC AGC TTC AGG ACC 550 840 Q, RE
(HinP1 I)		 9/57.5 light harvesting complex chlorophyll a/b binding protein 53 2e-07
2358f GTT AAC CCT CGA GGA GAC ATG GCT TCC ACA GTC CAC AAT CTG 330 330 Q, DGGE 22/0 phenylalanine tRNA synthetase 30 3.6
2393f ACA CGT CTG TCA TCT CAT GGG CCC GGC CGC CGG CGG CCC TGA ACC AGC ACA AGT ATC 280 350 S, DGGEI — BcDNA GH07269 34 0.99
2541f GCT GCT GAC ATG AGC TTT TG AAG TCC CAC ACA TCG AGG TAC 300 410 NPh — 6-phosphpfructo-2-kinase/ fructose-2,6-bisphosphatase 160 2-39
2610 CTG AGG GGA TGT GCA TGA C AAA CCA GCA GCC ACA TGA G 350 350 B, DGGE 6/50.9 metallothionein-like protein 40 1e-04
2615f CAC TCT TTA TTC TTG CCC TTC G TCG GTT AGG TAA CGA CTG GAC 320 400 Q, DGGE 11/49.5 P69 2-5A synthase I 33 1.6
2723 GCA CAC CAA AGC AGC AGC AAT GGG ACT TGC ACC TTC AG 550 850 P/A — actin depolymerizing factor 55 5e-11
2781f GAT GAT GCC CTG AAG AGC C ATG GAA CCA AAG GAG ATG CC 450 450 B, DGGE 8/64.7 glucose-induced repressor 43 0.002
2786f CTG CCA AGA CAG AGA AAC TGT C CCA TTG TCT GTT GTT GCC TC 300 300 NPh — transport protein 39 0.048
2889 ACG CCA GCT CTG ACT ACC AG GTT TCT TCT CGT GGT GCT CG 530 750 B, RE
(Rsa I)		 23/0 ABA/ ripening inducible protein 73 4e-13
2986 TCG CAG GTG AAG TTG TGA AG TCT AAA GGC CCA CCC TAA CTC 350 1,000 Complexh — histone H2A 40 0.005
cDNAs from xylem tissuee
8462 TGC CCC TAT TGT ACA CAG GTC GGA CCA TGC CAA CAT CAA AC 260 260 GUh — glutaredoxin 168 3e-41
8510 TTC AAG GCC GTT CCA TTC CAT TAA CCC AAA GGC CCA G 240 240 Q, DGGE 11/51.1 RNA-binding glycine rich protein 81 5e15`
8562 AAG ACG GCT GTG TGG TTC TG TAG CAG GAG ATG TTG GTT CCC 200 200 Complexh — chitinase 144 4e-21
8564 CAC CAG GGC AAA AAG TTG G GCA GTT ATA GGT TTC CTG GCC 230 230 B, DGGE 6/55 ribosomal protein 60S L2 147 4e-35
8565 ATT TGT GGC TGC GGA AAG CAC CAA GTA CAC CAC AAC ACC 200 220 Smearh — glucose-regulated 160 9e-43
8569 TCG ACC AGT GTT GCA GAG G TCG TCT CCC TCA TCA CCT TC 210 210 Q, DGGE 2/24 tubulin 242 1e-63
8598 CAC AGA TGG TGA CGG AGA AC GGT GAA CGC TTG TTG AAC G 190 190 Complexh — coatomer delta subunit 62 8e-15
8613 ATT CAC CCT TCC CCT GAC C GCC GGG CCC GGC CTT AGC CCA AAC AGG GAA AGC 210 210 B, DGGE 3/67.6 Embryogenesis-associated protein late embryogenesis LE14-goshi 113 5e-25
8643 TGG TTT CCA CTG TTG CAG C GAG TTT GAA GCC CTT GAC TGG 260 260 GUh — cysteine proteinase 98 3e-20
8650 CCC GGC CGC CGG CCC CTC TCA AGG AGC CGA ACA AGA CC CTT GGA TTT TCC CGT C 220 220 S, DGGEI — RNA binding protein 127 4e-29
8702 GTT GCA GAA AAG GGT GGC AGT CGC ACT TGC TCC AGT TC 290 350 Q, LV/
DGGE		 6/77.9 thioredoxin 69 3e-11
8704 TAA GGC ACA TGG CAA GCT C CAG AAG AAT GAG CTG GCA CC 250 250 NPh — pyruvate dehydrogenase 56 7e-16
8714 TGA ACT GCT GAA TCT TGG GC GAC GGC ATC TCA AGA ATA CCC 260 260 Smearh — initiation factor 5A-2 76 7e-15
8721 CCC GGC CGC CGG CCC TGC CCA AGT GCA ATC ATG CGC TTG CTC AGA TCA TGG 290 290 S, DGGEI — oligosaccharyl transferase 87 77e-17
8725 AGC GCT GAA TGA TGT CTT GG GCC GGG CCC GGC CCA AAC TTA CAC CAT GCT CG 260 260 Q, DGGE 9/1.0 COP9 complex subunit 4 106 8e-23
8728 CCA AAG CCC AAA TCC ATG CCA ATT TGC ACT TTG CCC 370 370 Complexh — aldolase plastid 60 3e-17
8744 TGC AGA TCC TAT CTT TGG GG CAA GGT CCT CGT ACC CAA AC 340 420 Complexh — cell wall glycosylatable polypeptide 67 7e-11
8758 GAG TTC GCA TCA CAC GTT ACC AAT TGC CCC ATT GAT TCT GG 350 350 Complexh — protein kinase guanine nucleotide binding 89 1e-17
8777 CCC GGC CGC CGG AAG AGC TTT GCA AGC GTG AG AAC GTC CAA ATC TGT CCA GG 170 170 NPh — ribosomal protein 60S L18A 199 1e-50
8790 TTG GGG AAG CAA GCA TTC CTT TGT TTC CAC CCT CCG AC 250 900 Complexh — hypothetical ORF 96 9e-20
8796 TCA GAG TCA GCT AGG CGT TG TTC ATC CAA GGC GTT GAA C 400 400 Complexh — ATP synthase vacuolar 77 5e-14
8886 TTC CGG AAG GTG TGG TGG AGT CAC TCC CTG TCA CCG AC 310 310 B, LV 7/81.4 kinase I nucleoside diphosphate spiol 78 3e-24
8887 TGG GGT TGG TGA GAT ACT GC CAT ATA TTG GGA AAA CGT TCG C 320 500 B, LV 4/89.1 RNA polymerase II subunit RPB10 homolog 73 1e-12
8898 GGG ATG GCA ACA ACA AAA AG ATG GGG GTG CAG CAT AAA C 330 1,400 B, DGGE 4/120 testis mitotic checkpoint 119 9e-27
8907 TCC TCA ACA GGC AGA CCT TC TGG GTG AGA CTT ACG AAC AGG 300 300 Q, DGGE 8/0 peroxidase cationic 67 6e-11
8917 CCT TTC AGA GGC TTG TTA GGG ACA TTG CGT TTC TGC GTA GG 310 550 Smearh — histone H3.2 149 9e-36
8939 ACG TGG ACG AGC AGT CAA AG AAC CAC GAG CTT GGC ATG 300 300 Q, DGGE 2/84.3 ribosomal protein 40S S16 195 2e-19
8962 TCC TGA AGA GGA GGA GGA GG AAC CAT GCA ATT GGA GCC 350 350 Smearh — kinetochore Skp1p homolog 69 2e-11
8972 TTG GTC CCC TTG TTG GAG GCC TCC ATT CGA CTC ACT TG 310 310 B, DGGE 6/18.3 protein 2 major intrinsic plasma membrane 136 1e-31
8993 CCA TCT CCA CTG ATG CGT AC GAA GAG CGG CAT ATC CGA C 250 600 Complexh — disease resistance response protein PEA 121 2e-27
9008 GGC GCC CGG TAA ACT GGG ATG GAT TGC TCT CGG ATA GGG CAA TAT GC 290 290 Q, DGGE 5/71.5 ATP-binding protein 242 2e-63
9022 CGG TGT GTT TCA TGT GCT G GGA TTT GCA TTT TGC ATG CC 210 210 B, DGGE 2/36.7 translation factor SUI1/GOS2 protein 88 2e-17
9034 AGC CCG GTC CAC TTA TCA AG TGC ACA CAG AAT CAG AAT TGG 300 300 Q, DGGE Not mappedh CAEEL 19.6 KD protein C23G10.2 in chromosome III 71 5e-12
9036 CAG GAC GAA TGA GAT ACC TGC GTC ATC CGA TAC AAC CTC AAT C 250 350 Q, DGGE 8/45.8 ribosomal protein L37 61 5e-20
9044 CGC GGC CCA ACT GGA GGA AAA GCA CGA C CAT CGC ATC AGT CAT ACT CAC C 280 280 B, DGGE 6/53.1 ribosomal protein 40S S27 105 1e-22
9047 CTA AAG GCT TTC CTA TGC GC GTC ATT TTT CCA CTT ATC CCT CC 300 300 NPh — cytochrome P450 71 6e-13
9050 GCG CCC AGC TCT GGA AGC TCT CTC C TTT TGA CAG TCT GAA ACG GC 250 250 Complexh — ABA and salt stress response 40g 73 6e-13
9053 GGC GGT GCA TGA TGA CGG CTC TAT G CCA CCG AAA TAT ATG CCT GTC 280 280 Q, DGGE 1/38.1 translationally controlled tumor homolog (TCTP) 108 2e-23
9055 ATA GGG CGC ACA ACA GCT C AGG AGC CTT CTT CTT TCC TGG 300 1,000 GUh — ribosomal protein 30S S13 89 5e-31
9061 GGC GCT CGG GCT GTA ACG AAT ATG C CCT CCA TCA CAG ACC AAC AG 250 250 NPh — Fe(II) ascorbate oxidase 69 1e-11
9064 GAG GGG AAC ACT AAT CAA GCG TGC CTC AAA TGC TTG CTT TC 270 270 Smearh — ribosomal protein L14 93 1e-18
9076 AGA ATT TAC TGG CCG CTC G CTC TAT TGC AAA AAT GTG CCA C 250 250 B, DGGE 11/39.1 Phosphoglucomutase 98 3e-20
9088 GCC CTT TTG GGT GTG CGT AGG AG AGC CCT GAA CAT AAT TTC AGG 290 290 NPh — DNA-damage-repair/toleration protein DRT102 64 7e-10
9092 GGC CGG GTC ACT GAC CTT AAC GTC CC AGC TAA AGT TGG CTG GCA TC 270 400 B, DGGE 5/43 nonspecific lipid transfer protein 89 7e-18
9098 GGC GGG GTG GGC TTG CTA TAA ATG C AGT GCA TCG TTC ACA ATT CTC 280 280 NPh — selenium-binding protein
9099 GGC CGG CAG ACA CAT TAA GCT GGG GTC C TTT CAG GGG AGT TGC AAT AAG 300 300 GUh — ribosomal protein S12 69 2e-11
9102 CCC AGA GAT CTT CCG CTA TG GGC CGC GGC AGA AAG GAG CAT TTC CCG AC 240 240 Q, DGGE 1/84.6 lacase 63 2e-10
9103 CGC GGG CTC TCA GGA GAC CAG TGA GAG C GCA TAG AGA GGA ACC TTT CCC 260 260 Smearh — ribosomal protein 60S L30 68 2e-11
9113 AGG AAA AGG TTC TCC AAG CG GCC GGC CGA CAG CTT AGG CAT TAC AGC CC 300 300 Q, DGGE 8/80.2 S-adenosylmethionine synthetase 2 95 2e-19
9123 CCG GGA CGT CTC TCT GCT ATC GTC G TTC CAA AGT TCC CAG GTC AG 280 280 NPh — amino peptidase 105 1e-22
9132 CGG CCG CTC CAT CTG GTG GGT TGA AG AAT CTA TGC CGT GGA ACA GC 270 270 Complexh — ribosomal protein L7 120 4e-29
9134 TAT GGT TGT GGA GAG GCT TG GAG GAA TCT TCT GCC ATT AGC 300 300 NPh — Profilin 107 4e-23
9136 GCC GGC CGG GCC CTT GTC CAA GAC TGT AAG CCG ATT TGA AAC ACA CAT CC 260 260 P, DGGEI — ribosomal protein S11 150 4e-36
9151 TAG TGA GCC CTG GAG CGT AC GCA GAA TCT CAG CAG CAA TG 290 290 Q, DGGE 7/20.1 cucumber basic protein, A blue copper protein electron transport 91 3e-18
9155 GCG CCC GGT TGG TGT TAG GCA GTC ATG G TGA AAT TCC CAG CCC ATA TC 260 550 P, DGGEI — EST Arabidopsis 80 2e-14
9156 GGC CCG CGC CCG TAA GCT TCG TGC AAC AGG AG GAC AAT CCC TCT AAA CCT CGC 280 400 Q, DGGE 9/13 ribosomal protein 40S S14 132 1e-30
9157 TTC CAG TTT CCC TGA GCA TC CCG CGG GCC GCG GCC AAT ACG CTG CTT AAT CGT GTC 275 275 B, DGGE 4/8.2 26S protease regulatory subunit 51 5e-06

a. For simplicity, only locus identity fields were reported for clone names.
b. Loblolly pine mapping populations used in this study and methods utilized to detected the DNA polymorphisms: B: base;
Q: qtl; P/A: Presence or absence of PCR products; LV: length variant; RE: PCR-RFLP; DGGE: denaturing gradient gel
electrophoresis.
c. LG: linkage group; cM: centimorgan; these values were also presented in Fig. 2.
d. Putative identities determined from BLASTN AND FASTA search of GenBank; Score: the score values obtained; E value:
expected values.
e. cDNA clones constructed from either seedling needle (clones #s < 3000) or xylem tissues (clones #s > 8000).
f. Primers were designed from sequences of seedling cDNA clones that were obtained by using an ABI 377 automated sequencing
apparatus.
g. GC-clamps were added to 5′ ends of one of the primers (GCs are distinguished as lower case; e.g., 0066).
h. PCR products dropped from further analysis for various reasons: Complex, Smear, NP (Non-polymorphic), GU (genetics un-
interpretable), Not mapped (could not be mapped)
i. Not polymorphic in the base and qtl mapping population but were polymorphic either in prediction (another loblolly pine
population, unpublished) or in slash pine (Brown et al. in prepartation).