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An extended map of the sugar beet genome containing RFLP and RAPD loci

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An updated map of sugar beet (Beta vulgaris L. ssp. vulgaris var ‘altissima Doell’) is presented. In this genetic map we have combined 248 RFLP and 50 RAPD loci. Including the loci for rhizomania resistance Rr1, hypocotyl colour R and the locus
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  Theor Appl Genet (1995) 90:189-193 9 Springer-Verlag 1995 E. Barzen 9 W. Mechelke - E. Ritter 9 . Schulte-Kappert F. Salamini n extended map of the sugar beet genome containing RFLP and R PD loci Received: 5 June 1994 / Accepted: 10 June 1994 Abstract An updated map of sugar beet (Beta vulgaris L. ssp. vulgaris var 'altissima Doell') is presented. In this ge- netic map we have combined 248 RFLP and 50 RAPD loci. Including the loci for rhizomania resistance Rrl, hypocotyl colour R and the locus controlling the mono- germ character M, 301 loci have now been mapped to the nine linkage groups covering 815 cM. In addition, the kar- yotype of some of the Beta vulgaris chromosomes has been correlated with existing RFLP and RAPD linkage maps. Key words Sugar beet 9 eta vulgaris 9 FLP 9 APD Introduction Restriction fragment length polymorphism (RFLP) maps of sugar beet (Beta vulgaris) have recently been published (Barzen et al. 1992; Pillen et al. 1992, 1993). The map pre- sented earlier by our group was based on 111 polymorphic RFLP loci and had a total length of 540 cM. It included the map positions of the genes M (polygerm vs. monogerm), R (hypocotyl colour) and Rrl (rhizomania resistance). In this paper we present an updated map that now covers 815 cM and is based on 301 loci distributed over all nine Beta chromosomes. The loci mapped have been revealed by RFLP probes and by randomly amplified polymorphic Communicated by G. Wenzel E. Barzen ([]) 9 Salamini Max-Planck-Institut ftir Z(ichtangsforschung, Carl-von-Linn6-Weg 10, D-50829 K61n, Germany W. Mechelke - E. Schntte-Kappert KWS, Institut f~ir Pflanzenz~ichtung, Kleinwanzlebener Saatzucht AG, Grimsehlstrage 31, D-37555 Einbeck, Germany E. Ritter Granja Modelo-CIMA, Km 366 de la N.I. Arkaute (Alava), Apartado Correos, 46, E-01080 Vitoria-Gasteiz, Spain DNAs (RAPDs; Williams et al. 1990). By considering the existing cytogenetical studies (Pillen et al. 1992, 1993; Wagner et al. 1992; Jung et al. 1992; Barzen et al. 1992; Lange et al. 1993; Uphoff and Wricke 1992; Salentijn et al. 1992; Jung and Herrman 1991; Jung et al. 1990; Na- kamura et al. 1991), we have correlated the karyotype of some of the Beta vulgaris chromosomes with RFLP/RAPD linkage maps already in existence. Materials and methods The mapping population, consisting of 49 plants produced from the cross between 2 F 1 plants (P1 and P2), was previously described by Barzen et al. (1992). The plants were maintained both vegetatively and by selfing to obtain sufficient leaf material for DNA extraction, RFLP and RAPD analysis. The plants were propagated by the KWS Kleinwanzlebener Saatzucht AG Einbeck, Germany. Methods used for the preparation of random genomic probes, DNA isolation, restriction digests, electrophoresis, blotting and hy- bridization were done as described by Barzen et al. (1992). Four hundred and forty random 10-met primers fiom Operon Technologies (Alameda, Calif., USA) were tested. Amplification re- actions were performed in a reaction volume of 25 ~tl containing: 20 mM TRIS-C1, pH 8.4; 50 mM KC1; 4 mM MgCI2; 200 gM each of dATR dCTR dGTP and dTTP (Pharmacia); 0.2 gM primer; 25 ng genomic DNA; 1.5 units ofTaq DNA polymerase (Gibco BRL); and overlaid with 2 drops of mineral oil. The amplification was performed in a Biometra DNA Thermal Cycler (G6ttingen, Germany) pro- grammed for 45 cycles, each one consisting of: 1 rain at 92~ 1.5 rain at 35~ and 2 rain at 72~ After the last cycle, the samples were incubated for 5 rain at 72~ and then kept at 20~ Amplifica- tion products were analyzed by etectrophoresis in 1.4% agarose gels, detected by staining with ethidium bromide and photographed under UV light. Linkage analysis was performed as described by Ritter et al. (1990). Each RFLP or RAPD fragment was scored as present or ab- sent for all genotypes, Pairs of fragments defining two po[ymorphic loci (A and B) were considered, and the allelic configuration at these loci was determined by observing whether the two fragments were present in both parents (configuration AB/AB) or in only one parent (AB/00). Recombination frequencies were calculated by the 'maxi- mum likelihood' method, and linkage groups were estabIished by the 'nearest neighbor' method. Linkage subgroups were joined by con- sidering loci with allelic fragments defining total linkage in repul- sion (Ritter et ah 1990).  190 236 a - 2 3a 4 5b 32a 37a 48b 56c 80b 8~ 163b 199 a 3a 11 18 48a 56b 78 843 86a 103a 20 a 109 190 a l19b 138 -119 a 146b 163c ~ 24i1481 a 1163 a 219 II 23 [_ 123 b 112 P=_ = 26 c 128- - 2----156 b, 159 137 b -- -~3 d, 62, 65, 1 72 a, 94 b 70-- 162 a~- -" 3 c, 162 b 174 b[_ - - 123 a " 148 b 147a 68 148a 203 155 122", 156a* 5a 15 137 a 59 - 199 a 72[ - 174 c 87 166~ 207a 5d * 17 * 37b * 38 *l 56a *] 57 * 58 * 913 * 103b* 107a* 131 * 150 *] 174 *l 176 *l 181a*] 186 *[ 192 *] 213 * 244* VI " -175" - -165 " -80a*, 88 - - 26 a 76a*: :115a*,210 9~." " 25a* 16 -39* 13a* 53 "121 133" 55___..~. ~ 104,184a 77a*~ -1178",199c* 98a*]: ~--'J147b* 1185 - - 66a*,246* - 21b*, 22b* - 28b - - 97b* ,1943* -158a* VII - 27c __~ 77b 170 - 2281 98b 188 [ 149 75 ~ -126 144"" - 234* 21a~ 22a 134 200b~ 32b, 64 95 96 I~ -273 1721 ~71 20S1'[19 61" 125" 145b* 231 * Fig. 1 Linkage map of sugar beet including RFLP and RAPD loci. The linkage groups labelled with arabic numerals are those that could be assigned to specific chromosomes. Loci with the same number but followed by different letters were revealed by the same genom- ic probe or primer. Map distances are given in centiMorgans. RFLP 3 13b =- 2373 R - 42 26b 69a. 129b 158b- 105 ~b 145a 152 180 ~ 93a 217b :83* VIII ~33 1102 1106 .4 - 25b 63a = 187 99~ 63b*l l-1201214b * 51 *~- 7223 222* I ~ ~ 230* - 168 1189 - 139 - 94 IV -114,243 2,4a 12a 1101 4~a]209 224 200 229 a 6 @73124 _L__dlll[167 154~ ~ 52b~0 [215 196 3d~233 Rr 1 ~216 1235 197 .L 11663 44, jr 157 208 ~J-183 a 153F 9a 901. 136 241 2 142 5c, 43 130 -28 a 7 L 132 204 184c IX 76b ~ 41 141 +247 85 ~ I 248~ 177 I 1 74[ 218"-I- 115b~ 160 I Z ~. W 225b I 35 173b 45, 54* ~ 182b [190b 40 I .-I-184b" 207bl ~245 I- [214c 220 1115a 5 9 9 a, 129 49 "~ 238a -117 - 181b*, 226 - 151 - 19a, 240a, 1983 143 22a 50, 212, 227 79 67b 67a, 97a 67c 6~ 66b loom ] loci are given in normal type and RAPD loci in italics. Rrl is the rhi- zomania resistance locus, R is the locus controlling hypocotyl colour and M is the locus controlling the monogerm/polygerm seed type. Loci showing a distorted segregation are indicated by asterisks Results and discussion An additional 165 random sugar beet genomic clones have been used to map 140 new RFLP loci on the existing RFLP map consisting of 111 loci (Barzen et al. 1992). By using the RAPD technique we were able to add an additional 50 loci. A total of 301 loci have now been mapped covering 815 cM [Fig. 1; units as defined by Kosambi (1944)]. Different DNA, primer, Mg 2§ and Taq DNA Polymer- ase concentrations were tested to establish a reproducible  polymerase chain reaction (PCR) protocol for RAPD anal- ysis in sugar beet. DNA concentrations of 7.5-50 ~ag were tested in reaction volumes of 25 or 50 gl. DNA, primer and enzyme concentrations had no evident effect on the num- ber and position of the PCR-amplified bands. The use of 4 mM Mg 2+ resulted in clearer amplification patterns than those observed with lower concentrations. According to our data, the optimal PCR reaction conditions for sugar beet are those reported in the Materials and methods. Out of the 440 RAPD primers screened with DNA from the two parents of our mapping population, 20% gave no amplification product; the rest yielded 1-10 bands in a size range of 100-2000 bp, of which 46% revealed no polymor- phism between the parental DNAs, while 34% produced 1-3 polymorphic bands. Primers revealing a polymor- phism were used for mapping. Forty-one RAPD loci seg- regated in the mapping population as dominant markers, predominantly with a segregation ratio of 1:1. At 9 loci a distorted segregation was observed. All 50 loci could be placed on the RFLP map (Fig. 1). The position of some loci defined by RAPDs was checked by RFLP analysis. Polymorphic amplified DNA fragments were excised from the agarose gel, labelled with [32p] and used as hybridization probes. In most cases the RFLP pattern obtained was quite complex, suggesting that the probe revealed the presence of repetitive sequences in 191 the genome. In the few cases where a RFLP pattern of low complexity was obtained, the polymorphic fragments could be mapped at the same position as their cognate am- plified products. Our results are consistent with findings in other species (Williams et al. 1990) and indicate that RAPDs can be fruitfully used to mark genomic regions which, due to the presence of repetitive DNA sequences, are difficult to access by RFLP analysis. The present state of the map In Table 1 we have summarized the available cytogeneti- cal data showing the correlation of the karyotype of Beta vulgaris chromosomes with linkage groups based on mu- tants, isoenzymes, RAPDs and RFLPs. In the table the numbering of chromosomes is based on the standard kar- yotypes of Bosemark and Bormotov (1971), as used by Romagosa et al. (1987) to identify their series of trisom- ics cytogenetically. The correspondence between the tri- somics of Romagosa et al. (1986) and those of Butterfass (1964) is based on assigning a specific karyotype to the tri- somics of Butterfass I, II, III, IV and VIII (Romagosa et al. 1986; recent personal communications of I. Romagosa and J.M. Lasa have confirmed these assignments). The genes reported in column 5 of Table 1 have been allocated Table 1 Summary of available cytogenetical data showing the correlation of the karyotype of Beta vulgaris chromosomes with linkage groups, based on isoenzyme, RAPD or RFLP analyses Numbering of Trisomics of Correspondence a Beta vulgaris Beta vulgaris between the chromosomes identified trisomics of based on the cytologically Romagosa et al. standard karyo- by Romagosa (1986) and type of Bose- et al. (1987) those of mark and Bor- Butterfass (1964) motov (1971) Genes allocated Genes linked to to chromosomes those allocated based on the to chromosomes trisomics of Butterfass (1964) Linkage groups corresponding to the trisomic series of Romagosa et al. (1987) Barzen Uphoff Wagner et al. and Wricke et al. (1992) (1992) (1992) 1 Type 1 I 2 Type 2 - 3 Type 3 II 4 Type 4 III 5 Type 5 IV 6 Type 6 - 7 Type 7 - 8 Type 8 VIII 9 Type 9 - Lapl b AK1 u R c, Got3 b, icdl a ye, B e, C f, Got3(2)g, VII o ii p iq R h, Est2 i. Fdp2 i Icdl(2) n Mdhl d Est5 , Rri j, Mdhl i, X k, III Z 1, Nb m, Gdh2 u . Acol d M i, Est31, Aco P, Fas i III S IIt aRomagosa et al. (1986); Nakamura et al. (1991) b Oleo et al. (1993) as cited in Lange et al. (1993) ~ Bunerfass (1968) d Lange et al. (1993) e Keller (1936); Owen and Ryser (1942); Owen et al. (1940); Abegg (1936) as cited in Abe et al. (1993) f Cited in Pillen et al. (1992; 1993) g Abe and Tsuda (1987); Wagner et al. (1992); Abe et al. (1993) h Pillen et al. (1992); Wagner et al. (1992); Abe et al. (1993) ! Wagner et al. (1992) J Barzen et al. (1992) k Pillen et al. (1993); Wagner et al. (1992) 1 Van Geyt et al. (1990) m Savitsky (1952; 1958) "Smed et al. (1989); Wagner et al. (1992); Abe et al. (1993) ~ R maps to this group p R maps to this group q R, lcdl, Got3(2), Est2, Fdp2 map to this group r Est5 maps to this group s M maps to this group t This group hosts an Aco locus most probably identical to the one mapped by Lange et al. (1993); M maps to this group u Abe et al. (1993)  192 to the four Butterfass trisomics I, II, III and IV - which have an extra chromosome corresponding to the karyotype of chromosomes 1,3, 4 and 5, respectively. The same genes allow the correlation of chromosomal karyotypes 3, 4 and 5 with available linkage groups based on isoenzymes, RAPDs and RFLPs. These three karyotypes correspond to the linkage groups I, III and II, respectively, of Wagner et al. (1992). Moreover, karyotypes 3 and 5 correspond to linkage groups VII and III, respectively, of Barzen et al. (1992), while karyotype 3 is most likely to be associated with linkage group II of Uphoff and Wricke (1992). The linkage groups of Fig. 1 are numbered as follows: group 3 and 5 according to their chromosomal assignment; and the remaining chromosomes as described by Barzen et al. (1992). In Fig. 1, the loci revealed by PCR are shown in italics and the loci with alleles segregating with abnor- mal ratios are followed by an asterisk. The finding of loci with alleles having a distorted segregation ratio in sugar beet is not new (discussed in Wagner et al. 1992 and Pil- len et al. 1993). In our map such loci seem to be located all along linkage group VI, at the end of linkage group 3 and at an intermediate map position of group VIII. A lower or higher than normal transmission of specific gamete types can be due to the action of self-incompatibility al- leles: four SI loci have been described in sugar beet (Lar- sen et al. 1977). The existence of gametic or zygotic lethal alleles has been reported for this species and can be an ad- ditional source of segregation distortion (Pillen et al. 1993). Structurally abnormal chromosomes (discussed in Barzen et al. 1992) may, however, also induce the skewed segregation of genetic markers. The possibility that such chromosomes were present in our mapping population could not definitely be excluded. We have hypothesized (Barzen et al. 1992) that a translocation may have been present in one parent of the cross. Moreover, local distur- bances of recombination were noted in several of our link- age groups, indicated by groups of markers which did not recombine. These were found on most chromosomes ex- cept 5 and VIII. Acknowledgements The authors thank Drs. C. Gebhardt and M. Heun for critical reading of the manuscript, and Dr. R. Schgfer-Pregl for statistical contributions. The technical assistance of N. Brinker and R. Stahl is greatly appreciated. References Abe J, Tsuda C (1987) Genetic analysis for isozyme variation in the section Vulgares, genus Beta. Jpn J Breed 37:253-261 Abe J, Guan G, Shimamoto Y (1993) Linkage maps for nine isozyme and four marker loci in sugarbeet (Beta vulgaris L.). Euphytica 66:117-126 Abegg FA (1936) A genetic factor for the annual habit in beets and linkage relationship. J Agric Res 53:493-511 Barzen E, Mechelke W, Ritter E, Seitzer JF, Salamini F (1992) RFLP markers for sugar beet breeding: chromosomal linkage maps and location of major genes for rhizomania resistance, monogermy and hypocotyl colour. Plant J 2:601-611 Bosemark NO, Bormotov VE (1971) Chromosome morphology in a homozygous line of sugar beet. Hereditas 69:205-212 Butterfass T (1964) Die Chloroplastenzahlen in verschiedenartigen Zellen trisomer Zuckerrfiben (Beta vulgaris L.). Z Bot 52:46-77 Butterfass T (1968) Die Zuordnung des Locus R der Zuckerrtibe (Hy- pokotylfarbe) zum Chromosom II. Theor Appl Genet 38:348-350 Jung C, Herrmann RG (1991) A DNA probe for rapid screening of sugar beet (Beta vulgaris L.) carrying extra chromosomes from wild beets of the Procumbentes section. Plant Breed 107: 275-279 Jung C, Kleine M, Fischer F, Herrmann RG (1990) Analysis of DNA from Beta procumbens chromosome fragment in sugar beet car- rying a gene for nematode resistance. Theor Appl Genet 79:663-672 Jung C, Koch R, Fischer F, Brandes A, Wricke G, Herrmann RG (1992) DNA markers closely linked to nematode resistance genes in sugar beet (Beta vulgaris L.) mapped using chromosome ad- ditions and translocations srcinating from wild beets of the Pro- cumbentes section. Mol Gen Genet 232:271-278 Keller W (1936) Inheritance of some major color types in beets. J Agric Res 52:27-38 Kosambi DD (1944) The estimation of map distances from recom- bination values. Ann Eugen 12:172-175 Lange W, Oleo M, De Bock TSM, D'Haeseleer M, Jacobs M (1993) Chromosomal assignment of three enzyme-coding loci (lcdl, Nad-Mdhl and Acol) using primary trisomics in Beet (Beta vul- garis L.). Plant Breed 111:177-184 Larsen K (1977) Self-incompatibility in B. vuIgaris L. I. Four ga- metophytic, complementary S-loci in sugar beet. Hereditas 85: 227-248 Nakamura C, Skaracis GN, Romagosa I (1991) Cytogenetics and breeding in sugar beet. In: Isuchiya T, Gupta PK (eds) Chromo- some engineering in plants: genetics, breeding, evolution, Part B. Elsevier, Amsterdam, pp 295-313 Oleo M, Lange W, D'Haeseleer M, De Bock TSM, Jacobs M (1993) Isozyme analysis of primary trisomics in beet (Beta vulgaris L.). Genetical characterization and techniques for chromosomal as- signment of two enzyme coding loci: leucine aminopeptidase and glutamate oxaloacetate transaminase. Theor Appl Genet 86:761-768 Owen FW, Ryser GK (1942) Some Mendelian characters in Beta vul- garis L. and linkages observed in the Y-R-B group. J Agric Res 65:153-171 Owen FW, Carsner E, Stout M (1940) Photothermal induction of flowering in sugar beets. J Agric Res 61:101-124 Pillen K, Steinrticken G, Wricke G, Herrmann RG, Jung C (1992) A linkage map of sugar beet (Beta vulgaris L.). Theor Appl Genet 84:129-135 Pillen K, Steinrficken G, Herrmann RG, Jung C (1993) An extend- ed linkage map of sugar beet (Beta vuIgaris L.) including nine putative lethal genes and the restorer gene X. Plant Breed 111:265-272 Ritter E, Gebhardt C, Salamini F (1990) Estimation of recombina- tion frequencies and construction of RFLP linkage maps in plants from crosses between heterozygous parents. Genetics 125:645-654 Romagosa I, Hecker RJ, Tsuchiya T, Lasa JM (1986) Primary tri- somics in sugarbeet. I. Isolation and morphological characteriza- tion. Crop Sci 26:243-249 Romagosa I, Cistue L, Tsnchiya T, Lasa JM, Hecker RJ (1987) Pri- mary trisomics in sugar beet. II. Cytological identification. Crop Sci 27:435-439 Salentijn EMJ, Sandal NN, Lange W, De Book TSM, Krens FA, Marcker KA, Stiekema WJ (1992) Isolation of DNA markers linked to a beet cyst nematode resistance locus in Beta patella- ris and Beta procumbens. Mol Gen Genet 235:432-440 Savitsky VF (1952) A genetic study of monogerm and multigerm characters in beets. Proc Am Soc Sugar Beets Technol 7:331-338 Savitsky VF (1958) Genetische Studien und Zt~chtungsmethoden bei monogermen Riiben. Z Pflanzenzficht 40:1-36 Smed E, Van Geyt JPC, Oleo M (1989) Genetical control and link- age relationships of isozyme markers in sugar beet (Beta vulga- ris L.). 1. Isocitrate dehydrogenase, adenylate kinase, phospho- glucomutase, glucose phosphate isomerase and cathodal peroxi- dase. Theor Appl Genet 78:97-104  Uphoff H, Wricke G (1992) Random amplified polymorphic DNA (RAPD) markers in sugar beet (Beta vulgaris L.): mapping the genes for nematode resistance and hypocotyl colour. Plant Breed 109:168-171 Van Geyt JPC, Smed E, Oleo M (1990) Genetical control and link- age relationships of isozyme markers in sugar beet (Beta vulga- ris L.). 2. NADP- and NAD-specific malate dehydrogenases, 6- P-gluconate dehydrogenase, shikimate dehydrogenase, diapho- rase and aconitase. Theor Appl Genet 80:593-601 193 Wagner H, Weber WE, Wricke G (1992) Estimating linkage relation- ship of isozyme markers and morphological markers in sugar beet (Beta vulgaris L.) including families with distorted segregations. Plant Breed 108:89-96 Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531-6535
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