Tree Genetics & Genomes (2005) 1: 31–40DOI 10.1007/s11295-005-0005-1

ORIGINAL PAPER

Ilga Porth . Caroline Scotti-Saintagne .Teresa Barreneche . Antoine Kremer . Kornel Burg

Linkage mapping of osmotic stress induced genes of oak

Received: 13 December 2004 / Revised: 7 March 2005 / Accepted: 18 April 2005 / Published online: 5 May 2005# Springer-Verlag 2005

Abstract Water stress affecting long-lived trees is an im-portant challenge in forestry. Due to global climate change,forest trees will be threatened by extreme conditions likeflooding or drought. It is necessary to understand differ-ences in stress tolerance within certain species and to in-vestigate putative relations on genomic level. In this study,osmotic stress induced genes of Quercus ssp. were posi-tioned on two genetic linkagemaps of oak. An intra-specificcross 3P*A4 of Quercus robur consisting of 88 offspringand an inter-specific cross 11P*QS29 of Q. robur and Q.petraea comprising 72 full-sibs were analyzed for the in-heritance of 14 loci represented by 34 individual single nu-cleotide polymorphisms. Seven genes in the intra-cross, aswell as other six genes in the inter-cross could be mappedand one gene could not be localised due to the severe dis-tortion of the segregation. The collection of expressed se-quences involved ribosomal proteins, members of theoxylase/oxygenase gene family, betaine aldehyde dehydro-genase, Dc3 promoter-binding factor, a putative member ofthe nodulin family, glutathione-S-transferase and proteinswith unknown functions. In the inter-cross, two linked mark-ers exhibited 89% deficiency of heterozygosity. Thirteengenes were positioned on ten different oak chromosomesand can serve as orthologous markers in comparative map-ping studies within Fagaceae.

Keywords Oak . Quercus ssp. . Osmotic stress . EST .Linkage map

Introduction

White oaks Quercus robur L. (pedunculate oak) andQuercus petraea (Matt.) Liebl. (sessile oak) are the twomain oak species growing in the Central European habitat.

Geographical maps indicating their natural spread showthat sessile and pedunculate oak are largely sympatric inWestern, Central and Southern Europe, althoughQ. petraeadoes not reach so far south and north and to ContinentalEastern Europe like Q. robur. Nevertheless, each speciesshows specific ecological limitations, which lead to diverg-ing distribution patterns and individual ecogeographicalranges (Führer 1998).

During their life cycle of up to 200 or more years, oaksmust cope with heterogeneous environmental conditions.Thus, genetic variation is an important determinant of theability of a forest tree population to survive temporarily andspatially diverse environments (Ziehe and Müller-Starck1991).

Q. robur is reportedly more sensitive to drought-induceddecline than Q. petraea (Führer 1998). It is assumed thatdifferences in physiological functions are responsible forthe varying adaptation potential. Lower water-use efficien-cy, for instance, of pedunculate oak could explain the ob-served declines in adult trees of this species after a period ofdrought (Ponton et al. 2002).

Generally, water stress response in plants is characterizedby a complex mechanism of cross-talks between variouscomponents of different pathways (Zhu 2002). Genes thatencode for putative protective proteins may play an im-portant role in genetic improvement of drought tolerance.Thus an allelic variation in the coding region of the genecan be selective in a particular environment defining a cer-tain ecotype. The chromosomal localisation of such genes,which are involved in the physiological response to environ-ment, facilitates breeding for desired traits such as droughtresistance.

Genomes of annual crops like barley (Hordeum vulgareL.) or wheat (Triticum aetivum L.) have been studied in-tensively concerning localisation of stress-related genes(Malatrasi 2002; Li et al. 1999; Cattivelli et al. 2002).

I. Porth . K. Burg (*)Division of Biogenetics and Natural Resources,Austrian Research Center Seibersdorf,2444 Seibersdorf, Austriae-mail: kornel.burg@arcs.ac.atTel.: +43-50550-3524Fax: +43-50550-3666

C. Scotti-Saintagne . T. Barreneche . A. KremerINRA BIOGECO,69 Route d’Arcachon,33612 Cestas, France

In forest trees, which are long-lived and late-fertile spe-cies compared to annually cultivated crops, genetic studiesand breeding processes are dependent on the long gener-ation period. The availability of highly informative markerssuch as simple sequence repeats (SSR) is much lower inwoody than in crop plants (Cervera et al. 2000). Hence, easilyavailable random amplified polymorphic DNA (RAPD)and amplified fragment length polymorphism (AFLP) mark-ers are often used to establish a map framework in tree spe-cies (Costa et al. 2000; Grattapaglia and Sederoff 1994).

The characteristics and advantages of cDNA restrictionfragment length polymorphism (RFLP) and expressed se-quence tag (EST) markers are that they are co-dominantand, because they are derived from expressed sequences,they exhibit high inter-specific homology. Thus, they pro-vide orthologous markers for aligning linkage maps andintegrate data sets from each parent into a sex-average con-sensus map, but they also help to establish broad synteniclinkage relationships amongst species or genera (Sewellet al. 1999; Cato et al. 2001; Chagné et al. 2003). Hence,interest is focused on saturating genetic linkage maps offorest trees with gene-based markers, which add to theinformation about genome organisation and structure ofwoody plants.

In this study, we aim at contributing to the informationabout the genome organisation in the Fagaceae family by(1) adding EST derived genes to the genetic linkage mapsof white oaks, (2) positioning putative adaptation-specificmarkers on chromosomes of an intra-specific (Barrenecheet al. 1998) and an inter-specific cross (Dussouchaud, 2002,unpublished data).

Twenty-five cDNAs originating from osmotic stressinduced ESTs formerly isolated from Q. petraea callus cellculture (Porth et al. 2005) were screened for sequencepolymorphisms at their 3′ end. Single nucleotide poly-morphisms (SNPs) were used to position the genes ontolinkage groups of the two available crosses. To the best ofour knowledge, we present here the first mapped activegenes on linkage maps of the Fagaceae family.

Materials and methods

Plant material

Genomic DNA originated from four adult oak trees (threeQ. robur 3P, A4 and 11P and one Q. petraea QS29). Thesetrees are parents of two full-sib families: one intra-specific

Table 1 Osmotic stress induced transcripts of oak. CF numbers are cDNA sequence entries; partial genomic sequences entries represent 3′UTR sequences for 1T57 and 2T3 and exon/intron sequences for 1T9, 1T15, 1T21, 1T62, 2T5, 2T14, 2T19 and 2T32 (AY numbers)

EST Genbank accession numbers BLASTx identities Species E value

Full length Partial genomic sequence

1T7 CF369276 Fiddlehead-like protein Gossypium hirsutum 0.01T9 CF369290 AY438654 Lipid transfer protein precursor G. hirsutum 2e−391T11 CF369263 Unknown protein Arabidopsis thaliana e−1191T12 CF369264 Oxidoreductase, 2OG-Fe(II) oxygenase family A. thaliana e−1141T15 CF369265 AY438655 TGF beta inducible nuclear protein Oryza sativa e−1031T21 CF369266 AY438656 60S ribosomal protein L10 Vitis riparia e−1101T24 CF369267 Unknown protein A. thaliana 3e−91T25 CF369268 Unknown protein A. thaliana e−471T26 CF369270 Ribosomal protein S20 A. thaliana e−501T41 CF369272 Pyruvate kinase A. thaliana 0.01T57 CF369273 AY438652 Dc3 promoter-binding factor-2 Helianthus annuus 6e−251T62 CF369274 AY438657 Betaine aldehyde dehydrogenase A. thaliana 0.01T69 CF369275 Unknown protein A. thaliana e−451T72 CF369277 40 S ribosomal protein S4 O. sativa 8e−872T3 CF369283 AY438653 mtN19-like protein A. thaliana e−1152T5 CF369286 AY438658 Plastid ribosomal protein L19 gene A. thaliana e−192T11 CF369278 Glutathione transferase A. thaliana 3e−492T12 CF369279 Small zinc finger-like protein TIM9 A. thaliana 2e−292T13 CF369280 Oxylase-like protein A. thaliana 5e−232T14 CF369287 AY438659 Thiazole biosynthetic enzyme precursor A. thaliana e−1312T18 CF369281 T5E21.12 A. thaliana 9e−302T19 CF369282 AY438660 NAD-dependent malate dehydrogenase Prunus persica e−1622T32 CF369284 AY438661 Cytosolic ribosomal protein S11 A. thaliana e−432T33 CF369271 Heat shock cognate protein 80 Lycopersicon esculentum 0.02T34 CF369285 60 S ribosomal protein L13a A. thaliana 4e−91

The sequence homology of the EST to the respective Genbank entry (BLAST identity) is supported by the expect value (E value) of the hit

32

Q. robur family (3P*A4) comprising 88 offspring and oneinter-specific Q. robur (female)×Q. petraea (male) family(11P*QS29) including a progeny of 72 full sibs. Bothcrosses were established at INRABIOGECO. To date, thesetwo crosses have been used for constructing genetic linkagemaps with various recently developed markers. A linkagemap of 3P*A4 has already been published (Barrenecheet al. 1998; Barreneche et al. 2004).

Expressed sequence tags

For this study, we selected osmotic stress induced ESTs thathad been isolated from Q. petraea callus cell culture bysubtractive hybridisation and differential screening. Cellshad been exposed to hyperosmotic stress for 1 h and 2 days,respectively. Twenty-five ESTs were sequenced to full-length cDNAs using unsubtracted cDNA libraries originat-ing from the stressed cells (Porth et al. 2005). Some of thesegenes were related to energy (glycolysis, citric acid cycle)and lipid metabolism, to ribosome structure and to tran-scription (as suggested by the results of the BLASTx searchin protein databases; http://www.ncbi.nlm.nih.gov/BLAST).Additionally, oak genes with significant homology to reac-tive oxygen scavengers, osmoprotectants and heat shockproteins were identified (Table 1).

Identification of SNP sites

Gene-specific PCR primers derived from these 25 osmoticstress related ESTs were designed using OLIGO primeranalysis software version 5.0 to amplify some hundreds ofbasepairs of genomic DNA in the four parents (Table 2).

For the two crosses, we screened for candidate SNPs inthe 3′ end of the genes of interest by sequencing the parents.

The PCR reaction assay for re-amplification was pre-pared according to the protocol of Qiagen’s HotStarTaqDNA polymerase kit. PCR consisted of: (1) 95°C 15 min,(2) 94°C 45 s, (3) Ta 45 s, (4) 72°C 1min, (5) go to step 2, 35times, (6) 72°C 10 min. The optimal annealing temperature(Ta) in a certain primer combination was determined byapplying a temperature gradient to a reaction (Table 2).Subsequently, sequencing reactions were performed usingABI Prism BigDye terminator cycle sequencing ready re-action kit [PCR: (1) 92°C 30 s, (2) 50°C 15 s, (3) 60°C 4min,(4) go to step one 24 times]. Purification of the reactionswas achieved by running them through Sephadex G50/TE8suspension filter columns. Samples were collected by cen-trifugation at 2,800 rpm for 2 min at room temperature.Sequence runs were always performed on an ABI PRISM3,100 genetic analyzer equipped with a 50-cm capillaryand POP-6 polymer. We used ABI Sequencing AnalysisSoftware 3.7 for sequence data analysis. The results of the

Table 2 Amplification and sequencing of 3′ end regions of 25 osmotic stress related genes in four parents of oak.

EST Name Forward Name Reverse Ta (°C) L (bp)

1T7 1T7:1546U26 tag ctt att tgg agg cta aag aga gg 1T7:1825L26 aga aga agc aaa cca tag gag taa ag 50 3201T9 1T9:417U20 aga tca gca cct cca cca ac 1T9:574L22 gcc gag gta cat gag gaa ata c 55 4841T11 cons1T11:880U24 cct agt ggc tat cat cca caa gat cons1T11:1393L21 aca gaa ggg ccc aat gta cac 58 5551T12 cons1T12:817U21 ggt ttg gac caa tga cga gta cons1T12:1303L26 aga aat ttg ggt tta ttc aga tat ca 57 5221T15 c1T15:611U21 cga cct tca ggg ttg cgg ttc c1T15:1082L26 taa aac ata agc ctt gac aat atc ag 55 6751T21 cons1T21:537U26 gtt ccc tgg tcg tca gaa gat tat tg cons1T21:849L26 cca caa aac aaa cac ctc caa aga ta 59 7701T24 cons1T24:415U21 aga aga agc acc acc gtt ata cons1T24:705L28 gtt atc atg aat ttg tta aac gat atg a 59 3251T25 c1T25:1049U26 gaa tct gtc ctc acc gtt ctc tat aa c1T25:1201L26 caa tga gca cca aga att cca tac ta 57 1871T26 cons1T26:355U24 caa cac ttg gga tag att tga act cons1T26:644L24 aat gaa aaa ccc ctc tta tct ttc 50 3221T41 1T41:U1690 gtg atg cgt ctg ttg tga aga tca ttg cons_gw1T41:2111L29 aaa ctg tat atg atg gca act cca act at 60 4501T57 1T57:U504 tgg ttt gct tca gcc tac aat gtg atg gw1T57L756 tga gca ttt tga ata cgt gtg tta tac t 50 2821T62 1T62:976U22 agg ctt ttc agg cag gaa tag t 1T62:1294L27 aat taa ata gtt tac tgc tcg tga tat 55 7051T69 cons1T69:322U24 atc tga ggg att cct tct gga gta cons1T69:592L24 ttt gga tcg tgg gta tta cat tct 50 3031T72 c1T72:579U22 aat tat taa ggc taa tga cac c c1T72:1078L24 agt ctg ata ctt ccc att gta gtg 55 5342T3 2T3:U1525 taa ggc tgc tgg att ctt aat gaa ttg gw2T3L2018 ttg cca aag acc aaa tgt gtg acc taa c 50 6302T5 c2T5:488U21 ttc aga tta agg agg cta gtg c2T5:847L21 aac att gct taa ccc cag tac 55 11302T11 c2T11:486U21 gcg aga gct tag ggt atc ttg c2T11:848L24 aaa cac tca gcc atc cct tta tag 55 3972T12 c2T12:291U21 tgt gcg tcg atg tgc tga gaa c2T12:707L26 tat gga gga tga tta tca tgt gaa tc 55 4442T13 c2T13:302U22 cat gca ctg cca atc tca gag a c2T13:605L26 ata att tgc ctc atc act aca taa ga 55 3342T14 cons2T14:930U21 ttg tgc ctg gga tga ttg tta cons2T14:1271L23 aaa gtt cca aag cca taa agt ac 59 8272T18 c2T18:259U24 tca gct aat tca gca acc aaa ttt c2T18:534L26 aca ata agt tga att ttt ttg agt aa 55 3062T19 cons2T19:939U23 aat gga gag tgg aag ata gtt ca cons2T19:1234L23 aac caa gat aac cat tat gaa tt 52 9992T32 c2T32:301U20 agt gcc aag atg gtc agg ac c2T32:661L26 cag gaa ctc caa tac aaa gat tta at 55 6082T33 2T33:U1544 tga aga aga agg ggt atg agg ttc tgt constr2T33:2368L27 cca cca atc tta aat acc tga cac gat 60 8242T34 cons2T34:620U21 aat tga ggg tta aag ctg aga cons2T34:916L28 aca tca caa att aag taa aaa atg tat c 52 331

Primer sequences in 5′ to 3′ orientation, annealing temperatures (Ta) for a PCR reaction and the resulting average product length (L) are shown

33

Tab

le3

Sum

marytableon

thelin

kage,segregatio

nandnature

oftheSNPmarkersanalyzed

Mapping

family

EST

Linkage

grou

pInform

ative

parent

Segregatio

nPrimer

nameSNaPshot

prim

ersequ

ence

SNPaEntry

code

Base

positio

nbDistance/

gene

(bp)

c

RegionEffecton

polypeptide

Typ

eHH:Hh:

hhχ2

3P*A

1T21

13P

1:1

46:42

0.2

1T21

U40

cagttt

tgaattgttattttt

aagtac

c/t

AY

590

−no

ne3P

1:1

42:46

0.2

1T21

L75

ctatcctaagaagatcaycctagtg

c/t

AY

667

−no

ne3P

1:1

46:42

0.2

1T21

L41

5gtttcaacttca

gatactcggt

g/t

CF

598

345

+Asp/Tyr

1T25

6A4,3P

1:2:121

:45:22

0.1

1T25

L12

4cacttc

ccaatttaccaggt

g/t

CF

1168

−−

none

1T57

4A4,3P

1:2:119

:44:25

0.8

1T57

U12

9cagtgctatgacatagtttca

tct

a/c

AY

151

0−

none

3P1:1

46:42

0.2

1T57

U17

9agtattatacatgtrtactcgtta

caatata/c

AY

207

56−

none

1T62

10A4

1:1

39:49

1.1

1T62

L62

7aatattgtttaa

taagatg

ttcttaac

ca/c

AY

141

0−

none

A4,3P

1:2:119

:45:23

0.5

1T62

L62

2ttt

aataagatgttc

ttaaccmaa

gaa/g

AY

146

5−

none

A4

1:1

39:49

1.1

1T62

L61

7agatgttcttaa

ccm

aagarcctg

a/g

AY

151

10−

none

1T69

8A4,3P

1:2:119

:47:22

0.6

1T69

L12

7aaaattgttccttactaggtatgtg

a/g

CF

444

0−

none

A4,3P

1:2:119

:47:22

0.6

1T69

L19

3gcattt

tcttt

cctctcctaa

g/t

CF

510

66−

none

2T5

33P

1:1

42:40

0.1

2T5L

46tgagg

tctgtggaagaaatgagtg

a/c

CF

787

−−

none

2T34

113P

1:1

40:48

0.7

2T34

U12

7ctgagttgcagttaa

atttctttag

c/t

CF

768

0−

none

3P1:1

40:48

0.7

2T34

L17

8gcagatgtaccctgcacaa

c/t

CF

793

25−

none

3P1:1

40:48

0.7

2T34

L20

4atgacatcttctacttgaaaaattg

a/g

CF

819

51−

none

11P*Q

S29

1T11

1011P

1:1

32:40

0.9

1T11L18

3mctccctacgaatgtaattg

tgtat

a/g

CF

1053

0+

none

11P

1:1

32:40

0.9

1T11U22

2mattctttagaactta

tgatgaagatactg/t

CF

1120

67+

Ala/Ser

1T12

11d

QS29

1:1

68:4

56.9**

1T12

L31

6mctcaactcttgtg

tacaacattct

a/t

CF

1127

0−

none

QS29

1:1

68:4

56.9**

1T12

L42

7mcaataacttcaacttctagccat

c/t

CF

1240

113

−no

ne1T

269

QS29

1:1

43:29

2.7*

1T26

U18

7gg

tagaaatatttgtcttatgttg

cac/t

CF

565

0−

none

11P

1:1

31:41

1.4

1T26

U22

8atatgcctcaacttt

gattgttc

a/g

CF

603

38−

none

1T57

4QS29

1:1

37:35

0.1

1T57

L87

attagtgaaagagtacaacagttt

gag/t

AY

770

−no

neQS29

1:1

37:35

0.1

1T57

U18

6agtattatacatgtatactcgtta

caa

tatca

a/c

AY

209

132

−no

ne

1T69

×11P

1:1

43:29

2.7*

1T69

L12

7aaaattgttccttactaggtatgtg

a/g

CF

444

0−

none

11P

1:1

43:29

2.7*

1T69

L19

3gcattt

tctttcctctcctaa

g/t

CF

510

66−

none

2T3

10QS29

1:1

33:37

0.2

2T3U

260

gagagagtactacttg

tataatatatg

tagaat

c/t

AY

221

0−

none

QS29

1:1

37:33

0.2

2T3U

279

aatatatgtaga

atycaccacctttt

a/t

AY

233

12−

none

QS29

1:1

33:37

0.2

2T3L

332

cactgcttc

aaaaagtta

cactact

a/c

AY

259

38−

none

QS29

1:1

37:33

0.2

2T3U

354

gtggtttca

agttcttgttatgg

a/g

AY

305

84−

none

2T11

711P

1:1

31:41

1.4

2T11U13

9aactgccctctgatggagga

g/t

CF

640

0+

Glu/Asp

QS29

1:1

38:34

0.2

2T11L19

4cttg

agtagaattaa

gccagactctgaa/c

CF

674

34+

Ile/Leu

2T13

1111P

1:1

32:40

0.9

2T13

U18

4aatgtgatggg

tattttt

cct

c/t

CF

504

0−

none

QS29

,11P

1:2:134

:34:4

25.2**

2T13

U24

7attaggtggtgtttt

attttg

aca/c

CF

570

66−

none

2T32

2QS29

1:1

37:35

0.1

2T32

U17

3gcactcaaacatccc

agc

a/g

CF

399

0+

none

QS29

,11P

1:2:114

:40:18

1.3

2T32

L32

0ttt

tgaaattaaccctaataacca

c/t

AY

298

128

−no

ne

34

sequence runs were checked with Genecodes Sequenchersequence assembly software, and the nucleotide sequencesfor the four parents were compared using DNASIS forWindows version 2.5.

Extension of the 3′ end sequence information

Some genes (1T41, 1T57, 2T33 and 2T3) required addi-tional sequence information at the 3′ end in order to planadequate reverse primers. Extension was done by usingGenomeWalker Kit (Clontech) with the following mod-ifications: Qiagen’s HotStarTaq DNA Polymerase was usedfor amplification of four different “genomic libraries” withgene-specific primers (1T41U1690: 5′-gtg atg cgt ctg ttg tgaaga tca ttg-3′, 1T57U504: 5′-tgg ttt gct tca gcc tac aat gtgatg-3′, 2T33U1544: 5′-tga aga aga agg ggt atg agg ttc tgt-3′,2T3U1525: 5′-taa ggc tgc tgg att ctt aat gaa ttg-3′). Theapplied touch-down PCR programme included the follow-ing steps: (1) 95°C 15 min, (2) 94°C 25 s, (3) 72°C 3 min,(4) go to step 2 seven times, (5) 94°C 25 s, (6) 67°C 3 min,(7) go to step 5, 32 times, (8) 67°C 7 min, end. Those PCRproducts that showed single bands on an ethidium bromidestained agarose gel (0.5%), were subsequently purified usingQiagen’s QIAQUICK PCR purification kit and subjectedto direct sequencing. The PCR product of 2T3(1525) wascloned (conventional T/A cloning, Invitrogen’s TOPO TAcloning kit) prior to sequencing. Sequencing reactions werecarried out as described above.

SNP detection

We used single nucleotide extension (SNE) reaction, amini-sequencing method, for direct readout of the genotype. Thefive-dye SNaPshot multiplex kit from Applied Biosystemsincludes four fluorescent dyes assigned to individual ddNTPs(A, C, G and T) and a fifth dye-labelled internal size stan-dard designed for small fragments (GeneScan-120 LIZ SizeStandard). The enzymatically purified (0.83 U ExonucleaseI, Escherichia coli from Epicentre; 1.67 U Alkaline Phos-phatase, Shrimp from Roche for 5 μl) PCR amplificationproduct was put directly into the SNaPshot assay and sub-jected to SNE reaction according to manufacturer’s instruc-tions. Dependent on the existence of polymorphisms usefulfor gene mapping in both parents of the two mapping fam-ilies (3P*A4, 11P*QS29), genotyping primers at certainsites were designed according to the distributor’s recom-mendations. In locus 2T34 of two cases, the same SNP sitewas analyzed by two different genotyping primers in the3P*A4 and 11P*QS29 crosses (Table 3). The enzymaticallypurified (1 U Alkaline Phosphatase, Shrimp from Roche)dye-labelled reaction products were separated on an ABIPrism 3,100 Genetic Analyser equipped with a 22-cm cap-illary and POP-4 polymer. The output file was analyzedwith GeneScan Analysis Software 3.5 using GeneScan-120LIZ size standard analysis parameter files. Genotyper 3.7software was applied to analyze the genotype of the mul-tiplexed reaction.M

apping

family

EST

Linkage

grou

pInform

ative

parent

Segregatio

nPrimer

nameSNaPshot

prim

ersequ

ence

SNPaEntry

code

Base

positio

nbDistance/

gene

(bp)

c

RegionEffecton

polypeptide

Typ

eHH:Hh:

hhχ2

2T34

11QS29

1:1

43:29

2.7*

2T34

U14

2ttt

gttacaatgttt

ctgtccg

c/t

CF

793

0−

none

QS29

1:1

43:29

2.7*

2T34

L19

1catgacatcttc

tacttg

aaaaattg

a/g

CF

819

26−

none

QS29

,11P

1:2:124

:36:12

4.0

2T34

L22

7ttt

gaaaacccaagtcagataa

g/t

CF

855

62−

none

Genotyp

ingprim

ersforpo

lymorph

icsites(nucleotides

y,r,m

indicatesiteswith

intheprim

ersequ

ence)andchangesin

theam

inoacidcompo

sitio

nof

thepo

lypeptidechaincaused

byalternativenu

cleotid

esas

wellassegregationdataof

theSNPinthemapping

family

areindicated(H

Hho

mozyg

otetype

1,hh

homozyg

otetype

2,Hhheterozygo

te);segregationratio

swere

tested

forMendeliansegregationby

thechi-square

good

ness-of-fitprocedure(χ

2)

−:Non

-cod

ingregion

(intron;

3′UTR),+:coding

region

,×:linkage

grou

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theinter-cross

*Distorted

segregationatp=

0.1

**Distorted

segregationatp≤

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datacertainsequ

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sitio

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sequ

ence

entriesin

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(entry

code

CFforDNAsequ

ence

andAYforgeno

micsequ

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entry,Table1)

c Relativedistancesof

SNPswith

inthecorrespo

ndinggene

dAssum

edlin

kage

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emapping

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Tab

le3(con

tinued)

35

Linkage mapping

JoinMap 3.0 software (Van Ooijen and Voorrips 2001) wasused to add SNPmarkers to the male and the female geneticlinkage maps of the two crosses.

Segregation data from 443 marker loci of the male par-ent and 429 loci of the female parent from the intra-cross3P*A4 as well as information about defined association ofmarkers (including RAPD, AFLP, SSR, SCAR and iso-zymes) to certain linkage groups (LGs) of oak was used(Barreneche et al. 1998 and unpublished data). Initially,the bulk of loci was grouped into 12 LGs corresponding tothe 12 chromosomes of Quercus by testing their indepen-dence. SNP markers were added according to their seg-regation type (Table 3), either to the male or the female(testcross marker) or to marker data of both parents (1:2:1inter-cross segregation). Significance of segregation distor-tion was tested by determining the probability of the chi-square distribution of genotype frequencies for one (1:1segregation) and two degrees of freedom (1:2:1), respec-tively. The final order of themarkers relative to each other inthe LG was determined by checking different thresholdLODs, using a recombination maximum of 0.4 and per-forming validation of the goodness-of-fit within a movingwindow of three adjacent markers (ripple function). Con-sensus LGs using the JoinMap option “combine groups formap integration”were calculated if the inter-cross segregat-ing SNPmarkers were available. Marker order was checkedwith respect to Barreneche et al. (1998), Barreneche et al.(2004), and finally Scotti-Saintagne et al. (2004).

In the inter-specific cross 11P*QS29, 286 loci were in-formative for the male map and 352 loci for the female map.Segregation data and information for this cross regardingthe association of framework markers to certain LGs werepreviously known (unpublished data). It has to be noted thatin both maps, around 65% of the loci were ungrouped atLOD=3.1, due to the low number of full sibs used formapping at this stage. Additionally, LG8was not defined byframework markers. SNPs were added to the maps as de-scribed for the intra-cross with LOD values as high as pos-sible. Pairwise recombination frequencies were calculatedto determine the most closely linked framework marker forthe SNP marker.

The genetic LGs were drawn using MapChart 2.1 soft-ware (Voorrips 2002).

Results

Sequence polymorphism

The four parents of the two mapping families have beenanalyzed for the presence of SNP variations in the selected25 putative osmotic stress modulated genes by sequencingthe appropriate amplified genomic regions (as described inMaterials and methods). Sequence analysis of the amplifiedgenomic fragments revealed the presence of introns in eightanalyzed loci (1T9, 1T15, 1T21, 1T62, 2T5, 2T14, 2T19,

2T32). TheGenbank entries of these genomic sequences arelisted in Table 1.

Fourteen out of the 25 selected loci yielded segregatingSNP sites in the mapping populations. Four genes (1T21,1T25, 1T62 and 2T5) yielded SNPs segregating in the intra-cross and seven genes (1T11, 1T12, 1T26, 2T3, 2T11, 2T13,2T32) were segregating in the inter-cross, whilst three loci(1T57, 1T69 and 2T34) yielded segregating SNP sites inboth mapping families. All loci except 1T25 and 2T5 wererepresented by at least two segregating SNP sites.

Altogether, 34 different SNP sites have been detected inthe 14 segregating loci: 17 nucleotide transitions and 17transversions. In the inter-specific cross (11P*QS29), tengenes identified by 23 SNPs, whilst in the intra-specificcross (3P*A4) seven genes identified by 15 SNPswere usedfor mapping (Table 3). As mentioned above, the segregationof three loci could be followed in both mapping popula-tions. In the case of locus 1T69 this applies to all, whilst inthe case of 2T34 two of three SNP sites yielded segrega-tion in both mapping populations. In locus 1T57 differentSNP sites yielded segregation in the two mapping families(Table 3).

As far as the location of the SNPs is concerned, 17.65%of the SNPs were discovered in introns, 64.7% in 3′ UTRregions and only 17.65% resided in coding regions of thegenes and could have an influence on the composition ofthe polypeptide chain. We identified four positions wherereplacement of amino acids occurred (Table 3).

Segregation and distortion

Most of the SNP sites yielded testcross type 1:1 segregationin both mapping families. However, about one third (fiveout of 15) of the markers segregated 1:2:1 in the intra-cross,and only three (out of 23) in the case of the inter-cross.

In locus 1T69, identical SNP sites showed different typesof segregation in the two crosses: 1:2:1 in the intra-cross and1:1 in the inter-cross, respectively. In the 2T34 locus thesegregation type for the two identical SNP was 1:1 in bothcrosses (Table 3).

Whilst most of the markers segregated according toMendel’s law, some were significantly distorted (Table 3).In the inter-cross, about one third of the sites showed dis-torted segregation, three of them at a significance level ofp=0.05 (1T12L316m, 1T12L427m and 2T13U247) andfive only at significance p=0.1 (1T69L127, -L193, 2T34U142,-L191 as well as 1T26U187). In the case of the 1T12 locus,both SNP sites showed a highly distorted segregation: ho-mozygotes were overrepresented (68 homozygotes+fourheterozygotes) for the two testcross markers 1T12L316 and1T12L427, resulting in a deficiency of heterozygosity by89% per SNP site. Concerning 2T13U247, whilst beingheterozygous in both parents the distribution of genotypesin the offspring was skewed to 34 heterozygotes, 34 homo-zygotes of type 1 (HH) and only four homozygotes of type2 (hh) (see Table 3). Thus this marker was classified ashighly distorted, because compared to the expected segre-

36

gation of 1:2:1 the site was highly deficient for one ho-mozygote genotype.

Mapping genes

For evaluating linkage and positioning EST-related SNPmarkers on LGs, the following strategies were applied: (1)construction of defined linkage maps (described in Mate-rials and methods) for adding markers, (2) calculation ofconsensus LGs for intercross segregating SNP markers(exclusively applied in the 3P*A4 mapping), (3) use ofpairwise linkages to framework markers of defined LGs(only used for the inter-specific cross).

In the intra-cross, all 15 SNP markers of seven ESTs(1T21, 1T25, 1T57, 1T69, 1T62, 2T5 and 2T34) could bemapped directly on LGs (Fig. 1a).

Generally, the low number of available frameworkmarkersin LGs of the inter-cross caused problems in mapping. Nev-ertheless, 18 markers of nine corresponding ESTs could bedirectly mapped to LGs. Two SNP markers of two differentloci (2T32L320 and 2T34L227) were placed as accessorymarkers next to their framework markers in the correspond-ing LGs using pairwise linkage calculations (Fig. 1b). Thetwo inter-cross markers, 2T13U247 and 2T34L227, despitesegregating 1:2:1, could only be located either on the femaleor on the male LG, respectively (possible cause: LG11 wasnot well defined by sufficient framework markers). In pair-wise linkage calculations, 2T11L194 showed linkage (LOD=

E-AAC/M-CTT-1360,0

E-AAG/M-CTT-872,9E-AAG/M-CTA-3303,6E-AAC/M-CTT-2304,3E-AAG/M-CTA-3375,8

E-AAC/M-CCT-21010,5

1T57U17912,51T57U12913,3

Y13-350/116,0

E-AAC/M-CTT-24618,5

E-AAC/M-CCT-18225,4E-AAC/M-CAC-21726,3

MSQ431,2

LG4

P-CCA/M-CAT-2540,0P-CCA/M-CAT-2580,2

E-AAC/M-CAA-496,8

E-AAC/M-CAC-34413,4

1T25L12416,6E-AAG/M-CTA-52817,6

B12-50019,7

N8-550/221,8

F20-1000/323,4

E-AAC/M-CTT-21723,6E-AAC/M-CTT-53224,3E-AAC/M-CTT-14225,0

E-AAG/M-CTA-42525,8

P-CCA/M-CAA-9726,5G2-900/2*29,6K11-1300/330,7

E-AAC/M-CAT-16232,8

quru-GA-1F0239,4

LG6

A10-1500/30,0

E-AAC/M-CCT-4211,5

E-AAC/M-CAA-2006,1

P14-4508,0

1T69L1271T69L193

11,5

E-AAC/M-CCT-17213,2

E-AAG/M-CTT-33016,0

E-AAC/M-CAT-14017,4

QrZAG5A20,1E-AAC/M-CTT-36521,3E-AAC/M-CAC-24221,5B11-150022,5

E-AAC/M-CAC-20226,1

ssrQpZAG11028,8

P-CAG/M-GCA-16631,3

E-AAC/M-CCT-61836,8

LG8

2T34L2042T34L178

2T34U1270,0

E-AAC/M-CAT-1561,5

E-AAG/M-CAC-623,7

QrZAG11111,1E-AAC/M-CAT-8811,4

N6-500/214,4

K20-900/215,5

B1-1200/317,2

B14-700/323,0

LG11f

2T5L460,0

I10-1400/3*4,6

N9-1000/37,1

Q13-1200/2

X20-1400/2*10,1

E-AAG/M-CTA-13325,4

E-AAG/M-CTT-34528,4

U19-1300/234,5

U1-550/236,8

LG3f

P-CCA/M-CAA-219/30,0

ssrQrZAG710,6

2T32U17317,9

B12-850/1***27,8ssrQpZAG3629,9

ssrQrZAG4434,6

E-AAC/M-CAA-29447,6

ssrQrZAG87***60,1

LG2m

E-ACG/M-AAT-449/10,0

1T57U186

1T57L8715,7

MSQ429,1

LG4m

P-CCA/M-CAA-124/20,0

2T11U13911,0

E-AAC/M-CAG-103/315,1

P-CAG/M-GGA-217/322,4

ssrQpZAG1/528,3

E-AAG/M-CTA-304/231,7

P-CCA/M-CAT-68/336,0

P-CAG/M-GCA-80/249,4

E-AAC/M-CTA-135/363,9

LG7f

P-CAG/M-GCA-156/20,0

1T69L193

1T69L12713,4

P-CCA/M-CAC-109/327,3

LGxf

E-AAC/M-CTT-125/30,0

mic-J-GA226,1

1T26U22812,3

ssrQpZAG1516,0

ssrQrZAG3122,9

E-AAG/M-CTA-191/3*28,4

MSQ1637,7

LG9f

Q14-2100/30,0

mic-J-GA227,6

1T26U18713,2

U19-850/318,4

ssrQpZAG1528,2

ssrQrZAG3138,5

LG9m

ssrQrZAG960,0

2T3U2602T3L33210,2

2T3U279

2T3U35415,8

LG10m

P-CCA/M-CAA-191/2*0,0

ssrQrZAG112,4

E-AAG/M-CTA-184/311,8

1T11U222m1T11L183m19,1

ssrQrZAG2523,8

ssrQrZAG6527,5

E-AAG/M-CTA-122/246,4

LG10f

P-CCAM-CAT-131/30,0

2T34L191

2T34U1428,9

ssrQrZAG111***18,4

LG11m

ssrQrZAG40,0ssrQrZAG180,2

2T13U18410,92T13U24711,4

E-AAG/M-CTT-217/516,8

N19-550/1*26,2

E-AAG/M-CTT-422/233,0

N13-580/337,0

ssrQrZAG11145,0

P-CCA/M-CAC-111/351,5

LG11f

(2T32L320)

(2T34L227)

LG1f

I14-550/10,0

E-AAG/M-CTT-148N7-550/2*

5,9

QrZAG206,2

R11-800/1*11,5

P2-1700/214,8I14-1300/220,0EMCs1321,1R12-400/124,1A2-900/325,0E-AAC/M-CAT-35926,0E-AAC/M-CTT-28026,3I14-1500/1

M12-700/326,8

E-AAC/M-CAT-33527,1E-AAC/M-CAT-35627,4E-AAC/M-CAA-7427,51T21L4151T21L75

1T21U4028,4

P14-300/231,5QrZAG10131,9Y13-2200/233,1quru-GA-0C1134,1E-AAC/M-CAC-10835,1E-AAC/M-CTT-13735,4QrZAG7436,1

LG10

ssrQpZAG3/620.0

Q14-1100/34.1E-AAG/M-CTT-4884.3

1T62L6228.91T62L617

1T62L6279.3

E-AAC/M-CAC-7010.7E3-500/311.5

E-AAG/M-CTT-38713.2E-AAC/M-CAA-33215.7QrZAG1117.7

E-AAC/M-CAA-25020.4

quru-GA-0M0524.4QrZAG6525.7A1-500/227.8

E-AAG/M-CTA-6329.3E-AAG/M-CTA-21229.5P-CCA/M-CAT-10229.6

E-AAC/M-CTT-9936.2

E-AAC/M-CAT-9739.8

E-AAC/M-CTT-1360,0

E-AAG/M-CTT-872,9E-AAG/M-CTA-3303,6E-AAC/M-CTT-2304,3E-AAG/M-CTA-3375,8

E-AAC/M-CCT-21010,5

1T57U17912,51T57U12913,3

Y13-350/116,0

E-AAC/M-CTT-24618,5

E-AAC/M-CCT-18225,4E-AAC/M-CAC-21726,3

MSQ431,2

LG4

E-AAC/M-CTT-1360,0

E-AAG/M-CTT-872,9E-AAG/M-CTA-3303,6E-AAC/M-CTT-2304,3E-AAG/M-CTA-3375,8

E-AAC/M-CCT-21010,5

1T57U17912,51T57U12913,3

Y13-350/116,0

E-AAC/M-CTT-24618,5

E-AAC/M-CCT-18225,4E-AAC/M-CAC-21726,3

MSQ431,2

LG4

P-CCA/M-CAT-2540,0P-CCA/M-CAT-2580,2

E-AAC/M-CAA-496,8

E-AAC/M-CAC-34413,4

1T25L12416,6E-AAG/M-CTA-52817,6

B12-50019,7

N8-550/221,8

F20-1000/323,4

E-AAC/M-CTT-21723,6E-AAC/M-CTT-53224,3E-AAC/M-CTT-14225,0

E-AAG/M-CTA-42525,8

P-CCA/M-CAA-9726,5G2-900/2*29,6K11-1300/330,7

E-AAC/M-CAT-16232,8

quru-GA-1F0239,4

LG6

P-CCA/M-CAT-2540,0P-CCA/M-CAT-2580,2

E-AAC/M-CAA-496,8

E-AAC/M-CAC-34413,4

1T25L12416,6E-AAG/M-CTA-52817,6

B12-50019,7

N8-550/221,8

F20-1000/323,4

E-AAC/M-CTT-21723,6E-AAC/M-CTT-53224,3E-AAC/M-CTT-14225,0

E-AAG/M-CTA-42525,8

P-CCA/M-CAA-9726,5G2-900/2*29,6K11-1300/330,7

E-AAC/M-CAT-16232,8

quru-GA-1F0239,4

P-CCA/M-CAT-2540,0P-CCA/M-CAT-2580,2

E-AAC/M-CAA-496,8

E-AAC/M-CAC-34413,4

1T25L12416,6E-AAG/M-CTA-52817,6

B12-50019,7

N8-550/221,8

F20-1000/323,4

E-AAC/M-CTT-21723,6E-AAC/M-CTT-53224,3E-AAC/M-CTT-14225,0

E-AAG/M-CTA-42525,8

P-CCA/M-CAA-9726,5G2-900/2*29,6K11-1300/330,7

E-AAC/M-CAT-16232,8

quru-GA-1F0239,4

LG6

A10-1500/30,0

E-AAC/M-CCT-4211,5

E-AAC/M-CAA-2006,1

P14-4508,0

1T69L1271T69L193

11,5

E-AAC/M-CCT-17213,2

E-AAG/M-CTT-33016,0

E-AAC/M-CAT-14017,4

QrZAG5A20,1E-AAC/M-CTT-36521,3E-AAC/M-CAC-24221,5B11-150022,5

E-AAC/M-CAC-20226,1

ssrQpZAG11028,8

P-CAG/M-GCA-16631,3

E-AAC/M-CCT-61836,8

LG8

A10-1500/30,0

E-AAC/M-CCT-4211,5

E-AAC/M-CAA-2006,1

P14-4508,0

1T69L1271T69L193

11,5

E-AAC/M-CCT-17213,2

E-AAG/M-CTT-33016,0

E-AAC/M-CAT-14017,4

QrZAG5A20,1E-AAC/M-CTT-36521,3E-AAC/M-CAC-24221,5B11-150022,5

E-AAC/M-CAC-20226,1

ssrQpZAG11028,8

P-CAG/M-GCA-16631,3

E-AAC/M-CCT-61836,8

A10-1500/30,0

E-AAC/M-CCT-4211,5

E-AAC/M-CAA-2006,1

P14-4508,0

1T69L1271T69L193

11,5

E-AAC/M-CCT-17213,2

E-AAG/M-CTT-33016,0

E-AAC/M-CAT-14017,4

QrZAG5A20,1E-AAC/M-CTT-36521,3E-AAC/M-CAC-24221,5B11-150022,5

E-AAC/M-CAC-20226,1

ssrQpZAG11028,8

P-CAG/M-GCA-16631,3

E-AAC/M-CCT-61836,8

LG8

2T34L2042T34L178

2T34U1270,0

E-AAC/M-CAT-1561,5

E-AAG/M-CAC-623,7

QrZAG11111,1E-AAC/M-CAT-8811,4

N6-500/214,4

K20-900/215,5

B1-1200/317,2

B14-700/323,0

LG11f

2T34L2042T34L178

2T34U1270,0

E-AAC/M-CAT-1561,5

E-AAG/M-CAC-623,7

QrZAG11111,1E-AAC/M-CAT-8811,4

N6-500/214,4

K20-900/215,5

B1-1200/317,2

B14-700/323,0

LG11f

2T5L460,0

I10-1400/3*4,6

N9-1000/37,1

Q13-1200/2

X20-1400/2*10,1

E-AAG/M-CTA-13325,4

E-AAG/M-CTT-34528,4

U19-1300/234,5

U1-550/236,8

LG3f

2T5L460,0

I10-1400/3*4,6

N9-1000/37,1

Q13-1200/2

X20-1400/2*10,1

E-AAG/M-CTA-13325,4

E-AAG/M-CTT-34528,4

U19-1300/234,5

U1-550/236,8

LG3f

A

B

P-CCA/M-CAA-219/30,0

ssrQrZAG710,6

2T32U17317,9

B12-850/1***27,8ssrQpZAG3629,9

ssrQrZAG4434,6

E-AAC/M-CAA-29447,6

ssrQrZAG87***60,1

LG2m

P-CCA/M-CAA-219/30,0

ssrQrZAG710,6

2T32U17317,9

B12-850/1***27,8ssrQpZAG3629,9

ssrQrZAG4434,6

E-AAC/M-CAA-29447,6

ssrQrZAG87***60,1

LG2m

E-ACG/M-AAT-449/10,0

1T57U186

1T57L8715,7

MSQ429,1

LG4m

E-ACG/M-AAT-449/10,0

1T57U186

1T57L8715,7

MSQ429,1

LG4m

P-CCA/M-CAA-124/20,0

2T11U13911,0

E-AAC/M-CAG-103/315,1

P-CAG/M-GGA-217/322,4

ssrQpZAG1/528,3

E-AAG/M-CTA-304/231,7

P-CCA/M-CAT-68/336,0

P-CAG/M-GCA-80/249,4

E-AAC/M-CTA-135/363,9

LG7f

P-CCA/M-CAA-124/20,0

2T11U13911,0

E-AAC/M-CAG-103/315,1

P-CAG/M-GGA-217/322,4

ssrQpZAG1/528,3

E-AAG/M-CTA-304/231,7

P-CCA/M-CAT-68/336,0

P-CAG/M-GCA-80/249,4

E-AAC/M-CTA-135/363,9

LG7f

P-CAG/M-GCA-156/20,0

1T69L193

1T69L12713,4

P-CCA/M-CAC-109/327,3

LGxf

P-CAG/M-GCA-156/20,0

1T69L193

1T69L12713,4

P-CCA/M-CAC-109/327,3

LGxf

E-AAC/M-CTT-125/30,0

mic-J-GA226,1

1T26U22812,3

ssrQpZAG1516,0

ssrQrZAG3122,9

E-AAG/M-CTA-191/3*28,4

MSQ1637,7

LG9f

E-AAC/M-CTT-125/30,0

mic-J-GA226,1

1T26U22812,3

ssrQpZAG1516,0

ssrQrZAG3122,9

E-AAG/M-CTA-191/3*28,4

MSQ1637,7

LG9f

Q14-2100/30,0

mic-J-GA227,6

1T26U18713,2

U19-850/318,4

ssrQpZAG1528,2

ssrQrZAG3138,5

LG9m

Q14-2100/30,0

mic-J-GA227,6

1T26U18713,2

U19-850/318,4

ssrQpZAG1528,2

ssrQrZAG3138,5

LG9m

ssrQrZAG960,0

2T3U2602T3L33210,2

2T3U279

2T3U35415,8

LG10m

ssrQrZAG960,0

2T3U2602T3L33210,2

2T3U279

2T3U35415,8

LG10m

P-CCA/M-CAA-191/2*0,0

ssrQrZAG112,4

E-AAG/M-CTA-184/311,8

1T11U222m1T11L183m19,1

ssrQrZAG2523,8

ssrQrZAG6527,5

E-AAG/M-CTA-122/246,4

LG10f

P-CCA/M-CAA-191/2*0,0

ssrQrZAG112,4

E-AAG/M-CTA-184/311,8

1T11U222m1T11L183m19,1

ssrQrZAG2523,8

ssrQrZAG6527,5

E-AAG/M-CTA-122/246,4

LG10f

P-CCAM-CAT-131/30,0

2T34L191

2T34U1428,9

ssrQrZAG111***18,4

LG11m

P-CCAM-CAT-131/30,0

2T34L191

2T34U1428,9

ssrQrZAG111***18,4

LG11m

ssrQrZAG40,0ssrQrZAG180,2

2T13U18410,92T13U24711,4

E-AAG/M-CTT-217/516,8

N19-550/1*26,2

E-AAG/M-CTT-422/233,0

N13-580/337,0

ssrQrZAG11145,0

P-CCA/M-CAC-111/351,5

LG11f

ssrQrZAG40,0ssrQrZAG180,2

2T13U18410,92T13U24711,4

E-AAG/M-CTT-217/516,8

N19-550/1*26,2

E-AAG/M-CTT-422/233,0

N13-580/337,0

ssrQrZAG11145,0

P-CCA/M-CAC-111/351,5

LG11f

(2T32L320)

(2T34L227)

LG1f

I14-550/10,0

E-AAG/M-CTT-148N7-550/2*

5,9

QrZAG206,2

R11-800/1*11,5

P2-1700/214,8I14-1300/220,0EMCs1321,1R12-400/124,1A2-900/325,0E-AAC/M-CAT-35926,0E-AAC/M-CTT-28026,3I14-1500/1

M12-700/326,8

E-AAC/M-CAT-33527,1E-AAC/M-CAT-35627,4E-AAC/M-CAA-7427,51T21L4151T21L75

1T21U4028,4

P14-300/231,5QrZAG10131,9Y13-2200/233,1quru-GA-0C1134,1E-AAC/M-CAC-10835,1E-AAC/M-CTT-13735,4QrZAG7436,1

LG1f

I14-550/10,0

E-AAG/M-CTT-148N7-550/2*

5,9

QrZAG206,2

R11-800/1*11,5

P2-1700/214,8I14-1300/220,0EMCs1321,1R12-400/124,1A2-900/325,0E-AAC/M-CAT-35926,0E-AAC/M-CTT-28026,3I14-1500/1

M12-700/326,8

E-AAC/M-CAT-33527,1E-AAC/M-CAT-35627,4E-AAC/M-CAA-7427,51T21L4151T21L75

1T21U4028,4

P14-300/231,5QrZAG10131,9Y13-2200/233,1quru-GA-0C1134,1E-AAC/M-CAC-10835,1E-AAC/M-CTT-13735,4QrZAG7436,1

I14-550/10,0

E-AAG/M-CTT-148N7-550/2*

5,9

QrZAG206,2

R11-800/1*11,5

P2-1700/214,8I14-1300/220,0EMCs1321,1R12-400/124,1A2-900/325,0E-AAC/M-CAT-35926,0E-AAC/M-CTT-28026,3I14-1500/1

M12-700/326,8

E-AAC/M-CAT-33527,1E-AAC/M-CAT-35627,4E-AAC/M-CAA-7427,51T21L4151T21L75

1T21U4028,4

P14-300/231,5QrZAG10131,9Y13-2200/233,1quru-GA-0C1134,1E-AAC/M-CAC-10835,1E-AAC/M-CTT-13735,4QrZAG7436,1

LG10

ssrQpZAG3/620.0

Q14-1100/34.1E-AAG/M-CTT-4884.3

1T62L6228.91T62L617

1T62L6279.3

E-AAC/M-CAC-7010.7E3-500/311.5

E-AAG/M-CTT-38713.2E-AAC/M-CAA-33215.7QrZAG1117.7

E-AAC/M-CAA-25020.4

quru-GA-0M0524.4QrZAG6525.7A1-500/227.8

E-AAG/M-CTA-6329.3E-AAG/M-CTA-21229.5P-CCA/M-CAT-10229.6

E-AAC/M-CTT-9936.2

E-AAC/M-CAT-9739.8

LG10

ssrQpZAG3/620.0

Q14-1100/34.1E-AAG/M-CTT-4884.3

1T62L6228.91T62L617

1T62L6279.3

E-AAC/M-CAC-7010.7E3-500/311.5

E-AAG/M-CTT-38713.2E-AAC/M-CAA-33215.7QrZAG1117.7

E-AAC/M-CAA-25020.4

quru-GA-0M0524.4QrZAG6525.7A1-500/227.8

E-AAG/M-CTA-6329.3E-AAG/M-CTA-21229.5P-CCA/M-CAT-10229.6

E-AAC/M-CTT-9936.2

E-AAC/M-CAT-9739.8

ssrQpZAG3/620.0

Q14-1100/34.1E-AAG/M-CTT-4884.3

1T62L6228.91T62L617

1T62L6279.3

E-AAC/M-CAC-7010.7E3-500/311.5

E-AAG/M-CTT-38713.2E-AAC/M-CAA-33215.7QrZAG1117.7

E-AAC/M-CAA-25020.4

quru-GA-0M0524.4QrZAG6525.7A1-500/227.8

E-AAG/M-CTA-6329.3E-AAG/M-CTA-21229.5P-CCA/M-CAT-10229.6

E-AAC/M-CTT-9936.2

E-AAC/M-CAT-9739.8

Fig. 1 a Positioning osmotic stress related ESTs to single female (f)and combined linkage maps of Q. robur intra-cross 3P*A4. SNPmarkers (indicated in bold) were mapped to the reference linkagegroups with the following LOD scores: 1f/5,0; 3f/4,5; 4combined/6,0;6combined/5,0; 8combined/7,0; 10combined/6,0 and 11f/7,0. Mark-ers displaying significant segregation distortion are denoted by anasterisk. b Positioning osmotic stress related ESTs to female (f) andmale (m) linkagemaps of inter-crossQ. robur×Q. petraea 11P*QS29.Corresponding SNP markers (indicated in bold) were mapped to thelinkage groups with the highest LOD score possible: 2m/3,2; 4m/4,1;

7f/1,7; xf/3,1; 9m/4,4; 9f/4,5; 10m/6,7; 10f/3,2; 11m/7,2 and 11f/2,7.SNP markers added in brackets are related to markers of thecorresponding ESTs (LOD>6) but to other framework markers onlyloose relation was observed (for 2T32L320: LOD<2; for 2T34L227:LOD<4). Markers displaying significant segregation distortion aredenoted by asterisks. P...PstI/MseI AFLP marker; E...EcoRI/MseIAFLP marker; Qr, MSQ, ssr, mic, quru, EMCs13...microsatellites; A,B, E, F, G, I, K, M, N, P, R, Q, U, X, Y...RAPDs; B11-1500, B12-500,P14-450...SCARs; 1T, 2T...SNPs

37

3.7) to a marker of the male inter-cross map which had notbeen related to any LG yet (possible cause: for LG7m therewas no strong LOD>3-linkage between ssrQpZAG1/5 and“nearest” AFLP markers).

Thus, seven (Fig. 1a) and nine genes (Fig. 1b) werepositioned on LGs for 3P*A4 and 11P*QS29, respectively.Therefore, by using two different crosses, we were able tolocate 13 different osmotic stress-related genes on chromo-somes of oak. Three genes could be investigated in bothcrosses; two of them (1T57, 2T34) were located, as ex-pected, on the corresponding LGs (Fig. 1a and b). The third(1T69) was unrelated in the inter-cross, because there wereno SSR markers defined for this LG (Fig. 1b). Due to thehighly significant segregation distortion of its SNP sites,EST 1T12 could not be related to any LG in Quercus.Comparative mapping, however, allowed its localisation onthe corresponding LG of LG11 of oak in Castanea sativa(Casasoli, personal communication) (Table 3).

On LG10 of the inter-cross, we observed that SNP sitesbelonging to a putative single locus segregated like twodistinct loci. The four testcross markers separated by 5.5cMinto two+two tightly linked markers (U260/L332 andU279/U354, respectively) on the male LG10. Thereforethese markers within locus 2T3 (concerning sequence sim-ilarity putatively related to Medicago truncatula nodulingene MtN19) suggest the presence of two closely relatedmembers of a gene family with so far unknown function intrees but putatively located in the plasma membrane, asassumed by Fedorova et al. (2002). Indeed, sequencing the3′ end of EST 2T3 yielded two dissimilar (80% sequencehomology) 3′UTRs (data not shown) supporting the idea ofa gene family.

Discussion

Polymorphisms

Generally, SNPs and sequence-based indels represent aninexhaustible source of polymorphic markers for high-resolution genetic mapping of traits and association studiesbased on candidate genes (Berger et al. 2001; Cho et al.1999).

For maximizing—in a given set—the number of genes,which could be located on the genetic linkage map of oak,we combined the following strategies: (1) search for poly-morphisms in the highly variant 3′ end region of a gene(Cato et al. 2001; Rafalski 2002; Bhattramakki et al. 2002;Iida et al. 2001); (2) map in “multiple” populations in orderto raise possibility for marker segregation (Sewell et al.1999). As expected, the inter-cross provided more poly-morphic markers suitable for mapping (63% of total SNPs).By choosing 3′UTR, we intended to avoid co-amplificationof genes, which have related coding regions (Brady et al.1997).

The choice of screening mainly the 3′ UTR regions ofgenes not only gives us a greater chance to find polymor-phisms for mapping purposes, but also reduces the pos-sibility of discovering replacement polymorphisms (see

Table 3). Dependent on their positions within the polypep-tide chain, these polymorphisms may lead to altered proteinfunction if this specific irregular allelic variation is ex-pressed. Although non-replacement polymorphisms do notcontribute to amino acid changes, they are supposed to af-fect gene function at transcriptional or translational level,through splicing or RNA stability (Gibson andMuse 2001).We may assume that this can obviously be the case whennatural selection for a certain genotype is significant.

Gene mapping and marker distortion

In the intra-cross, out of the seven mapped genes two locicould be located on LGs with a LOD value of 7 (1T69;2T34), two with a value of 6 (1T57; 1T62), two with 5(1T21; 1T25), and at least one gene was positioned on therespective LG with a LOD score of 4.5 (2T5). Thus, wefound a good statistical support for the localisation of thenewly added markers. For the inter-cross, however, the lowstarting number of markers which could be effectivelygrouped into 12 LGs forced us to reject the mapping strat-egy we used for the intra-cross (see Materials and methodsand below).

In this study also a higher rate of marker distortion wasobvious for the inter-cross; however, marker distortion ininter-specific linkage analysis is quite frequent (Ky et al.2000). We found three significantly (p≪0.05) distorted andfive SNP sites distorted at a weaker significance level(p=0.1). Classification of all the distorted sites provided uswith the following picture: any deviation from the Mende-lian law of segregation was exclusively found in the inter-specific cross, i.e. 35% of all SNP markers segregating inthis cross. All of them were skewed towards an excess ofhomozygote genotypes (see Table 3). For the bulked frame-work markers of the intra-cross (for both female and malemaps), approximately 20% showed distortion at p=0.05(28% at p=0.1). This high percentage of distortion is char-acteristic for pedunculate oak and was related to high geneticloadwithin this species (Barreneche et al. 1998). Segregationdistortion was observed to the same extent for frameworkmarkers in the inter-cross. The unequal segregation of locimay be the consequence of the selection processes at thegamete or zygote stage.

Concerning the identity of the genes housing highly sig-nificant distorted SNP sites, we found an interesting detail:both genes belonging to the oxylase/oxygenase gene family(1T12; 2T13) were involved (Tables 1 and 3). Both sites inEST 1T12 were so highly distorted that the calculated het-erozygote deficiency reached nearly 90%.Consequently, wecould not use these markers in mapping, but they becameinteresting tools for population analysis (Porth, personalcommunication).

The distribution of genotypes in the offspring concerningone SNP of EST 2T13 (U247) segregating 1:2:1 may alsosuggest that too few individuals had been genotyped in thatcase. Hence this collection of individuals displayed “dis-torted” segregation.

38

In the intra-cross 3P*A4 one third of SNP markersshowed the 1:2:1 segregation type, compared to only 13%in the inter-cross 11P*QS29 (Table 3). Consequently, as ahigher portion of heterozygosity was evident for the parentsof 3P*A4, in this cross the characteristics are inherited morefrequently from both parents compared to 11P*QS29 (e.g.for 1T69). Since mostly testcross markers are used to buildfemale and male maps separately (Barreneche et al. 1998),co-dominant markers segregating 1:2:1 are important toidentify homologies which represent important bridges tointerconnect both maps to a consensus map. However, be-cause the standard error of the recombination fraction be-tweenmarkers segregating 1:2:1 is much higher than for 1:1segregating markers, their position on the linkage map isless precise (Ritter et al. 1990). Thus pairs of markers ofintermixed segregation provide less information. This mayexplain the genetic distance observed for 1T57U129 and1T57U179 and between 1T62L622 and the two other sitesof this locus on LG4 and LG10, respectively, as well as theproblem in locating 2T32L320 and 2T34L227 directly onthe respective LGs. A larger number of individuals wouldbe crucial to study co-segregation, which is certainly truefor all of these cases. For 11P*QS29, in general, a largermapping family is required in addition to a larger number offramework markers to locate testcross as well as intercrossco-dominant markers more precisely. Generally, the ob-served “distortion” in the segregation of SNP sites (exceptEST 1T12) may be attributed to the low number of geno-typed individuals.

Localisation of stress genes

The main objective of this study was to find linkage and tolocalise the putative candidate genes for drought/salt tol-erance in Quercus on the respective linkage groups. How-ever, the 88 offspring for the intra-cross used in this surveywere sufficient to show linkage, but more genotyped indi-viduals are required to achieve more precise marker order.In the present frameworkmap, accessorymarkers publishedby Barreneche et al. (1998) were also included; however, ahigher density of markers would require more individualsto obtain a precise marker order.

In the inter-cross, besides the low number of pedigree(72), we faced the following problems: (1) only few mark-ers per LG existed, (2) the LOD score for map building wasoften below 3, and (3) one LG had not been defined yet.Therefore, for gene mapping it was crucial to find stronglinkages to SSR markers, which were important determi-nants of LGs (Barreneche et al. 1998). Thus, it becamemoreimportant to relate an SNP marker in the inter-cross to adefined LG than to exactly locate it within a group of severalmarkers (e.g. LG10m, LG11m, LG4m).

For genemapping, it would be sufficient to define an ESTby one SNP marker, which could be linked to anothermarker defining a LG. In our case, all investigated SNP sitesexcept three (1T12L316, -L427 and 2T11L194) provideddirectly or indirectly (via linked SNP markers) significantlinkage information (LOD>3) to informative framework

markers, which enabled us to relate the respective ESTs toLGs of oak. Thus, we were also able to reveal differencesin segregation by using multiple SNP sites within a locus.Ten different LGs of oak were associated with SNP mark-ers: 1, 2, 3, 4, 6, 7, 8, 9, 10 and 11. For this set of 13 differentgenes we found no tight clustering of osmotic stress relatedloci on linkage groups of oak, although three genes werelinked to LG10 and two genes were positioned on LG11.This was due to the fact that segregation was found in dif-ferent mapping families (1T11, 1T62 and 2T3) and to thelow number of linked framework markers (2T3). Howev-er, within a recent approach to align corresponding linkagegroups of Quercus and C. sativa, ten of the presented andmapped ESTs in this work were also used for comparativemapping (Cassasoli, personal communication). The se-quence identity of the amplified regions exceeded 90%.Two interesting details concerning marker localisation andclustering in C. sativa are noted: the two ESTs (1T11 and1T62) were found to be closely linked (<1.5 cM) on thecorresponding chestnut LG, onto which also EST 2T3could be positioned; ESTs 1T12 and 2T13, the two differentmembers of the 2OG-Fe(II) oxygenase family, were locatedon the same chromosome in Castanea (Cassasoli, personalcommunication).

In the inter-cross, with the help of EST 2T13, it waspossible to define the female LG11. The marker composi-tion of this LG in the inter-cross was previously unknown,but is nowdetermined by three SSRmarkers (i.e. ssrQrZAG4,ssrQrZAG18 and ssrQrZAG111) well established in LG11of the intra-cross (Barreneche et al. 2004).

Recently, Porth et al. (2005) presented results for osmot-ically treated plantlets that indicated significant differencesbetweenQ. petraea andQ. robur in the expression of genesassumed to enhance water-stress tolerance. In the presentstudy, three of the identified genes (1T62, 2T11 and 2T13),which were up-regulated in sessile oak, showed segrega-tion in the two mapping families and mapped to three dif-ferent LGs. Concerning the chromosomal localisation ofthe two remaining candidate genes (1T9 and 2T33), addi-tional mapping populations are required to achieve markersegregation.

A common strategy to identify sequence variations ingenes of interest is to compare natural populations as theyprovide the benefit of maximum polymorphism. However,we used parents of controlled crosses to detect polymor-phisms we could apply in linkage mapping of our genes.Nevertheless, it is interesting to investigate the allelic dis-tribution of these SNPs in the natural populations of Q.robur and Q. petraea to: (1) investigate putative selectionfor specific genotypes in the inter-specific cross (e.g. 1T12)and (2) check the allele frequency distribution of multipleSNP sites within a locus (Porth, personal communication).For allele frequency estimations in mixed oak stands, it isworthwhile to use genetic markers that have already beenrelated to chromosomes of oak. Thus, it is possible to mon-itor the extent of genetic differentiation of the two speciesdetermined for these markers distributed along the genome(Scotti-Saintagne et al. 2004).

39

Acknowledgements This study was carried out with financial sup-port from the Commission of the European Communities, Commu-nity research programme “Quality of Life and Management of Livingresources” (Project OAKFLOW QLK5-2000-00960).

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