Vol 5 (1996): 209-217. A linkage map of spring turnip rape based on RFLP and HARD markers Pirjo K. Tanhuanpää Agricultural Research Centre ofFinland, Institute of Crop andSoil Science, FIN-31600 Jokioinen, Finland Juha P. Viikki Boreal Plant Breeding, FIN-31600 Jokioinen, Finland H. JohannaViikki Agricultural Research Centre ofFinland, Institute ofAnimal Production, FIN-31600 Jokioinen, Finland A linkage map of spring turnip rape (Brassica rapa ssp. oleifera) was conslructed from an F 2 population of a cross J04002 x 5v3402. The map contained 22 RFLP loci, 144 RAPDs, one microsatellite, and one morphological marker (seed colour). All ten B.rapa linkage groups could be identified and the total map distance was 519 cM. A proportion of the markers (13%), most of which were located in two linkage groups, showed segregation distortion. Key words: DNA polymorphism, microsatellite, segregation distortion ntroduction The development of highly polymorphic DNA markers has facilitated the construction of gen- etic linkage maps. During the last few years linkage maps have been developed for many plant species, e.g. in the genus Brassica for B.oleracea (Slocum et al. 1990, Kianian and Quiros 1992,Landry et al. 1992), B.napus (Lan- dry et al. 1991, Ferreira et al. 1994, Uzunova et al. 1995), and B.rapa (Song et al. 1991, Chyi et al. 1992,Teutonico and Osborn 1994). The most commonly used type of DNA marker in linkage studies has been restriction frag- ment length polymorphism (RFLP). Recently de- veloped marker types based on use of the polymerase chain reaction (PCR) such as ran- dom amplified polymorphic DNA (RAPD), have several advantages over RFLPs. RAPD analysis is easy to perform and rapid, and does not re- quire the use of radioactivity. In addition, be- cause only minute amounts of crude template DNA are needed, it is possible to use rapid small- scale DNA extraction methods. A disadvantage is that the dominant nature of RAPD markers can cause problems if an F 2 intercross popula- tion is used. In such cases, estimation of recom- bination frequency is very inefficient between repulsion phase markers (Ott 1985) and, there- © Agricultural and Food Science in Finland Manuscript received April 1996 209 AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=rCiaJt4FZVasTOwg.MF9vnUPQlba3blc5_HxXYg.1fvdHzZ4mN2v8t3EIO-bYiWMGsmhkKXi585z6VcQN1L88I82aekBNDg6ARRo1EOy49WNOsOpdz9Q8RVgSCQMWsLydOwQkpiKGxOzCQMfe2CR0vrwIMzoG7o9O2xaMDETpkaSdY9ylgiaD9mKSOL8Euw8X2PH5uJI56m1GS9DZWuFlB349aSJpZLDegpFhl_DGigvZDqwOEiwZ5jMwqaTWAr-Am5UeLRHsXigA5kxS31ufoYxVWEuPZzGwDEawrNb4pyukzZezBRNwH6wsL7nOc8YKKajpK5kK6YIx-M4UCx8dwbaNWqGZVbcZkl9-M4IpWESep07ng Tanhuanpää, P.K., Viikki, J.P. & Viikki, H.J.: A linkage map ofspring turnip rape fore, two maps including only coupling phase markers have to be constructed. Existing B.rapa linkage maps are mostly composed of RFLP markers. Our aim here was to construct a linkage map of spring turnip rape (B.rapa ssp. oleifera) consisting mainly of RAPD markers. RFLP markers were used to in- tegrate our map with the existing B.rapa map (Teutonico and Osborn 1994). Material and methods Plant material The F 2 mapping population was derivedby self- pollinating five F, individuals from a cross be- tween two individualsofrepeatedly selfed spring turnip rape lines J04002 and 5v3402. The link- age data are mostly based on 77 F 2 individuals; 28 additional plants were scored to confirm link- ages between some markers. DNA of the plants was extracted by a method slightly modified from that of Dellaporta et al. (1983), as describedby Tanhuanpää et al. (1993). Markers RFLP analysis was performed using standard methods (Maniatis et al. 1982) with restriction enzymes EcoRI or ///«dill as described by Tan- huanpää et al. (1994). The F 2 progeny was screened with 24 DNA clones from B.rapa or B.napus (Teutonico and Osborn 1994) and two PCR-amplified genomic sequences of Brassi- caceae: the Brassica self-incompatibility gene SLG-8 (Dwyer et al. 1991), and the 5-enolpyruvyl- shikimate-3-phosphate synthase (EPSPS) gene from B.napus (Gasser and Klee 1990). RAPD primers were either synthesised on an Applied Biosystems 392 DNA/RNA Synthes- izer (Table 1) or purchased from Operon Techno- logies (Alameda, California, USA). RAPD ana- lysis was performed as described in Tanhuanpää et al. (1995a) with minor modifications. Putat- Table 1. RAPD primers used to analyse the F 2 progeny of the B.rapa ssp. oleifera cross, J04002 x 5v3402. In addi- tion, primers from Operon Technologies were used. Primer Sequence 5’ to 3’ 10 G€T GCT CGA GT 11 CGT CCT TAA GC 14 GCA CTG TCG AC 19 CGC TCT AGA CC 20 TGC CAG TTA CG 25 GCG TGT AGG CT 26 GGA ATC TCG GT 33 CCG CTT AGT TC 45 AGA CGA TGT AC 63 GAC CGT GAG AC 65 ACG TGC ATG G 72 TGG ACT CGA G 74 GCT GAC TCG AG 75 CGA ACC TGA TC 76 ATC GTC GAT GC 77 GCT AGC TAC TG 78 AGT CGA CTT C 90 ACG CTA GAC CT 93 GGT ACT CGA CT 101 ATG CGT CAG TC 102 TGA TCG ACT CG 103 CGT TCG AGT CT 105 TGC ATC GTA C 107 GAC TCG AGA C 110 ACG CCG TAC G 111 TCG GAA GGA C 112 GGA CAC TAC T 117 GCG CAA GTG AA 118 CGT CGC TGT T 123 ACT GAG CGT G 127 CAG CTC AGG CT 129 GTC CAC GTA GC 130 ACT CTG GCA G 134 GAC TGT GCA T 137 CTA CAT GCA CG 138 GTC CAC AGA T 140 ACG CTA TGA C 141 CTG ATC TGC A 146 GCT TCA TCG TG 147 CGT TCA CCT C 148 CCG ACT TCC A 149 TGC CAG TCT CC 164 AGA AAT GGG G ive allelism of two RAPD markers was invest- igated by hybridisation using one of the RAPD bands as a probe. 210 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 209-217. Table 2. B.napus microsatellites used to search for polymorphism between the parents of the B.rapa ssp. oleifera cross, J04002 and 5v3402. indicates no identifiable amplification. locus product repeat flanking sequences size sequence 5' to 3' range (bp) MB4» 71 (TG) |0 TGT TTT GAT GTT TCC TAC TG GAA CCT GTG GCT TTT ATT AC MBS" (AT),GT(AT) 4(GT) g AAC ATC TTT TTG CGT GAT AT AAT AGC ATT GAA GCC TTA C X64257" - < ATA)„ GTC TGC TCT CCA GAA CTA CTG TAC CTT TGG TTT CGG X 61097b - (CT) n AAC GAC CCT TTT CCG TCA GGC CGC TCA CAT TTG TAT 12AC 314 (GA)„(AAG)4 GCC GTT CTA GGG TTT GTG GGA GAG GAA GTG AGA GCG GGA AAT CA 35D C 222-234 (GA)„ GCA GAA GGA GGA GAA GAG TTG G TTG AGC CGT AAA GTT GTC ACC T 38A< 155 (TG)„ TGG TAA CTG GTA ACC GAC GAA AAT C ACG CTG TCT TCA GGT CCC ACT C 59A1< - (CA) n TGG CTC GAA TCA ACG GAC TTG CAC CAA CAA GTC ACT AAA GTT 12k' 277 (TAA)S(GA), GCC CAC CCA CCT TCT TGT CCT CCC TTC ATC CAA ACT CCT CCT CGT 8381 C 196 (GA) n GCC TTT CTT CAC ACC TGA TAG CTA A TCA GGT GCC TCG TTG AGT TC 92A1 C - (A)2g ACC GCC CGT GAC CAA A CCC ACC CCG TTA ACA TAT AAG TC 9Bl 204 (GA)2 „ GAC CGT GGA AGC AAG TGA GAA TG CCA AGC TTA TCG AGC CAT CCC 25C2C 132 (GA) m AAA CCT CCT CAA AAA CCC CTA AAC G TCC CCT CTT TCC TCT CTC TCT AGG C 19AL (GA)8 CAC AGC TCA CAC CAA ACA AAC CTA C CCC CGG GTT CGA AAT C a Lagercrantz et al. (1993) b Microsatellites from the EMBL and Genßank databases c Kresovich et al. (1995), Dr A. Szewc-McFadden, pers. comm. PCR programs used are those in the respective articles, microsatellites from databases amplified with the program described by Lagercrantz et al. Microsatellites are simple DNA sequences consisting of repeated nucleotide motifs, and show extensive polymorphism due to the occur- rence of different numbers of repeat units. The microsatellites (Table 2) were amplified in PCR using a pair of flanking primers, one primer of each pair labelled with fluorescein. The ampli- fied products were visualised with ALF DNA Sequencer (Pharmacia). One morphological marker, seed colour, which exhibits dominant inheritance (’brown’ dominant over ’yellow’), was scored visually in the F 2 population. Nomenclature RFLP probes and the respective loci (Fig. 1) were named according to Teutonico and Osborn 211 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Tanhuanpää, P.K., Viikki, J.P. & Viikki, H.J.: A linkage map ofspring turnip rape (1994): with the prefix WG (genomic DNA clones from B.napus cv ’Westar’), TG (genomic DNA clones from B.rapa cv ’Tobin’) or EC (cDNA clones from B.napus cv ’Westar’). RAPD loci (Fig.l) were named by the primer: self-synthesised primers with plain numbers, and Operon primers with a letter and a number. Dif- ferent polymorphic markers produced by the same primer were assigned with a small letter following the number of the primer (Table 3). The microsatellite marker on the map has the prefix MS. The nomenclature of ten B.rapa linkage groups (LGI-LG10) follows that on the previ- ous map (Teutonico and Osborn 1994), the groups being identified by the common RFLP loci. Unassigned groups were named with cap- ital letters (A-C, Fig. 1). Statistical analysis Because the inbred lines J04002 and 5v3402 contained residual heterozygosity, the F, seed was not uniform. Some marker loci were homo- zygous in some of the five F, individuals, lead- ing to genetically uniform (with respect to these loci) F 2 progeny which had to be omitted in the linkage analysis. Therefore, the number of seg- regating individuals within the pooled F 2 popu- lation varied from locus to locus. Goodness-of-fit to the expected F 2 segrega- tion at marker loci was tested by chi-square ana- lysis. Linkage relationships were evaluated by the MAPMAKER 3.0 computer program (Lander et al. 1987). Markers were grouped with a LOD score of 4.0 and a maximumrecombination frac- tion of 0.4 as linkage criteria. On a few occa- sions, the LOD score threshold for linkage was decreased to 2.0 to include additional RFLP loci (indicated with a dashed line in Fig. 1) on the map. Map distances in centiMorgans were com- puted by Haldane’s mapping function. Separate linkage analyses were performed for data set A (dominant markers originating from Jo4002) and data set B (dominant markers from 5v3402). Codominant markers were present in both data sets. The map was built in two phases. First, a framework map was constructed from data set A, using only those markers that could be or- dered with a LOD score difference > 3.0 (in some cases 2.0) in favour of the best map. To build up the final linkage map, all the other markers linked to each group with a LOD score > 4.0 were placed to the side of the closest framework locus (markers from data set A and codominant markers to the left and markers from data set B to the right). Results A high level of DNA polymorphism was ob- served in the mapping population: 67% of the Fig.l. Linkage map of B.rapa ssp. oleifera constructed from the F 2 population of a cross, J04002 x 5v3402. For grouping markers, a LOD score threshold of 4.0 was used, except for TGIHI2 and WGIG6, which were attached to the framework using a LOD score of 2.0 (indicated with a dashed line). For ordering, a LOD score difference >3.0 (wider line) or >2.0 (LGs 4,6, 7, slim line) in favour of the best map was used. Dominant RAPD markers on the framework and on its left side are derived from J04002 (data set A), on the right side from 5v3402 (data set B). Marker distances are shown in centimor- gans; for markers not included in the framework, two point map distances between the marker and the nearest framework locus are shown (LG9 includes four markers, 65a, 93a, 147b, 140d, which did not show linkage to any framework markers but only to markers from data set B). Linkage groups are named after the previous B.rapa RFLP map (Teutonico and Osbom 1994); the orientation of groups LGI, 6, 7 and 8, where only one locus is common with the previous map, is arbitrary. Codominant markers are underlined, loci common with the previous map printed in italics. The nomenclature of markers is described in Material and methods. Loci exhibiting aberrant segregation are indicated with *(P