E. Ritter et al.UHD linkage map of Pinus pinaster Original article Towards construction of an ultra high density linkage map for Pinus pinaster Enrique Ritter a* , Ana Aragonés a , Torsten Markussen b , Virginie Acheré c , Santiago Espinel a , Matthias Fladung b , Sandra Wrobel b , Patricia Faivre-Rampant c , Sylvain Jeandroz c and Jean-Michel Favre c a NEIKER, Apartado 46, Vitoria, Alava, 01080, Spain b BFH, Institute for Forest Genetics, Sieker Landstrasse 2, Grosshansdorf, 22927, Germany c UMR UHP-INRA Plant-Microbes Interactions, Faculté des Sciences, BP 239, 54506 Vandœuvre-lès-Nancy, France (Received 16 August 2001; accepted 22 February 2002) Abstract – Two parental linkage maps have been constructed from the P. pinaster reference population (0024 × C803) based on AFLP, SSR and EST markers. Although segregating polymorphism was low due to a high degree of homozygosity in the parents, 12 linkage groups with 26 to 46 markers each were obtained for each parent. The availibility of 70 anchor points based onfragmentscommontoboth parents and based on co- dominant SSR and EST markers allowed to determine homologous chromosomes for both maps and to construct one integrated map. Total ge- nome length of the integrated map is around 2000 cM including 1182 markers. Since some of the EST and SSR markers are also mapped in different pine species, association of linkage groups of our reference population with those of other published maps was possible. AFLP, SSR, EST markers / genetic mapping Résumé – Vers la construction d’une carte génétique ultra-haute densité chez Pinus pinaster. Deux cartes génétiques ont été construites à partir d’un croisement intra-spécifique de P. Pinaster (0024 Landes × C803 Corse) avec des marqueurs AFLP, SSR et EST. Malgré un faible po - lymorphisme dû à la forte homozygotie des parents, 12 groupes de liaison comprenant 26 à 46 marqueurs ont été obtenus pour chacune des car - tes. La présence de 70 points d’ancrage déterminés à partir des fragments communs aux deux parents a permis d’identifier les chromosomes homologues des deux cartes et de construire une carte consensus d’une longueur totale d’environ 2000 cM et comprenant 1182 marqueurs. La présence de quelques marqueurs EST et microsatellites déjà cartographiés chez différentes espèces de pins a permis d’aligner un certain nombre de groupes de liaison avec cette carte de pin maritime. marqueurs AFLP, SSR, EST / cartographie génétique 1. INTRODUCTION Several linkage maps have been produced in a variety of forest species including Pinus. They are based on different marker types such as RFLPs in Pinus taeda [6, 19] and RAPDs in Pinus pinaster [4] and Pinus radiata [7]. Also AFLP maps are available for Pinus pinaster [4] , P. radiata [2] and Pinus edulis [22]. Recently a high-density map of P. pinaster has been constructed [5]. Other marker types such as proeins and isozymes are integrated in these maps [4, 6, 15] as well as EST [3] and SSR markers [8] which may be useful for aligning different maps. In order to apply DNA marker technology in breeding of coniferous species a project has been initiated with the aim of constructing an ultra-high-density linkage map (UHD map) of Pinus pinaster based on several thousands AFLP markers and numerous published microsatellites (SSR). The reference map will be used for comparative genome and QTL analyses in different genetic backgrounds. It is the aim to align other Ann. For. Sci. 59 (2002) 637–643 637 © INRA, EDP Sciences, 2002 DOI: 10.1051/forest:2002049 * Correspondence and reprints Tel.: 34 945 121 381; fax: 34 945 281 422; e-mail: eritter@neiker.net published linkage maps in forest species with this reference map. Based on a reduced number of markers, comparative ge - nome and QTL analyses will be performed in different pine species and related gymnosperms. In this paper we present the first project results, which consist of an integrated linkage map derived from two P. pinaster genotypes and some associations of linkage groups of our map with those of different pine species. 2. MATERIALS AND METHODS 2.1. Plant material The F1 reference population of P. pinaster descended from the cross 0024 Landes × C803 Corsica. The parental trees had been se - lected in cooperation by INRA-Pierroton and AFOCEL SW re - search stations. The cross was performed in 1987 and the 129 resulting progeny trees were established in the field by AFOCEL in 1990. A total of 80 out of the 129 progeny genotypes, established in the AFOCEL experimental stands of Troussas and Arsague (Landes; SW, France), were used for linkage mapping. 2.2. Molecular methods Genomic DNA was extracted from needles using the DNeasy Plant Kit from Qiagen with slight modifications of the supplier’s protocol. AFLP analysis was performed according to [23] using EcoRI/MseI adapters. Preamplification was performed with one se- lective nucleotide and specific amplification with 3 selective nu- cleotides (+1/+3 amplification). Also +2/+4 amplifications were performed. Amplification products were separated on 6 or 8% dena- turing polyacrylamide gels. Different techniques were used for de- tecting amplification products. AFLP fragments were detected on a LI-COR 4200-S1 DNA sequencer using primers labelled with the fluorescent infrared dye IRD800 (LI-COR, Lincoln, Nebraska, USA) or on a ALFexpressII (Amersham Pharmacia Biotech, Ger - many) with Cy5-Amidite labelled primers (MWG-Biotech, Ger - many). Analysis was performed according to the manufacturer instructions in each case. SSR primers developed in different species were analyzed for polymorphism and segregation. SSR developed in Pinus pinaster and P. halepensis [13] in P. strobus ([9, 10]; http://dendrome.ucdavis.edu/Data/echt_ssr_primers.html), in P. radiata and P. sylvestris ([11, 20, 21]; http://dendrome.ucdavis.edu/ Data/hardssr.html) were used for this purpose. Furthermore, several other as yet unpublished SSR primers from Pinus radiata were ob - tained from Gavin Moran (CSIRO, Australia) and from Craig Echt (Forest Research Inc., New Zealand) and from Picea abies from Giovanni Vendramin (IMGPF, Italy). SSR analysis was performed as described by [13] or based on the information given in the men - tioned WEB pages. EST primers were obtained from the informa - tion provided on http://www.pierroton.inra.fr/genetics/pinus/ primers.htm. EST analysis was performed according to [12]. 2.3. Data analysis and linkage mapping Polymorphic DNA fragments were scored for presence or ab - sence in parents and F1 progenies. Linkage analysis between marker fragments, estimation of recombination frequencies, and determina - tion of linear order between linked loci including multipoint linkage analysis and the EM algorithm for handling missing data were per - formed as described in [16, 17]. The MAPRF program [17] was ap - plied for the computational methods. Firstly, linkage groups were constructed based on fragments specific to either parent. Linked fragments were arranged into linkage groups using a minimum, commonly accepted LOD threshold of 3.0 between consecutive markers. Subsequently, fragments common to both parents were in - tegrated into linkage groups as anchor points as described in [16]. Only common markers linked with recombination frequencies of zero to at least one parent (LOD > 6) and linked with a minimum LOD threshold of 3.0 to the other parent were considered for this purpose. 3. RESULTS 3.1. Generation of polymorphic DNA markers Nearly 300 different primer combinations (PCs) were ana - lyzed for the generation of AFLP-fragments. More than 100 fragments were often produced with specific primer combi - nations, with from one to 25 segregating fragments in the mapping population. However, some primer combinations produced no segregating fragments. This was generally the case for primer combinations with high AT contents. In gen- eral, better quality of gels were obtained with the +2/+4 am- plification system. A total of 239 AFLP primer combinations were used for the molecular analyses and generated 1740 segregating frag- ments. Thus on average, 7.3 polymorphic bands per primer combination were obtained. Approximately 39% of the seg- regating fragments were specific for either one parent of the cross, while 22% of the fragments were present in both par - ents. Around 16% of the fragments showed significant devia - tions (α > 5%) from the expected segregation ratios. Furthermore, a total of 120 SSR and 30 EST primer pairs were used in this study. Amplification products were ob - tained in most cases after adapting the particular PCR condi - tions in each case. However, as with AFLP markers, a low degree of polymorphism between parental alleles together with a large degree of homozygosity (i.e. non segregating polymorphic fragments) was observed. Only 21 SSR and 10 EST markers showed one or more segregating bands. 3.2. Construction of linkage maps Initially, individual linkage maps of 12 linkage groups each were obtained for the two parents of the mapping popu - lation. Their characteristics are summarised in table I. Details of the maps, parental AFLP profiles as well as the obtained polymorphisms are displayed on the project WEB page (http://www.neiker.net/UHDfor). Linkage groups of the P1 map (parent 0024) contained 26 to 46 individual and common markers each and were between 107.8 and 180.1 cM in length. The total P1 map length (female parent 0024) was 638 E. Ritter et al. 1736 cM. The P2 map (male parent C803) was 1942 cM in length and made up of linkage groups with 23 to 41 markers each. The size of the linkage groups varied between 115.1 and 190.5 cM. Further 217 fragments were linked with recombination frequencies of zero to other fragments in these linkage maps and have not been not considered in these counts. Linkage to mapped markers on linkage groups was appar - ent for 206 additional markers. However, these were highly distorted or consisted of common fragments linked to other fragments with reduced LOD values and could not be placed in a single interval with high certainty. Since the standard er - rors of the estimates of the recombination frequencies were high, these 206 markers are included in this map as so called “associated markers”, anchored to the marker with the high - est probability. Based on the integration of 70 markers common to both parents and codominant markers like SSRs and ESTs into linkage groups for both parents, it was possible to assign all 12 homologous chromosomes for P1 and P2, and to obtain in this way an integrated consensus map with a total of 759 markers (table I, figure 1). Linkage groups of the inte - grated map varied between 123.2 and 191 cM in length and contained between 45 and 74 markers each. Considering the 217 markers linked without recombination to other displayed markers and the 206 associated markers, an integrated map of 1182 markers was achieved with an average of 99 markers per linkage group. 3.3. Associations of linkage groups between the reference map and other published maps The SSR and EST markers amplified one or two loci each with variable number of alleles. A total of 14 SSR (19 loci) and 7 EST markers (7 loci) could be placed on the reference map (figure 1). Since some of them were also mapped in other pine species, an association of several linkage groups from our reference population with those of other published maps was possible. The summarized results are shown in table II. 4. DISCUSSION 4.1. The generation of segregating polymorphic DNA markers The pine genome is known to be relatively large and con - tains large amounts of repetitive elements [24]. Thus a highly increased number of AFLP amplification products can be ex - pected. It is also well known that increased AT contents in the selective nucleotides leads to a higher number of amplifica - tion products. However, the resolution of the gel is limited so that different amplification products may comigrate, hiding in this way possible segregating polymorphisms. Therefore using PCs with lower AT content and/or increasing the num - ber of selective nucleotides in the primers to 4, potentially re - sults in less amplification products, but in higher segregating polymorphisms of variable number of bands with good qual - ity. UHD linkage map of Pinus pinaster 639 Table I. Characteristics of the P. pinaster maps from the cross 0024 × C803. P1 – MAP (0024) P2 – MAP (C803) Integrated Map LG Length IM CM TM Length IM CM TM Length IM CM TM AP 1 160.4 35 9 44 173.3 27 10 37 175.6 62 12 74 7 2 153.2 28 12 40 171.1 28 10 38 168.9 56 13 69 9 3 131.8 26 9 35 174 25 6 31 176.5 51 9 60 6 4 151.2 27 10 37 190.5 23 7 30 175 50 11 61 6 5 146.3 27 8 35 187.3 30 7 37 179.3 57 11 68 4 6 107.8 20 6 26 153.7 19 4 23 151.5 39 6 45 4 7 121.5 24 7 31 116.7 23 6 29 123.2 47 8 55 5 8 135.3 22 7 29 130.5 22 5 27 142.5 44 8 52 4 9 180.1 38 8 46 181.5 27 8 35 191 65 10 75 6 10 148.7 33 7 40 115.1 24 7 31 156.5 57 9 66 5 11 149.2 27 8 35 175.1 24 12 36 182.8 51 13 64 7 12 150.3 28 7 35 172.7 32 9 41 171.4 60 10 70 6 Total: 1735.8 335 98 433 1941.5 304 91 395 1994.2 639 120 759 70 Mean: 144.7 27.9 8.2 36.1 161.8 25.3 7.6 32.9 166.2 53.3 10 63.3 5.8 + 217 markers linked with recombination frequencies of zero to other markers + 206 associated fragments: Total Markers: 1182 Legend: LG = linkage group; IM = individual markers (parent specific); CM = markers common to both parents; TM = total number of markers for linkage group; AP = number of anchor points. 640 E. Ritter et al. 8/2 77/11 8.4 148/8 18.7 222/10 19.9 39/6 221/5 240/3 22.5 96/1 27.5 268/7 30.0 229/4 33.4 NZPR386a 35.1 RSO1D5 56/10 152/4 35.2 152/18 38.3 239/3 39.2 NZPR823 43.8 140/5 44.5 157/4 158/7 57/9 189/6 49.1 58/8 53.1 111/4 54.6 156/3 62.5 138/2 64.6 76/5 75.0 149/14 75.9 157/2 80.9 228/2 84.3 159/2 239/1 86.1 244/4 89.7 62/5 94.0 92/9 96.1 215/7 96.9 216/7 101.0 78/7 101.9 152/14 198/2 104.0 14/7 105.7 149/3 107.8 212/10 110.6 66/11 111.4 31/1 152/15 116.7 213/2 122.9 59/1 64/1 147/5 126.0 63/8 131.6 ASO1F3 136.1 225/2 138.2 NZPR0413 140.1 182/2 140.5 237/3 142.8 263/4 152.5 190/9 153.5 23/8 35/14 153.6 143/5 154.5 183/2 155.6 185/3 157.7 149/8 158.3 255/5 159.8 156/2 160.2 150/2 163.0 54/18 164.1 143/9 165.8 92/8 165.9 4/2 168.7 77/4 175.6 8/23 0.0 257/5 7.3 62/6 15.4 69/5 18.1 221/7 21.9 148/1 24.6 68/1 25.8 58/13 150/1 179/8 42.8 61/4 264/9 44.0 73/1 49.2 151/14 52.2 253/6 52.4 229/3 55.0 171/7 57.5 183/4 61.4 159/15 61.9 191/1 63.4 35/17 63.8 138/3 146/3 66.2 105/5 67.5 157/3 72.1 45/7 146/9 72.4 155/5 NZPR1702a 73.5 54/15 66/2 73.6 212/6 75.8 254/8 82.2 164/2 89.2 137/5 250/3 89.3 35/18 91.8 164/3 95.8 159/13 97.0 78/1 103.3 77/13 106.7 54/2 81/1 108.3 8/5 92/11 57/7 110.4 107/6 112.2 139/1 113.7 148/5 120.0 14/5 182/6 120.1 267/2 126.6 53/2 130.6 77/1 135.8 29/3 158.6 52/2 162.5 33/10 163.8 ITPH4516a 166.3 40/12 172.7 188/7 176.5 109/9 0.0 Lg:1 10/2 2.8 39/3 228/4 224/2 5.7 216/2 9.4 241/5 22.3 238/4 25.2 152/12 158/12 25.3 NZPR006 27.5 56/11 36.5 212/7 43.1 54/10 234/4 56/12 58/11 52.1 23/4 23/7 150/9 54.0 RTPEST11 220/2 55.9 54/7 56.7 11/1 60.1 40/9 236/8 64.5 92/6 65.6 257/10 74.6 216/6 78.9 151/16 80.5 222/8 82.0 8/6 84.1 216/3 88.4 181/3 254/4 89.4 145/6 93.8 235/1 96.5 52/5 96.7 241/2 101.2 59/4 102.7 33/8 104.2 28/1 104.8 139/2 139/4 105.9 146/5 108.6 -E28072 109.5 54/6 23/6 113.2 54/1 116.1 230/11 117.6 244/9 122.1 63/11 159/9 241/8 123.4 71/5 124.7 156/4 129.6 31/3 130.6 231/8 132.1 56/8 232/6 136.1 244/11 136.3 188/4 142.4 61/7 143.8 -E87909 144.1 242/6 152.2 99/4 156.9 22/2 168.9 31/2 0.0 Lg:2 Lg:3 245/1 0.0 179/13 1.6 56/9 10.8 20/5 222/3 228/6 14.7 ITPH4516b 27.5 152/2 145/3 27.6 60/3 29.6 237/1 30.6 142/6 153/3 30.7 76/20 37.8 100/8 40.5 97/1 41.3 140/3 42.6 245/5 50.1 175/5 58.8 245/8 59.5 34/2 60.4 56/3 151/4 61.5 NZPR386b 70.1 231/7 75.2 76/21 81.9 215/13 83.1 148/4 84.0 58/10 191/3 84.5 190/11 85.3 147/16 85.8 195/11 89.0 112/8 91.9 159/14 100.7 7/8 59/6 79/1 255/7 108.7 189/3 112.8 232/2 118.6 98/2 132.0 111/13 133.8 222/2 138.5 143/10 142.3 57/4 144.5 34/4 145.8 92/2 147.1 142/8 154/3 147.4 8/1 148.7 58/5 151.3 75/6 154.1 23/1 111/2 157.1 106/8 161.9 35/22 167.3 109/3 167.6 145/5 175.0 Lg:4 FRPP94a 0.0 221/3 5.1 267/12 11.7 167/1 18.1 145/4 19.7 92/4 149/1 31/4 21.1 58/1 23.0 PR092a 25.0 236/4 27.0 241/3 28.0 53/6 29.9 FRPP94b 31.9 238/1 40.0 41/8 58/2 75/3 150/4 43.8 111/14 49.6 255/6 53.9 189/2 54.6 241/4 58.8 66/7 112/3 62.9 187/5 78.1 169/3 81.7 231/4 82.3 56/7 231/3 86.2 256/1 90.7 152/7 93.0 PPA8 241/9 96.2 8/3 198/5 96.3 159/8 97.7 157/5 99.2 162/4 99.8 181/2 103.5 236/6 105.2 60/2 115.1 63/2 230/12 115.7 158/14 126.0 253/5 127.2 53/4 129.6 35/6 59/9 129.7 105/2 130.8 171/8 131.3 240/10 135.7 189/5 137.2 148/2 144.6 151/6 229/2 144.7 8/22 148.5 182/3 150.4 152/1 150.5 104/11 230/5 151.8 249/2 156.1 30/8 254/3 163.3 142/4 148/6 165.7 97/3 179.3 Lg:5 145/2 0.0 222/6 1.2 69/6 11.0 271/2 14.9 52/8 26.2 222/9 27.5 50/1 31.2 147/1 31.6 233/1 34.2 152/10 35.5 166/1 36.9 61/8 40.0 69/2 60/6 43.2 157/1 44.2 39/1 239/5 46.0 170/2 257/6 17/5 37/1 48.1 92/10 50.2 3/1 52.4 152/6 54.5 267/9 59.4 62/4 62.1 188/1 66.6 240/2 74.5 8/9 80.8 252/4 82.3 215/1 86.0 63/9 88.2 NZPR1078 143/8 149/13 94.6 181/4 95.9 32/4 63/3 97.3 105/8 100.0 67/2 106.6 255/2 109.6 224/5 111.8 107/9 120.5 13/4 139.9 76/4 151.5 Lg:6 UHD linkage map of Pinus pinaster 641 241/11 0.0 77/8 2.7 54/4 13.7 76/14 14.3 188/3 21.1 170/5 22.7 171/1 24.0 52/4 171/2 29.6 169/1 32.2 175/4 34.9 145/7 36.3 182/1 230/7 232/1 39.1 40/3 40.4 198/4 41.7 152/8 43.2 58/7 46.6 76/9 47.3 148/10 49.8 218/2 51.5 4/1 12/6 55.9 252/2 62.5 158/1 68.8 154/2 73.8 76/12 79.8 114/10 82.7 55/2 76/6 83.9 39/7 108/9 ISSR9 138/4 239/6 87.0 188/2 245/7 88.8 58/3 66/1 93.6 253/2 98.3 242/4 100.6 143/13 221/2 234/1 103.0 189/1 104.3 149/6 149/7 105.6 63/6 106.9 151/8 109.3 241/10 110.6 68/4 111.1 30/5 112.6 104/5 117.3 214/6 123.2 Lg:7 257/9 0.0 80/1 153/4 9.7 147/2 220/5 15.2 77/12 17.1 57/10 19.2 73/3 25.5 56/4 59/5 143/3 39/2 28.3 255/4 30.6 76/11 37.8 76/8 39.7 35/20 146/2 41.7 55/4 44.3 34/5 46.9 145/8 50.3 144/2 52.9 75/1 54.8 102/1 56.7 102/4 57.4 106/2 59.7 8/4 39/2 198/12 65.7 90/3 68.4 253/4 68.6 158/10 71.5 212/15 73.8 190/13 74.2 112/7 85.7 7/2 98.5 101/14 99.4 142/12 230/16 101.2 230/17 102.3 155/4 103.0 8/10 104.4 180/4 106.8 190/10 109.0 69/1 117.3 181/6 120.4 147/11 127.0 147/17 130.5 33/1 158/3 146/7 132.2 35/1 140.0 17/4 142.5 Lg:8 198/9 0.0 66/3 4.9 143/17 149/9 6.3 232/4 10.4 147/12 16.4 30/6 173/4 23.0 155/7 25.6 104/1 26.5 147/20 31.8 92/7 147/6 32.9 249/3 33.1 150/8 229/7 35.1 231/6 37.1 146/12 45.7 35/12 47.4 PR092b 52.6 78/2 57.4 190/5 58.0 147/18 63.0 164/1 63.2 33/11 234/5 65.0 154/6 71.9 14/6 72.9 57/6 257/7 79.6 254/5 83.3 4CL 85.5 142/7 158/9 87.3 225/3 91.1 139/3 94.9 143/2 98.9 142/3 236/7 99.3 54/12 104.0 155/1 105.0 159/6 109.3 55/3 110.1 190/4 111.1 61/5 112.9 76/19 115.5 57/3 120.0 185/2 122.8 178/4 124.8 241/6 126.8 254/6 127.7 190/6 131.5 262/1 135.1 53/3 136.9 80/6 140.6 139/6 143/7 146.6 142/1 148.4 10/1 111/5 149.4 143/6 150.1 159/12 228/8 153.4 35/4 160.0 63/4 160.1 33/2 54/16 161.3 253/7 164.2 234/8 166.4 183/3 171.0 141/1 171.9 59/12 173.2 6/4 179.2 90/2 183.4 65/1 191.0 Lg:9 57/11 0.0 154/5 3.7 222/4 10.0 158/6 11.3 92/3 13.8 152/9 18.9 90/1 171/3 25.6 244/5 36.7 159/1 39.1 172/4 44.1 80/8 236/5 48.3 144/1 49.8 59/8 50.5 165/1 54.2 152/16 152/17 216/1 218/3 220/1 57.0 158/11 63.3 156/6 63.7 80/9 69.8 147/14 75.6 8/25 77.3 8/14 194/6 79.0 141/3 80.9 7/4 81.4 105/3 81.9 108/2 82.9 162/3 86.1 4/6 167/5 86.2 41/4 93.6 59/11 225/5 95.2 181/7 97.4 143/4 99.6 169/6 100.6 151/9 111.3 173/6 111.5 182/5 114.6 146/1 117.9 102/3 142/10 122.4 49/2 123.8 228/3 125.8 172/6 127.5 195/9 128.1 261/2 129.5 152/11 130.7 259/3 130.9 58/9 132.0 235/3 133.2 191/2 134.0 65/4 134.5 262/3 135.8 63/1 138.2 147/10 140.1 34/3 141.4 101/12 143.4 60/7 252/1 149.4 30/1 156.5 Lg:10 23/5 0.0 60/5 10.0 150/6 65/2 16.4 103/8 17.9 171/4 19.8 151/10 27.2 32/1 32/2 29.8 222/7 30.0 148/9 32.3 100/1 34.7 66/6 141/2 153/1 35.9 136/1 45.4 266/1 46.4 40/10 146/15 48.5 56/1 52.7 236/3 59.7 100/2 63.3 80/10 65.2 153/2 194/1 83/4 230/8 69.8 188/6 70.8 82/1 120/6 73.1 14/8 105/7 78.4 8/17 79.7 160/3 94.0 59/10 95.9 68/2 100/5 107/2 148/7 97.8 236/2 99.3 242/3 109.3 101/8 110.7 225/1 112.7 66/9 113.8 222/1 168/3 234/6 118.9 208/1 120.2 FRPP91 127.3 68/3 221/6 4/5 71/4 143.9 227/1 144.6 7/5 146.8 63/7 69/8 NZPR472 155/2 150.6 54/17 151.9 171/5 157.1 80/7 166.0 158/2 167.4 180/6 182.8 Lg:11 104/9 0.0 103/2 11.4 7/12 17.0 244/13 18.5 149/10 19.9 35/19 21.2 150/7 23.7 140/2 24.9 7/10 139/8 241/7 26.3 97/4 27.5 17/7 28.2 187/3 31.0 35/21 32.0 83/3 38.2 76/1 39.8 236/1 41.2 140/1 238/3 41.9 66/10 RPS-160 159/3 46.0 53/7 57/8 47.9 138/5 50.9 149/12 143/11 147/19 58.0 234/2 59.1 147/15 66.8 212/8 71.2 253/3 76.5 249/1 82.8 254/1 84.1 239/4 86.5 176/3 86.8 240/8 87.6 145/9 89.9 105/6 91.9 31/6 146/10 92.3 184/4 93.4 240/6 99.2 187/6 105.8 212/4 110.5 NZPR1702b 112.9 AF028073 116.6 241/1 118.1 151/12 118.7 150/5 122.1 188/5 122.7 143/16 125.5 63/5 125.9 59/3 128.9 224/1 134.8 146/13 137.1 158/13 158/16 144.9 89/1 151.6 230/1 151.7 33/12 58/6 152.9 238/2 154.2 250/1 260/1 156.7 190/7 159.4 216/4 163.6 101/13 168.2 209/6 171.4 Lg:12 Figure 1. Integrated map of Pinus pinaster Cross: 0024 × C803. Positions in cM [Kosambi units] are given on the left of the linkage group bars. Marker names and fragment numbers on the right of the bar. Markers common to both parents are underligned. Most of them represent anchor points. EST and SSR markers are indicated in bold. For details on marker names and fragment numbers including parental AFLP profiles see http://www.neiker.net/UHDfor. Independently of these findings, the unexpected low de - gree of polymorphism of AFLP, SSR and EST markers ob - served in our progeny is surprising considering the well marked differentiation between the original provenances of the parents [14] and the similar level of genetic diversity en - countered in P. pinaster and other Pinus species [9, 20]. Many polymorphic fragments exist between the parents of our mapping population, which represent different ecotypes from Landes and Corsica, respectively. However, a large de - gree of homozygosity exists, since parent specific fragments do not segregate. This increased homozygosity is probably due to a low degree of biodiversity, which exist at the specific sites (i.e., trees are quite different between sites but very sim - ilar within a site). 4.2. Arrangement of DNA markers into linkage maps The analysis of segregating DNA markers established twelve independent linkage groups for the P. pinaster geno - types 0024 and C803, respectively (i.e., lateral markers were not statistically linked to any other lateral marker of any other linkage group). These 12 linkage groups may correspond to the 12 chromosomes of the haploid pine genome (2n = 2x = 24). Moreover, the presence of common markers made it possible to identify all homologous chromosomes in each parent. With several common markers present in the same order on chro - mosomes of both parents, it is possible to combine the infor - mation of markers from different individuals as described in [17]. In this way the number of markers available per chro - mosome can be increased. The total length of linkage maps did not differ between the parents of the mapping population and is in agreement with other linkage maps obtained in this species. Our P. pinaster reference map represents one of the maps with the highest number of markers in forest species. 4.3. Alignment with other Pinus maps Alignment between different linkage maps can be achieved, if identical markers have been used in these maps 642 E. Ritter et al. Table II. Locations of mapped SSR and EST markers in our P. pinaster reference map and in other published Pinus maps. No Name Type Origin Location in reference map Location in other published maps 1 4CL EST P. taeda Lg 9 P. abies Lg6 (1) 2 AFO28073 EST P. taeda Lg 12 P. pinaster Lg8 (2) 3 ASO1F3 EST P. pinaster Lg 1 P. pinaster Lg4 (2) P. abies Lg4 (1) 4 E28072 EST P. taeda Lg 2 P. pinaster Lg5 (2) 5 E87909 EST P. taeda Lg 2 P. pinaster Lg8 (2) 6 PPA8 EST P. pinaster Lg 5 P. pinaster Lg10 (2) 7 RSO1D5 EST P. pinaster Lg 1 P. pinaster Lg7 (2) 8 FRPP91 SSR P.pinaster Lg 11 P. pinaster Lg9 (3) 9 FRPP94a/b SSR P.pinaster Lg 5/Lg 5 P. pinaster Lg5 (3) 10 ISSR9 ISSR Lg 7 – 11 ITPH4516a/b SSR P.halepensis Lg 3/Lg 4 P. pinaster Lg3 (3) 12 NZPR0413 SSR P. radiata Lg 1 P. pinaster Lg4 (2) P. radiata Lg4 (4) 13 NZPR1702a/b SSR P. radiata Lg 12/Lg 3 P. pinaster Lg8/Lg11(2) P. radiata Lg10 (4) 14 NZPR1078 SSR P. radiata Lg 6 P. pinaster Lg2 (2) P. radiata Lg2 (4) 15 NZPR386a/b SSR P. radiata Lg 1/Lg 4 P. radiata Lg2 (4) 16 NZPR472 SSR P. radiata Lg 11 P. pinaster Lg1 (2) P. radiata Lg1 (4) 17 NZPR006 SSR P. radiata Lg 2 P. radiata Lg5 (4) 18 NZPR823 SSR P. radiata Lg 1 P. pinaster Lg5 (2) P. radiata Lg5 (4) 19 PR092a/b SSR P. radiata Lg 5/ Lg 9 P. radiata Lg3 (5) 20 RPS-160 SSR P.strobus Lg 12 – 21 RTPEST11 SSR P. taeda Lg 2 P. pinaster Lg5(2) (1) http://www.pierroton.inra.fr/genetics/Picea/ (2) http://www.pierroton.inra.fr/genetics/pinus/primers.html and Chaumeil P., Développement de marqueurs hypervariables (microsatellites) chez le pin maritime (Pinus pinaster Ait.) et ap- plications en génétique, 2001, DEA Biologie Forestière, Université de Nancy (several markers are only cited in the DEA but will be published on this web site). (3) Mariette et al., 2001. (4) P. radiata map aligned with P. taeda reference population [1]; Phil Wilcox and Craig Echt, personal communication. (5) Devey et al., 1999. and if comigrating bands map to identical positions. SSR and EST markers are mainly codominant, highly polymorphic and represent powerful tools for different genetic analyses. Since they seem to be conserved among species and to a cer - tain degree also within families, they have been used for map - ping and alignment of linkage maps in several forest species [1, 3, 8, 13]. We have evaluated numerous SSR and EST markers in our study and several could be used to associate linkage groups in different parents (table II). However, the low level of polymorphism of EST and SSR markers ob - served in our reference population has led to association of linkage groups between maps. Since this goal is crucial for the usefulness of our map, additional SSR/EST primers will be evaluated in order to achieve a complete alignment. 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Ritter et al.UHD linkage map of Pinus pinaster Original article Towards construction of an ultra high density linkage map for Pinus pinaster Enrique Ritter a* , Ana Aragonés a , Torsten. breeding of coniferous species a project has been initiated with the aim of constructing an ultra- high- density linkage map (UHD map) of Pinus pinaster based on several thousands AFLP markers and numerous. one or more segregating bands. 3.2. Construction of linkage maps Initially, individual linkage maps of 12 linkage groups each were obtained for the two parents of the mapping popu - lation. Their