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RFLP markers for sugar beet breeding: chromosomal linkage maps and location of major genes for rhizomania resistance, monogermy and hypocotyl colour

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RFLP markers for sugar beet breeding: chromosomal linkage maps and location of major genes for rhizomania resistance, monogermy and hypocotyl colour
  The Plant Journal(1992) 2 4), 601-61 1 RFLP markers for sugar beet breeding: chromosomal linkage maps and location of major genes for rhizomania resistance monogermy and hypocotyl colour E. Barzen', W. Mechelke', zyxwvut . Ritter3, J.F. Seitzer2 and F Salamini'9' Max- Planck-lnstitut fur Zuchtungsforschung, Carl- von- Linne- Weg 10, W-5000 Koln 30, Germany, KWS, lnstitut fur Pflanzenzuchtung, Kleinwanzlebener Saatzucht AG, GrimsehlstraBe 3 1, W-3352 Einbeck, Germany, and 3Granja Modelo - CIMA, Km 366 de la N.I. Arkaute (Alava), Apartado Correos, 46, E-0 7080 Vitoria-Gasteiz, Spain Summary An RFLP linkage map for the nine chromosomes of sugar beet (Beta vulgaris L. zyxwvutsr sp vulgarisvar. altissima Doell) was constructed by using a segregating popula- tion from a cross between two plants which were heterozygous for several agronomically interesting characters. One hundred and eleven RFLP loci have been mapped o nine linkage groups using 92 genomic markers. The current RFLP map covers a total length of 540 cM. Evidence for the existence of a major gene for rhizomania resistance zyxwvutsr Rrl) s given, together with its map position on linkage group IV in the interval between loci GS44 and GS28a. The presence of an RFLP fragment at the GS3dlocus s, until now, the best molecular marker for rhizomania-resistant genotypes in segregating populations of sugar beet; GS3d is linked o Rrl with 6.7 cM. The gene zyxwvu M ontrolling the polygerm/monogerm seed type, has been mapped on linkage group IX in a distal position at 4.2 cM from the locus GS7. The gene Rcontrolling the hypocotyl colour maps to linkage group VII and does not recombine with the RFLP locus GS42. The inheritance of a group of RFLP loci revealed the possible presence of a translocation in the population used to establish the map. The data presented are discussed in relation to the possibility of using RFLP markers in sugar beet breeding. Introduction A novel contribution of molecular genetics to plant breed- ing is the development of diagnostic DNA markers to assist the selection of superior genotypes of agricultural Received 17 January 1992; revised 9 March 1992. For correspondence fax +49 221 5062 413). plants. RFLP markers revealing sequence polymorphisms on the DNA level are easy to score, phenotypically neutral and enviromentally stable. When they are closely as- sociated in a linkage map to genes relevant for the expres- sion of agronomically interesting traits, they can be used to reduce, in segregating populations, the number of genotypes which have to be grown and evaluated in the field (reviewed in Tanksley et a/., zyx 989; Gebhardt and Salamini, 1992). In this paper we report on the present state of our RFLP linkage map for sugar beet (Beta vulgaris). As agronomi- cally important characters like monogermy, hypocotyl colour and resistance to rhizomania were segregating in the population of plants used to develop the map, we could locate the corresponding genes on the sugar beet genome near RFLP markers. These markers can now assist the selection of these traits. The relevance of the data presented or sugar beet breeding can be understood considering that diploid and triploid hybrids of this crop are widely cultivated n Europe. Their breeding is based on a complex crossing system where 2n mate sterile mono- germ tines are used as female parents n combination with 2n or 4n males (Hecker and Helmerick, 1985; Poehlman, 1987; Sneep et a/., 1979), and selection based on hypo- cotyl colour is applied to cross different parents avoiding emasculation (Poehlman, 1987; Sneep et a/., 1979). Moreover, the introduction of the genetic resistance to rhizomania into currently cuftivated genotypes is proving to be the only availabfe tool to counteract this serious disease of sugar beet (Barocka and Ross, 1985 . Two sources of rhizomania resistance were carried by fines R01 and R02, both derived putatively rom B. vulgaris spp. maritima (Figure 1). Of the other lines used, R01, N01, nd NO2 were monogerm with green hypocotyl; R02 was multigerm with red hypocotyl; lines N01, NO2 and NO3 were susceptible to rhizomania. Two F, hybrid plants were used to generate the basic population A used in mapping (Pl and P2 in Figure I , and both plants were rhizomania resistant, with P1 monogerm having a green hypocotyl and P2 multigerm having a red hypocotyr. Two further populations were considered B and C in Figure 1) which were obtained by selfing P1 and P2, respectively. The line R03 was derived from setfing of population C48, a multi- germ strain with resistance to rhizomania derived from B. vulgaris spp. marifima provided by R.T. Lewellen, USDA, Salinas, CA. Line R04 is multigerm with a moderate resistance to rhizomania. All genotypes mentioned are diploid (2n = 18). 601  602 E. Barzen et al. Line R01 Monogerm Resistant to rhizomania Green hypocotyl colour Line NO1 Monogerm Susceptible to rhizomania Green hypocotyl colour Resistant to rhizomania from Beta maritima) Red hypocotyl colour Line R02 Multigerm Line NO2 Monogerm Susceptible to rhizomania Green hypocotyl colour I ine R03 Ori inated by I zyxwvutsr SjA Salinas. Resistant to rhizomania Line NO3 Multigerrn Susceptible to rhizomania zyxwvutsrqpon :J F1 resistant plants selected Line R04 Multigerm Resistant to rhizornania Figure 1. Origin of plant material and crossing schemes. Results The srcin of the four-way cross central to this study (population A) is summarized in Figure 1, together with the breeding information illustrating accessory crossing ex- periments and the description of the several genetic strains involved. A major gene contributes to rhizomania resistance The test for rhizomania resistance was performed with 48 out of the 49 plants of population A by evaluating the viral concentrations in roots of greenhouse-infected plants. The three peaks in Figure 2A correspond to plants with high (I), medium 11) or no (Ill) level of resistance. Peaks II and 111 were clearly separated. If the threshold between peaks I and II was set at a value of 0.4 reading units, the number of plants under the three peaks were 14, 22 and 12, respectively. This distribution approaches he theoreti- cal ratio 1:2:1 zyxwvuts x2 = 0.49) expected for a single gene for resistance segregating in the cross, with both parental plants being heterozygous for the resistance gene. If the number of plants under peaks I and II was compared with that of plants classified under Ill, a perfect 3:l ratio was obtained (36:12). These data are compatible with the following conclu- sions: i) a single major gene for rhizomania resistance is segregating in population A; (ii) the two resistant plants used as parents for population A carry resistance alleles of the same gene. This would indicate that the two sources of resistance to rhizo- mania (R01 and R02) have a common origin. An RFL P fragment co-segregates with rhizomania resistance The finding of a genetic segregation in accordance with the existence of a major gene for rhizomania resistance indicated a search for RFLP fragments linked to it was necessary. Such a linked RFLP marker would indicate not only the genetic position of the gene in a RFLP linkage map of sugar beet, but also supports the existence of such a major genetic factor responsible for low levels of virus in sugar beet roots. Therefore the segregation of 171 RFLP fragments were monitored in population A. Out of the srcinal 49 plants of this population, resistance and RFLP data were available or 47. The marker GS3 hybridizes with seven polymorphic fragments in Taql-digested DNA of population A. One out of these seven fragments (Figure 3) was present in 34 rhizomania-resistant plants and absent in 10 susceptible genotypes. This fragment behaved in crosses as an allele of the RFLP locus GS3d which was linked to the rhizomania resistance ocus (see below). The resistant plants making up peaks I and II of Figure 2A showed, with the exception of plant 44, the presence of the fragment (Figure 3). On the contrary, among the 12 susceptible genotypes (peak 111 in Figure 2A), only plants 45 and 49 had the fragment. The three exceptional plants 44, 45 and 49 are considered as recombinants. Both parents of population A (P1 and P2 in Figure 3) were characterized by the presence of the allelic fragment of the locus GS3d. Subsequently, populations B and C were also analysed for an association between the fragment GS3d and rhizo- mania resistance. As shown in Figure 1, the populations B and C descended from selfing the rhizomania-resistant F,  RFLP markers for sugar beet breeding 603 n zyxwv rel. virus concentration Ado5) Figure 2. Distribution of plants of population A, B and C into classes of rhizomania resistance. The viral concentration in root tissues tested by ELISA is given in relative absorbance units. Dashed boxes represent plants of populations B and C as reported in Table 1. plants which, upon crossing, generated population A. The viral concentration of the 47 plants of population B, when analysed by ELISA, gave a bimodal distribution Figure zyxwv 8 . his distribution did support a segregation ratio of 3:l when, as for population A, an ELISA value > 2.5 reading units was considered for susceptible plants 37 resistant and 10 susceptible: zyxwvuts 2 = 0.35). The five plants classified in this population as the most resistant were all charac- terized by the presence of the allelic fragment of the locus GS3d Table 1). Moreover, out of the six plants classified as the most susceptible, five did not show the fragment; one, however, had it. In population C the average level of rhizomania re- sistance was higher than in populations A and B. The distribution of the 90 plants among resistance classes was again bimodal, but with a lower than expected number of plants falling in the susceptible group Figure 2C). When the ELISA value for resistance was set, as in population A, at 1.4 reading units, only six plants out of 90 were clas- sified as susceptible x2 = 16.1). The RFLP analysis of a sample of plants from population C is reported in Table 1. As in the case of populations A and zy   he 12 most resistant and three of the four most susceptible plants were associated with the presence and the absence, respectively, of the GS3d fragment. As found in population B, one plant, considered to be susceptible by ELISA, did show this fragment. Beside the resistant parent P1, srcinated from the cross R01 zyxwv   N01, six additional S2-lines out of the same cross were selected based on their rhizomania resistance: in all of them the GS3d allelic fragment was present in the RFLP pattern these and other results which follow in this section are not shown). Two lines with exceptional re- sistance to viral infection were, moreover, isolated from the cross R02 x N01: they were also characterized by the presence of the fragment. In a sample of 10 lines derived by selfing of the resistant breeding source R02, the frag- ment was present in nine of them. The line N03, a breeding line with high potential sucrose yield but susceptible to rhizomania, did not show the GS3d allelic fragment. R03, an independent source of resistance to rhizomania derived from B. vulgaris spp. maritima, developed by Lewellen and Skoyen 1987), also showed the RFLP frag- ment. These RFLP analyses support the hypothesis that in genotypes derived from B. vulgaris spp. maritima a major gene is responsible for the degree of rhizomania re- sistance. We have assigned the symbol Rrl to the re- sistance allele of the major gene conditioning rhizomania resistance. The current RFLP map of the nine linkage groups of B. vulgaris RFLP analysis of the 49 plants of population A was carried out with a total of 135 molecular markers isolated from a genomic Pstl library. The library was constructed starting from the diploid sugar beet line R04. Thirty percent of the genomic markers tested did not reveal polymorphisms when the DNAs of the segregating population were re- stricted with the enzymes Alul, Rsal and Taql. Based only on informative probes, 171 segregating fragments com- prised the data set for constructing the linkage maps of  604 E. Barzen et al. plant no. resistance classification z  5 6 7 Figure zyxwvutsrqpon   Segregation of marker GS3. Resistance R) or susceptibility S) o rhizomania s indhabove he corresponding lane below the plant number. Genomic DNAfrom 49 plants of population A numbered 1-34 and 36-50) was digested with Taql, separated and hybridized as described in Experimental procedures. The hybridizing fragments are numbered from 1 to 7. Only the lower part of the autoradiogram is shown. Fragment 7 reveals the RFLP marker GSSd, fragments 5 and 6 segregate independently from the rhizomania resistance gene. Plants 44, 45 and 49 are presumably recombinants between the DNA sequence hybridizing to the probe and the putative resistance ocus. Plants 17 and 23, indicated by an asterisk, were not evaluated n the resistance est. P1 = single plant from population B R01 x N01); P2 = single plant from population C R02 x N02). Table 1. Association of rhizomania resistance and presence of the RFLP fragment GS3d in the segregating populations B and C. Resistance/susceptibility is expressed by low/high concentration of virus in sugar beet roots as measured by ELlSA Population No. of plants Plants classified as the most Plants classified as the most Presence +) vs. absence -) of tested susceptible by ELlSA resistant by ELlSA the RFLP fragment GS3d ~ ~~ B 47 2.92 (0.14) 2.86 (0.19) 2.92 (0.23) 2.80 (0.49) 2.72 (0.63) 2.69 (0.64) C 90 1.82 (0.83) 1.66 (0.52) 1.52 (0.97) 1.41 (0.64) 0.70 0.37) 0.72 (0.49) 0.71 (1.02) 0.63 (0.82) 0.30 (0.20) 0.16 (0.14) 0.13 (0.18) 0.12 (0.11) 0.11 (0.02) 0.11 (0.09) 0.10 (0.08) 0.10 (0.09) 0.07 (0.05) 0.07 (0.03) 0.07 (0.05) 0.07 (0.05) 0.05 (0.09) + + + + + + + + + + + + + Standard errors are given in parentheses.  GS37a zyxwvutsrqpo   I -- GS 77a -- GS 13~ -- GS 39b GS 25a GS 76a -- GS 80a GS 48b GS 56c GS 80b GS 86b GS 66c GS 666 zyxwvutsrqpo @b zyxwvuts   GS 26c f GS 23 GS 3c GS 70a GS 65 GS 72al I GS 15 GS59 S62 kGs87 S5d zyxwvut   S 68 GS 72b GS 77a GS 13c GS 39b GS 25a GS 76a GS 80a r’ I I I I GS88 GS26a GS5a GS66a GS 17 GS 376 ~ GS 38 GS 56a GS 57 GS 71a GS 27a GS 19a GS 79 GS 13c GS 26b GS 29c GS 60 zyxwvut   RFLP markers for sugar beet breeding 605 @ GS 92 GS 73 GS 89a GS 19b GS 716 GS 3d GS 32b, GS 64 GS 22a GS 75 GS 67b GS 77b fg GS 13b GS 10 GS 896 IGS29b GS9 I GS zyxw 0~ GS 16 I GS52b GS53 GS 24 GS 6 GS 44 zyx r 1 GS 5c, GS 43 GS 39a GS 55, GS 90 GS 13a GS 28a 5 cM zyx   GS 42, R GS 69a GS 636 S51 GS 45, GS 54 GS 61, GS 696 GS 83 GS 766 zy   GS 85 GS 58 GS 91a Figure 4. RFLP linkage map of sugar beet. Map distances are given n cM. RFLP loci with the same number but followed by different letters are revealed by the same marker. Rrl = rhizomania resistance locus; zyxwvutsrqpon   = locus controlling hypocotyl colour; MM = locus controlling the monogerm/polygerm seed type. The two different maps reported for chromosome VI aand b) may be explained by a translocation as discussed in the text. The nomenclature for the genomic clones is GSna, GSnb, etc. with n being the marker number and letter indicating the existence of more than one locus revealed by the same RFLP marker. Marker probes are given in normal type, RFLP loci in italics. sugar beet chromosomes. Linkage groups were estab- lished as described by Ritter et a/. (1990). When neces- sary, multipoint estimates were used to find the best order of linked loci. Out of the 171 fragments considered, 159 were mapped to 11 1 loci distributed over nine linkage groups (Figure 4). Twelve polymorphic fragments could not be assigned to specific positions on the nine linkage groups. The total length of our linkage map, not taking into account the presence of a chromosome abnormality n the mapping population (see below), is 540 cM (units as defined by Kosambi, 1944). Parent-specific RFLP alleles segregating with a 1 1 ratio were 47 for P1 and 72 for P2.
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