Molecular Breeding 13: 251–261, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
251
An integrated genetic linkage map of pepper (Capsicum spp.) Ilan Paran1,5,*, Jeroen Rouppe van der Voort2,5, Véronique Lefebvre3,5, Molly Jahn4,5, Laurie Landry4, Marco van Schriek2, Bahattin Tanyolac1, Carole Caranta3, Arnon Ben Chaim1, Kevin Livingstone4, Alain Palloix3 and Johan Peleman2 1Department
of Plant Genetics and Breeding, Agricultural Research Organization, The Volcani Center, Bet Dagan, P. O. Box 6, 50250, Israel; 2Keygene Genetics, P. O. Box 216, 6700 AE Wageningen, The Netherlands; 3Institut National de la Recherche Agronomique (INRA), Unité de Génétique et d’Amélioration des Fruits et Légumes, Domaine Saint-Maurice, BP94, F84143 Montfavet Cedex, France; 4Department of Plant Breeding, Cornell University, Ithaca NY 14853, USA; 5These authors contributed equally to the work described in this paper; *Author for correspondence (e-mail:
[email protected]; fax: 972-3-9669642) Received 24 January 2003; accepted in revised form 31 October 2003
Key words: Capsicum, Integrated linkage map, Molecular markers
Abstract An integrated genetic map of pepper including 6 distinct progenies and consisting of 2262 markers covering 1832 cM was constructed using pooled data from six individual maps by the Keygene proprietary software package INT_MAP. The map included: 1528 AFLP, 440 RFLP, 288 RAPD and several known gene sequences, isozymes and morphological markers. In total, 320 anchor markers 共common markers in at least two individual maps兲 were used for map integration. Most anchor markers 共265兲 were common to two maps, while 27, 26 and 5 markers were common to three, four and five maps, respectively. Map integration improved the average marker density in the genome to 1 marker per 0.8 cM compared to 1 marker per 2.1 cM in the most dense individual map. In addition, the number of gaps of at least 10 cM between adjacent markers was reduced in the integrated map. Although marker density and genome coverage were improved in the integrated map, several small linkage groups remained, indicating that further marker saturation will be needed in order to obtain a full coverage of the pepper genome. The integrated map can be used as a reference for future mapping studies in Capsicum and to improve the utilization of molecular markers for pepper breeding.
Introduction Saturated genetic maps consisting of molecular markers were constructed in recent years for most crop plants 共Paterson et al. 2000兲. These maps are being used for the determination of synteny relationships, gene tagging, marker-assisted selection and for gene cloning. For pepper 共Capsicum spp.兲, several genetic maps have been published in the past 15 years. Most maps were constructed using F2, BC1 or doubled haploids 共DH兲 populations from interspecific crosses of C. annuum x C. chinense or from intra-specific crosses of C. annuum and were based
on RFLP, RAPD and AFLP markers. The first interspecific map with a wide genome coverage that contained 85 loci was constructed by Tanksley et al. 共1988兲. A later study using a cross with the same C. chinense parent and a different C. annuum parent allowed the construction of a more detailed map containing 192 markers 共Prince et al. 1993兲. The most comprehensive pepper-tomato comparative map was constructed by Livingstone et al. 共1999兲, who mapped over 1000 loci in 13 linkage groups using the same cross as in Prince et al. 共1993兲. In addition to RFLP markers, PCR-based markers, mostly AFLP, were added to the map 共Livingstone et al. 1999兲. Another
252 map from a C. annuum x C. chinense cross was created by Kang et al. 共2001兲, which contained mostly AFLP markers and pepper-based RFLP markers. The use of only a few markers common to the other published maps, did not allow the alignment of the latter map with the previous ones. Most recently, a BC2 population from a cross of C. annuum x C. frutescens was used to construct an RFLP-based map of 92 markers 共Rao et al. 2003兲. In addition to these inter-specific maps, several intra-specific C. annuum maps have been reported. An integrated map containing 85 markers derived from three partial maps constructed with DH lines was reported by Lefebvre et al. 共1995兲. Most recently, an update of the C. annuum map constructed from the integration of three individual maps containing 543, 630 and 208 marker loci each, including RFLP, AFLP and known function genes was reported by Lefebvre et al. 共2002兲. Lastly, an intra-specific C. annuum map from the cross of Perennial and Maor, which consists of 177 AFLP and RFLP markers was constructed by Ben Chaim et al. 共2001a兲. The maps described above have been used mostly to map disease resistance genes 共e.g., Ben Chaim et al. 2001b; Caranta et al. 1997; Grube et al. 2000; Jahn et al. 2000; Lefebvre and Palloix 1996; Thabuis et al. 2003兲 and for mapping of quantitative trait loci 共QTLs兲 for fruit-related traits such as fruit weight and fruit shape 共Ben Chaim et al. 2001a; Rao et al. 2003兲 and color genes 共Kang et al. 2001; Lefebvre et al. 1998; Thorup et al. 2000兲. Because each map was composed of a different marker set, the ability to extrapolate linkage information from population to population was limited. The creation of a single resource by the integration of these maps will greatly enlarge the repertoire of markers for any mapping project in Capsicum. Similar maps have been reported recently for several crop species such as Brassica oleraceae 共Sebastian et al. 2000兲, Brassica napus 共Lombard and Delourme 2001兲, sunflower 共Gedil et al. 2001兲, lettuce 共Jeuken et al. 2001兲, tomato 共Haanstra et al. 1999兲 and barley 共Qi et al. 1996兲. In this study we describe the integration of six pepper maps including previously unpublished mapping data into a single Capsicum map that contains 2262 markers. This integrated map will serve as a foundation for further studies of genome structure and function in this genus.
Materials and methods Mapping populations Six mapping populations were used to construct the integrated map 共Table 1兲. One map 共coded EGOD兲 done at Cornell University was based on an inter-specific F2 cross of C. annum Nu Mex RNaky x C. chinense PI 159234 共Livingstone et al. 1999兲. Two maps developed at the Volcani Institute were based on an intra-specific F2 derived from C. annuum cv Maor x C. annuum cv Perennial 共Ben Chaim et al. 2001a兲 and from an inter-specific BC1 cross of C. annuum 100/63 x C. chinense PI 152225 共EGOT and EGOE, respectively兲. Three intra-specific C. annuum maps were contributed by INRA 共Lefebvre et al. 2002兲. The first map 共HV兲 was constructed from a DH population from the cross of H3 and Vania, the second 共PY兲 from a DH population from the cross of Perennial and Yolo Wonder and the third 共YC兲 from an F2 population from the cross of Yolo Wonder and Criollo de Morelos 334 共CM334兲. The codes for the Cornell and Volcani maps are internal Keygene ones; the codes for the INRA maps are the same as in Lefebvre et al. 共2002兲. Map integration Maps were integrated using the Keygene proprietary software package INT_MAP 共Peleman et al. 2000兲. The software integrates separate genetic linkage maps into a single consensus map. As is the case for input files, drawmap files 共.dm1 files; Van Ooijen 1994兲 provide the program with data on marker orders and pair-wise genetic distances. Map integration consisted of three separate steps. Firstly, common markers between at least two maps were defined as anchor markers and were used to link corresponding linkage groups on the individual maps. Secondly, the consensus order of the anchor markers was calculated from their relative positions on each individual map. The consensus sequence begins with the most common marker and adds the most proximate, most common markers one at a time. The consensus position was calculated as a weighted average of the individual map distances between new markers and previously positioned markers. Two variables should be set prior to map integration: the minimum fraction of overlap between the groups 共e.g., three out of 10 anchor markers present on linkage group 1兲 and the maximum allowed disagreement in cM-distance be-
a ⫽ Livingstone et al. 1999; b ⫽ unpublished data; c ⫽ Ben Chaim et al. 2001a; d ⫽ Lefebvre et al. 2002. 1
15 2262 288 440 1528 Integrated map
YC
EGOE EGOT HV PY
3
3
13
1832
d 736 21 183 93 151 F2
52
38
–
–
13
b c d d 1559 1264 1086 1294 50 32 27 23 13 13 15 13 397 405 490 616 – 3 – – – 4 66 203 308 331 389 320 83 BC1 180 F2 98 DH 94 DH
89 67 35 92
2 17
NuMex RNaky ⫻ PI159234 100/63 ⫻ PI 152225 Maor ⫻ Perennial H3 ⫻ Vania Perennial ⫻ Yolo Wonder Yolo Wonder ⫻ CM334 EGOD
350 Parents Code
75 F2
303
– – – 1
672 –
14
Total No. markers No. Morphol. No. Isozyme No. RAPD No. RFLP Type and pop. No. size AFLP Cross
Table 1. List of the attributes of the six populations and maps used to construct the integrated map.
No. linkage groups
43
No. gaps ⬎ 10 cM
1439
Map length 共cM兲
a
Ref.1
253 tween two adjacent anchor markers. For the pepper integrated map, the latter two variables were set to 0.30 and 10 cM, respectively. Common markers which deviated from these criteria were excluded from map integration. Thirdly, all unique markers were positioned on the integrated map by means of intra- and extrapolation. The accuracy of their estimated position is limited to the interval between the nearest anchor markers from the respective individual map. A detailed description of the map integration process is given in the Appendix. Readers who are interested to apply the software for map integration are requested to contact Keygene at
[email protected]. The marker names in the integrated map are identical to the names used in the original maps. Mostly RFLP and AFLP markers were used as anchor markers. Common RFLP markers were identified by the use of the same clone name, while common AFLP markers were identified by the use of the same primer/enzyme combination and an identical mobility of the anchor marker on reference gels that included AFLP fragments from the parents of the individual maps.
Results and discussion The pepper integrated map is composed of a total of 2262 markers, mostly AFLP 共1528兲, tomato and pepper RFLP 共440兲, RAPD 共288兲 and a few isozymes and morphological markers 共Table 1, Figure 1兲. These markers are distributed in 13 linkage groups with a total map length of 1832 cM. Linkage group 7 defined by other published maps was split into two subgroups that could not be integrated because of a lack of common markers. Sub-group 7a included markers from the C. annuum x C. chinense EGOD and EGOE crosses, while sub-group 7b included markers from the other four C. annuum intra-specific crosses. Despite the integration of markers from six different maps, several small linkage groups from the individual maps remained unlinked after integration. These included linkage group B of 12 cM in EGOD, one linkage group of 20 cM in EGOE, three linkage groups of 15, 20 and 40 cM in HV and one linkage group of 16 cM in YC. Chromosomes 1 and 8 could not be separated from each other in the C. annuum x C. chinense EGOD map because of pseudolinkage as a result of reciprocal translocation involving these two chromosomes 共Livingstone et al. 1999兲. In the C.
254
Figure 1. Integrated genetic linkage map of pepper. Only anchor markers 共common markers between at least 2 individual maps兲 are shown. Map distances are based on the complete integrated map 共www.plbr.cornell.edu/psi; www.keygene-genetics.com/html/public_maps.htm兲. Marker types are: tomato RFLP clones 共TG, CT, CD兲, pepper RFLP clones 共PG兲, AFLP markers named according to the primer combination followed by the size of the amplified fragment, RAPD markers labeled according to the primer name followed by size of the amplified fragment, and known function genes 共GC兲. Letters behind RFLP loci names represent multiple bands per RFLP probe.
255
Figure 1. Continued.
256 annuum intra-specific EGOT map, chromosome 8 appeared as a large distinct linkage group 共Ben Chaim et al. 2001a兲. However, the top part of the chromosome including 12 markers that span a 66 cM region from TG176 to E48/M48-F-206 in EGOT 共Ben Chaim et al. 2001a兲 was not included in the integrated map because we could not obtain an unequivocal assignment of this segment to either chromosome 8 or chromosome 1. Similarly, most of the linkage group P8 that was unlinked to P1 in the intraspecific HV, PY and YC crosses 共interval GC36 to TG 281, Lefebvre et al. 2002兲, was integrated into the P1 chromosome from interspecific crosses in the present map. Therefore, chromosome 8 remained the smallest chromosome in the integrated map and included only 46 markers that covered a distance of 62 cM. Secure identification and splitting of pepper chromosomes 1 and 8 will require the separate intraspecific mapping within C. annuum and C. chinense of a larger set of common markers. Map integration improved the coverage of the genome as was demonstrated by comparing the number of gaps of at least 10 cM between adjacent markers in the individual and in the integrated maps 共Table 1兲. The integrated map contained only 15 gaps while in individual maps, the number of gaps ranged from 21 to 50 per map. It is noted that the YC map which showed the lowest number of gaps, covers only part of the pepper genome 共map length of only 736 cM兲. The total length of the integrated map 共1832 cM兲 was greater than the length of each of the individual maps 共Table 1兲. For some chromosomes 共e.g., 6, 7 and 9兲, map expansion occurred by adding markers within the chromosome and due to larger distances in intraspecific crosses. However for most other chromosomes 共e.g., 2, 4, 5, 10, 11 and 12兲, the distal parts of the most populated individual map 共EGOD兲 were extended by adding markers from other crosses. Similar map expansion due to the addition of markers to the distal parts of three individual maps was observed in the consensus map of Brassica napus 共Lombard and Delourme 2001兲. Single copy markers such as RFLP are the most robust markers for map integration. The validity of using co-migrating multi-locus AFLP markers for alignment of genetic maps has been proved in barley 共Waugh et al. 1997兲 and potato 共Rouppe van der Voort et al. 1997兲. Furthermore, the possibility of introducing a mapping error because of including nonorthologous co-migrating AFLP markers was minimized in the current study because such markers were
Table 2. Common markers among individual maps used for map integration. Population
EGOE
EGOT
HV YC PY Total
EGOD EGOE EGOT HV YC PY
76
56 40
18 24 77
18 5 19 28
32 11 49 60 64
129 109 161 140 75 133
discarded according to the threshold setting in the integration procedure. Therefore, in addition to common RFLP markers, AFLP markers with the same mobility were used to anchor the pepper maps. In total, 320 markers were available for map integration; these included 172 AFLP markers that were amplified by 35 common primer combinations and 148 RFLP markers 共Figure 1兲. Because of space limitations, only the anchor markers are included in Figure 1; the complete integrated map can be viewed at www.plbr.cornell.edu/psi or at www.keygene-genetics.com/ html/public_maps.htm. The majority of anchor markers 共265兲 were common to two maps, while 27, 26 and 5 markers were common to three, four and five maps, respectively. The number of common markers for each pair of the individual maps ranged from 5 to 77 and the total number of common markers used for integration from each cross ranged from 75 to 161 共Table 2兲. Thirty-nine markers were not included in the integrated map because of erroneous grouping or major linkage position discrepancies. These discrepancies could result from reasons such as scoring errors, co-migration of different loci and mapping of different members of gene families. The average marker density of the integrated map is 1 marker per 0.8 cM. This density is improved compared to 1 marker per 2.1 cM in the most dense individual maps 共EGOD and PY兲. However, the distribution of the markers along the chromosomes was unequal due to clustering of markers in the putative centromeric regions as was observed most clearly in the EGOD map 共Livingstone et al. 1999兲. In each chromosome, 15-86% of the markers were concentrated in one major cluster. The most extreme clustering occurred in chromosome 11 where 224 out of 261 markers 共86%兲 were located within a single 20 cM region. Similar centromeric clustering, in particular of AFLP markers, has been observed in genetic maps of other plants such as maize, tomato and barley 共Vuylsteke et al. 1999; Haanstra et al. 1999; Qi et al. 1998兲.
257 Conclusions
on the basis of presence/absence of markers but also indicate where differences between lines are located in the genome. Additional marker saturation for the remaining unlinked regions by methods such as bulked segregant analysis 共Michelmore et al. 1991兲 will be required to join these groups to the integrated map.
The integrated map of pepper allows the improved availability of molecular markers for applications in breeding and genome analysis because of better coverage. The integrated map extends linkage groups defined by each of the individual maps and reduces gaps within these maps. Furthermore, integrating a large number of different marker types into a single map increases the opportunity to find informative markers in any part of the genome in various backgrounds. The present map that contains mostly anonymous AFLP and RFLP markers can be used as a reference for future mapping studies in Capsicum in which SSR, SNP and known genes will be added. Furthermore, integration of a large number of AFLP loci in the map that are amplified by few primer combinations allows the comparison of breeding lines not only
Acknowledgments This research was partly supported by The United States–Israel Binational Agricultural Research and Development Fund Grant No. IS- 3225-01C, USDA IFAFS Award No. 2001-52100-113347 and by the Association Nationale de la Recherche Technique, France, grant CIFRE No 99001.
Appendix Table A1. An example is given for the top of chromosome 1 where the first five anchor markers are given together with the original linkage group name 共group兲 and position 共pos兲 on the six individual maps as well as their averaged position and deviation from their positions on their original maps eg0e
eg0d
eg0t
Marker
group
pos
TG24 E33/M49-F-530
# # eg0e00_1
eg0d00_1 20.6 eg0d00_1
E41/M49-F-290 E38/M61-F-81 CT131
group
pos
HV
group
pos
18.6 g0t00_1b 25.8 g0t00_1b
35.2 eg0d00_1
group
pos
group
PY pos
0.0 7.0 . 7.7 7.4
g0t00_1b g0t00_1b # eg0e00_1
YC
39.8
YC_8a
100.8
avg_pos 共sigma兲
group
pos
PY_1b
22.8 0.0 6.4
共 1.4兲 共 0.4兲
PY_1b PY_1b PY_1b
18.3 6.5 25.6 6.6 41.7 20.7
共 6.9兲 共 2.8兲 共 0.0兲
Table A2. Map integration starts with the most common marker, in this case marker CT131 that occurs in four populations. The corresponding position on their individual maps is taken as identical, thus having a standard deviation 共⫽sigma兲 of 0. The integrated map position 共avg_pos兲 of this marker initially becomes 0. Step 1 eg0e
eg0d
Marker
group
pos
group
pos
CT131
# eg0e00_1
35.2
eg0d00_1
39.8.
eg0t
HV
YC
PY
group pos
group pos
group
pos
YC_8a
100.8 PY_1b
group
pos
avg_pos
共sigma兲
41.7
0.0
共0.0兲
258 Table A3. In a next round marker E33/M49-F-530 is integrated because it occurs on three maps and it is closest to the already positioned marker CT131. On map EG0E and EG0D the distance is 14.6 and 14.0 respectively so the averaged map position becomes ⫺ 14.3 共and sigma ⫽ 0.4兲. Step 2 eg0e Marker
E33/M49-F-530 CT131
eg0d
eg0t
HV
group
pos
group
pos
group
pos
# eg0e00_1 # eg0e00_1
20.6 35.2
eg0d00_1 eg0d00_1
25.8 39.8
g0t00_1b
7.0
group
YC pos
group
PY pos
group
YC_8a 100.8
pos
PY_1b 41.7
avg_pos
共sigma兲
⫺ 14.3 0.4 0.0 共0.0兲
Table A4. Next marker TG24 is positioned on average position ⫺ 20.7 from three distances 7.2, 7.0 and 18.9 on maps EG0D, EG0T and PY respectively. The distance is calculated based on the proximate marker from the corresponding map i.e., marker E33/M49-F-530 for maps EG0D and EG0T and marker CT131 for map PY. The average is weighted by the distance from that proximate anchor marker. Step 3 eg0e Marker
group
eg0d pos
group
eg0t pos
group
HV pos
TG24
#
eg0d00_118.6
g0t00_1b 0.0
E33/M49-F-530 CT131
# eg0e00_120.6 6 # eg0e00_135.2
eg0d00_125.8 eg0d00_139.8
g0t00_1b 7.0
group
YC pos
group
YC_8a
PY pos
100.8
共sigma兲
group
pos
avg_pos
PY_1b
22.8
⫺ 20.7 共1.4兲
PY_1b
41.7
⫺ 14.3 共0.4兲 0.0 共0.0兲
Table A5. Next marker E38/M61-F-81 is considered. From map EG0T the position is calculated as ⫺ 14.3 ⫹ 0.4 ⫽ ⫺ 13.9. From map PY it gets ⫺ 20.7 ⫹ 2.8 ⫽ ⫺ 17.9. The weighted average now equals ⫺ 15.8. Step 4 eg0e Marker
group
eg0d pos
group
eg0t pos
TG24
#
eg0d00_118.6
E38/M61-F-81 E33/M49-F-530 CT131
# eg0e00_120.6 # eg0e00_135.2
eg0d00_125.8 eg0d00_139.8
group
HV pos
group
YC pos
group
PY 共sigma兲
group
pos
avg_pos
g0t00_1b 0.0
PY_1b
22.8
⫺ 20.7 共 1.4兲
g0t00_1b 7.4 g0t00_1b 7.0
PY_1b
25.6
PY_1b
41.7
⫺ 15.8 共2.8兲 ⫺ 14.3 共0.4兲 0.0 共0.0兲
YC_8a
pos
100.8
Table A6. Finally marker E41/M49-F-290 gets position ⫺ 13.9 ⫹ 0.3 ⫽ ⫺ 13.6 from map EG0T and ⫺ 20.7 ⫺ 4.5 ⫽ ⫺ 25.2 from map PY. The weighted average results in ⫺ 19.9. Step 5 eg0e Marker
TG24 E41/M49-F-290 E38/M61-F-81 E33/M49-F-530 CT131
group
eg0d pos
group
eg0t pos
#
eg0d00_118.6
# eg0e00_120.6 # eg0e00_135.2
eg0d00_125.8 eg0d00_139.8
group
HV pos
group
YC pos
group
PY pos
g0t00_1b 0.0 g0t00_1b 7.7 g0t00_1b 7.4 g0t00_1b 7.0 YC_8a
100.8
group
pos
avg_pos
共sigma兲
PY_1b PY_1b PY_1b
22.8 18.3 25.6
PY_1b
41.7
⫺ 20.7 ⫺ 19.9 ⫺ 15.8 ⫺ 14.3 0.0
共1.4兲 共6.9兲 共2.8兲 共0.4兲 共0.0兲
259 Table A7. Then the average positions are recalculated to positive figures and reshuffled. Step 6 eg0e Marker
eg0d
group
pos
eg0t
group
TG24 # E41/M49-F-290 E38/M61-F-81 E33/M49-F-530 # eg0e00_1 20.6 CT131 # eg0e00_1 35.2
pos
eg0d00_1 18.6
eg0d00_1 25.8 eg0d00_1 39.8
HV
group
pos
g0t00_1b g0t00_1b g0t00_1b g0t00_1b
0.0 7.7 7.4 7.0
YC
group
pos
PY
group
pos
YC_8a
100.8
group
pos
avg_pos
共sigma兲
PY_1b PY_1b PY_1b
22.8 18.3 25.6
PY_1b
41.7
0.0 0.8 4.9 6.4 20.7
共1.4兲 共6.9兲 共2.8兲 共0.4兲 共0.0兲
Table A8. This reshuffeling enables the improvement of marker order according to their individual map position. A marker can be replaced as long as it does not conflict with individual map positions. In this way marker E33/M49-F-530 could be repositioned to the ‘second’ position. Step 7 eg0e Marker
TG24 E33/M49-F-530 E41/M49-F-290 E38/M61-F-81 CT131
group
eg0d pos
# # eg0e00_120.6
# eg0e00_135.2
group
eg0t pos
eg0d00_118.6 eg0d00_125.8
HV
group
pos
YC
group
pos
PY
group
pos
g0t00_1b 0.0 g0t00_1b 7.0 g0t00_1b 7.7 g0t00_1b 7.4
eg0d00_139.8
YC_8a
100.8
group
pos
avg_pos
共sigma兲
PY_1b
22.8
PY_1b PY_1b PY_1b
18.3 25.6 41.7
0.0 6.4 0.8 4.9 20.7
共1.4兲 共0.4兲 共6.9兲 共2.8兲 共0.0兲
Table A9. Starting from this order a check is performed on consecutive marker position. The individual marker positions should not deviate more than the parameter ‘maximum allowed disagreement between adjacent anchor markers’ in the ‘wrong’ direction. None of the markers conflicts on this criterion. Finally the average position is recalculated to an ascending list where negative ‘jumps’ are corrected to the previous marker position ⫹ 0.1 cM. Step 8 eg0e Marker
TG24 E33/M49-F-530 E41/M49-F-290 E38/M61-F-81 CT131
group
eg0d pos
# # eg0e00_120.6
# eg0e00_135.2
group
eg0t pos
eg0d00_118.6 eg0d00_125.8
HV
group
pos
YC
group
pos
PY
group
pos
g0t00_1b 0.0 g0t00_1b 7.0 g0t00_1b 7.7 g0t00_1b 7.4
eg0d00_139.8
YC_8a
100.8
group
pos
avg_pos
共sigma兲
PY_1b
22.8
PY_1b PY_1b PY_1b
18.3 25.6 41.7
0.0 6.4 6.5 6.6 20.7
共1.4兲 共0.4兲 共6.9兲 共2.8兲 共0.0兲
Table A10. Single markers can now be added. A start is made with markers on top of the chromosomes. For example, TG55a, the first marker on top of chromosome 1 is also the first marker on the EG0D map. The distance between TG55a and anchor marker TG24 on the EG0D map is 18.6 cM. TG55a is localized at a position of ⫺ 18.7 relative to marker TG24 共18.6 * 1.01 where 1.01 is a correction factor for the relative lengths of the separate maps兲. Based on this minimum value of ⫺ 18.7, the anchor markers are repositioned: Step 9 eg0e Marker TG55a TG24 E33/M49-F-530 E41/M49-F-290 E38/M61-F-81 CT131
group
eg0d pos
# # # eg0e00_120.6
# eg0e00_135.2
group
eg0t pos
eg0d00_1 0.0 eg0d00_118.6 eg0d00_125.8
eg0d00_139.8
group
HV pos
group
YC pos
group
PY pos
g0t00_1b0.0 g0t00_1b7.0 g0t00_1b7.7 0t00_1b 7.4
group
pos
PY_1b 22.8
YC_8a 100.8
PY_1b 18.3 PY_1b 25.6 PY_1b 41.7
final
pos
18.7 25.1 25.2 26.3 39.4
0.0 共0.0 ⫹ 18.7兲 共6.4 ⫹ 18.7兲 共6.5 ⫹ 18.7兲 共6.6 ⫹ 18.7兲 共20.7 ⫹ 18.7兲
260 Table A11. The position of markers which occur in an interval between two anchor markers is determined based on the relative position of both anchor markers on their individual maps. Their integrated position is obtained from interpolation between the closest anchor markers. An example is given for marker “X” which occurs solely on the PY map: Step 10 eg0e Marker E38/M61-F-81 Marker X CT131
group
eg0d pos
group
eg0t pos
group
HV pos
group
YC pos
group
PY pos
group
pos
final pos
25.6 30.5 41.7
26.3
100.8
PY_1b PY_1b PY_1b
g0t00_1b 7.4 # eg0e00_1 35.2
eg0d00_1 39.8
YC_8a
39.4
Table A12. Position Marker X ⫽ 共30.5-25.6兲/共41.7-25.6兲 * 共39.4-25.3兲 ⫹ 25.3 ⫽ 29.4 eg0e Marker E38/M61-F-81 Marker X CT131
group
eg0d pos
group
eg0t pos
group
HV pos
group
YC pos
group
PV pos
group
pos
final pos
100.8
PY_1b PY_1b PY_1b
25.6 30.5 41.7
26.3 29.4 39.4
g0t00_1b 7.4 # eg0e00_1 35.2
eg0d00_1 39.8
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