RAPD markers linked to microspore embryogenic ability in Brassica crops

In order to identify the markers linked to microspore embryogenic ability in Brassica crops, RAPD segregation analyses were performed in a microspore-derived (MD) population and a F₂ population derived from F₁between ‘Ho Mei’ (high responsive parent in microspore embryogenesis) and ‘269’ (low responsive parent) in Chinese cabbage, and between ‘Lisandra’ (high responsive parent) and ‘Kamikita’ (low responsive parent) in oil seed rape. After 230 and 143 primers were screened, a total of 148 and 52markers were detected to be polymorphic between the parents in Chinese cabbage and oilseed rape, respectively. Twenty-seven percent of the markers in the MD population showed a significant segregation distortion in both crops. Of the markers showing segregation distortion in the MD population, 71–75% of the markers followed the expected Mendelian segregation ratio in the F₂ population. When the relationships between such distorted markers and microspore embryogenesis of the F₂ population were examined, 7 and 3 markers were identified to be associated with embryogenic ability in Chinese cabbage and oilseed rape, respectively. These markers showed additive effects on embryo yields, and the plants having more alleles of the high responsive parent produced higher embryo yields. These markers maybe useful in marker-assisted selection for improving microspore responsiveness straits in Brassica crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of RAPD and SCAR markers linked to northern leaf blight resistance in waxy corn (Zea mays var. ceratina)

Exserohilum turcicum causes northern corn leaf blight (NCLB), an important disease occurring in maize producing areas throughout the world. Currently, the development of cultivars resistant to E. turcicum seems to be the most efficient method to control NCLB damage. Marker-assisted selection (MAS) enables breeders to improve selection efficiency. The objective of this work was to identify random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) markers associated with NCLB resistance. Bulked segregant analysis (BSA) was used to search for RAPD markers linked to NCLB resistance genes, using F₂ segregating population obtained by crossing a susceptible inbred ‘209W’ line with a resistant inbred ‘241W’ line. Two hundred and twenty-two decamer primers were screened to identify four RAPD markers: OPA07₅₂₁, OPA16₄₅₇, OPB09₅₂₀, and OPE20₅₃₆ linked to NCLB resistance phenotype. These markers were converted into dominant SCAR markers: SCA07₄₉₆, SCA16₄₂₀, SCB09₄₆₄, and SCE20₄₂₉, respectively. The RAPD and SCAR markers were developed successfully to identify NCLB resistant genotypes in segregating progenies carrying NCLB resistant traits. Thus, the markers identified in this study should be applicable for MAS for the NCLB resistance in waxy corn breeding programs.     

Identification and mapping of a quantitative trait locus controlling extreme late bolting in Chinese cabbage (Brassica rapa L. ssp. pekinensis syn. campestris L.) using bulked segregant analysis

DNA markers linked to a locus controlling an extreme late bolting trait, which was originally found in a local cultivar of a non-heading leafy vegetable,‘Osaka Shirona Bansei’ (Brassica rapa L. ssp. pekinensis syn. campestris L.) were identified using bulked segregant analysis. A doubled haploid (DH) line, DH27, which is a progeny of ‘Osaka Shirona Bansei’, shows extreme late bolting, and bolts without vernalization. DH27 was crossed with a normal bolting DH line, G309. The plantlets of the parents, F₁ and F₂, were vernalized and then grown in a greenhouse. The bolting time of F₂ plants showed a continuous distribution from 19 to 231 days after vernalization (DAV), suggesting the effects of a few major genes and polygenes. Possible linkage markers for this trait were screened by modified bulked segregant analysis (BSA). The BSA using four bulks suggested that a 530-bp RAPD band RA1255C was linked to a locus controlling the bolting trait. The RAPD band was cloned and used as a probe to detect RFLP. The fragment detected a single locus, BN007-1,the segregation of which in the F₂ population matched that of RA1255C. Three other RAPDs were found to be linked to BN007-1. A quantitative trait locus(QTL) affecting the bolting time was detected around BN007-1 using MAPMAKER/QTL. Since the difference between bolting times of both the parental genotypes in the F₂ was 138 days, these markers may be useful for a marker-assisted selection (MAS) in the breeding program for late bolting or bolting-resistant cultivars in B. rapa crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of a RAPD marker linked to a brown planthopper resistance gene in rice

We report the tagging of a brown planthopper (BPH) resistance gene (Bph–1) in rice using RAPD and RFLP markers. The Korean rice variety ‘Gayabyeo’ has dominant duplicate genes including Bph–1 conferring resistance to biotype 1 of BPH. Bulked segregant RAPD analysis was employed for rapid identification of DNA markers linked to resistance genes. For tagging these two genes, an F2F3 population from a ‘Gayabyeo’ × ‘Nagdongbyeo’ cross was developed and evaluated for BPH resistance. Three bulked DNAs from two groups of homozygous BPH resistant (each for Bph–1 and the other unknown gene) and homozygous susceptible F2 plants were analyzed by RAPD using 140 random oligomers. One primer, OPD–7 yielded a 700-bp fragment that was present in Gayabyeo and resistant F2 plants (homozygous for Bph-1 locus) but absent in Nagdongbyeo and susceptible F2 plants. Cosegregation of this marker with Bph-1 was verified using an F2 population segregating for Bph-1. Chromosomal regions surrounding the Bph-1 were examined with additional RFLP and microsatellite markers on chromosome 12 to define the location of the RAPD marker and Bph-1. Use of this RAPD marker could facilitate early selection of resistant lines for BPH. This revised version was published online in July 2006 with corrections to the Cover Date.     

Segregation and linkage analysis of DNA markers in microspore derived and F2 populations of oilseed rape (Brassica napus L.)

Summary The segregation of RFLP and RAPD markers was compared in two oilseed rape (Brassica napus L.) breeding populations from the cross Topas x R4, the latter being a low linolenic mutation line. A total progeny of 68 F₂ and 40 microspore derived plants were studied with 25 markers. The results indicated a significant excess of Topas alleles at five RAPD loci in the microspore derived population. This suggests that genomic regions which probably affect microspore culture ability do not have identical distribution in the two population types.     

Detecting and estimating segregation distortion and linkage between glufosinate tolerance and blackleg resistance in Brassica napus L.

Summary The segregation and linkage between glufosinate (transgenes ‘Rf3’ and ‘T177’) and blackleg resistance genes in canola (Brassica napus L.) were assessed using F₁ microspore-derived doubled haploid (DH) populations from four crosses including reciprocals, two involving the transgene ‘Rf3’ and the other two involving the transgene ‘T177’. To relax the assumption of no segregation distortion required for the conventional analysis of segregation and linkage, we employed Bailey's analysis that allows detecting segregation distortion at linked loci. The significant departures from the 1:1 segregation were detected in the crosses involving the transgene ‘T177’ but not in the crosses involving the transgene ‘Rf3’. The apparent deficit of the herbicide tolerant DH lines in the crosses with the transgene ‘T177’ is likely due to differential selection against the gametes carrying ‘T177’ during microspore culture. The linkage was strong between blackleg resistance and the transgene ‘Rf3’ but weak or absent between blackleg resistance and the transgene ‘T177’, suggesting that the two transgenes are probably inserted into distant regions of the genome. The observed linkage offers an opportunity to develop new canola cultivars with both glufosinate tolerance conferred by transgene ‘Rf3’ and blackleg resistance.     

Segregation distortion in doubled haploid lines of barley (Hordeum vulgare L.) detected by simple sequence repeat (SSR) markers

A total of 147 simple sequence repeat (SSR) markers (including86 barley and 61 wheat microsatellite markers) were tested for their segregation in a doubled haploid (DH) and an F₂ population of barley. The DH population consisted of 71 doubled haploid lines, developed from F₁ plants of a cross between Tadmor and WI2291using isolated microspore culture technique. A genetic linkage map consisting of 43 microsatellite markers was constructed using the DH population. Particularly on chromosome 4H microsatellite markers showed distorted segregation ratios. Segregation of DH lines based on molecular markers were compared with segregation of 92 F₂ lines from the same cross. The proportion of loci deviating from the expected monogenic segregation ratios in the DH population was significantly higher (19/43loci, 44%) than in the F₂ population (7/43 loci, 16%). The deviation was biased towards the WI2291 parent alleles. In line with this observation, WI2291 was found to perform better than Tadmor in regenerating green plantlets with the isolated microspore-culture technique. This revised version was published online in July 2006 with corrections to the Cover Date.     

Different recombination frequencies in wheat doubled haploid populations obtained through maize pollination and anther culture

This study compared the meiotic recombination frequency between wheat doubled haploid (DH) populations obtained through two different methods, maize pollination (MP♀) and anther culture (AC♂). The comparison was based on a genetic linkage analysis, performed with DNA markers. Thirty-five polymorphic markers (15 SSR, 15 AFLP, 5 RAPD) were screened in MP♀ and AC♂ doubled haploids populations, derived from the same hybrid genotype (F₁ of ‘Eta’ × ‘Darkhan 15’). Nine linkage groups, comprising 35 loci (the MP♀ lines) and 31 loci (the AC♂ lines), were constructed. The linkage groups in both DH populations showed identical orders of markers, except for one group mapping to chromosome 6B. The MP♀ and AC♂ linkage maps differed significantly in recombination frequencies for corresponding intervals. In total, the AC♂ linkage map (495.5 cM) was 40.5% longer than the MP♀ map (352.8 cM), indicating a significantly higher meiotic recombination rate in pollen mother cells. The enhancement in recombination was visible in five of nine linkage groups, and in 7 intervals between individual loci out of 19 compared. Moreover, for 6 other intervals a lack of linkage was observed in the AC♂ population, as compared to the MP♀ map.     

Potato Virus Y Resistance Gene, Ry chc, Mapped to the Distal End of Potato Chromosome 9

Summary A Japanese leading cultivar ‘Konafubuki’ has an extreme resistance gene Ry _chc to Potato virus Y (PVY) in a simplex fashion. To reduce complexity of the highly heterozygous autotetraploid genome and map the Ry _chc gene, haploid plants were parthenogenetically induced from ‘Konafubuki’. The ratio of 7 resistant: 5 susceptible haploids confirmed the single dominant, simplex genotype of ‘Konafubuki’. A selected resistant haploid 98H20-5 was crossed with a susceptible diploid, resulting in 119 F ₁ plants. Using RFLP and RAPD markers, Ry _chc was mapped to the most distal end of the chromosome 9 where the recovery of recombinant genotypes was extremely reduced in this mapping population possibly due to the linked inferiority factor. The location of Ry _chc is different from those of the other extreme resistance genes to PVY, but possibly resides in one of resistance gene clusters.     

Sequence characterized amplified region markers tightly linked to the dwarf mosaic resistance gene <Emphasis Type="Italic">mdm1 (t)</Emphasis> in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Maize dwarf mosaic is one of the devastating and wide spread viral diseases in the world. The present investigation was carried out to develop DNA markers closely linked to the resistance gene mdm1 (t). Linkage between the markers and phenotypes was confirmed by analyzing an F₂ population obtained from a cross between a resistant parent ‘Huangzaosi’ and a susceptible parent ‘Mo17(478)’. Four AFLP markers were found in the maize dwarf mosaic resistant plants. By using (BSA) bulked segregant analysis, two of the four AFLP markers were transformed into Sequence-characterized amplified regions markers (SCARs), nominated Rsun-1 and Rsun-2. The two amplified fragment length polymorphism (AFLP) markers, RHC-1and RHC-2, from the amplification products of primer combination E-AGC/M-CAA and E-AGC/M-GAA, showed linkage with the mdm1 (t) gene in a genetic distance 1.6 and 2.0 cM, respectively. The results indicate that the new SCAR markers will be valuable to distinguish resistant plants from susceptible plants in plantlets growing in seedbeds. The markers developed in this study are suitable for marker-assisted selection for maize dwarf mosaic resistance.     

Identification of RAPD markers linked to fusarium crown and root rot resistance (Frl) in tomato

In tomato ( Lycopersicon esculentum Mill.) a single dominant gene ( Frl) on chromosome 9 confers resistance to fusarium crown and root rot (crown rot) incited by Fusarium oxysporum f. sp. radicis-lycopersici. To identify randomly amplified polymorphic DNA (RAPD) markers linked to Frl, crown rot susceptible and resistant tomato lines were screened for polymorphisms using 1000 random 10-mer primers and three reliable RAPD markers were found linked to Frl (UBC #'s 116, 194, and 655). A codominant polymorphic PCR marker of TG101, a restriction fragment length polymorphic (RFLP) marker linked to Frl, was developed to facilitate the linkage studies. Using TG101 and the four RAPD markers, on a Frl segregating backcross population of 950 plants indicated that all belong to the same linkage group. The polymorphic allele order was found to be TG101 – 655 – 116 – 194 – Frl. UBC 194 was found to be 5.1 cM from Frl in this population. Furthermore, it was the only marker found in the resistant genotypes ‘Mocis’ and Fla 7226, whereas resistant genotypes ‘Momor’, Ohio 89-1, and Fla 7464 all had UBC 194 and UBC #'s 116, 194, and 655. This revised version was published online in July 2006 with corrections to the Cover Date.     

Segregation distortion of Brassica carinata derived black rot resistance in Brassica oleracea

Three segregating F₂ populations were developed by self-pollinating 3 black rot resistant F₁ plants, derived from across between black rot resistant parent line 11B-1-12 and the susceptible cauliflower cultivar ‘Snow Ball’. Plants were wound inoculated using 4 isolates ofXanthomonas campestris pv. campestris (Xcc) race 4, and disease severity ratings of F₂ plants from the three populations were scored. A total of 860 arbitrary oligonucleotide primers were used to amplify DNA from black rot resistant and susceptible F₂ plants and bulks. Eight RAPD markers amplified fragments associated with completely disease free plants following black rot inoculation,which segregated in frequencies far lower than expected. Segregation of markers with black rot resistance indicates that a single, dominant major gene controls black rot resistance in these plants. Stability of this black rot resistance gene in populations derived from 11B-1-12 may complicate introgression into B. oleracea genotypes for hybrid production. This revised version was published online in July 2006 with corrections to the Cover Date.     

A proposed mechanism for loss of heterozygosity in rice hybrids

A phenomenon, loss of heterozygosity (LOH), was discovered in hybrid plants involving a selected plant (named AMR) of the Chinese rice cultivar ‘ZhongxinNo. 1’ as one parent. In these hybrids and some of their progenies, somatic variations were manifested by molecular genotypes and/or morphological phenotypes in vegetative parts of the same plant. Random amplified polymorphic DNA (RAPD) markers for the parents have been followed through the F₃ generation. RAPD markers were uniformly present or absent in all plants within some or all F₂ panicle rows derived from F₁ hybrids involving AMR. In contrast, RAPD markers segregated in the Mendelian manner for dominant markers in panicle rows derived from control hybrids. Certain F₂ panicle rows from F₁hybrids involving the special rice became fixed for all assayed RAPD markers. Genotype fixation was confirmed by molecular assays and field observations of the F₃ progenies. We propose a new biological mechanism, called ‘assortment mitosis,’ as being responsible for the observed phenomenon. The use of this mechanism in plant hybrids allows the development of uniform progenies as early as the F₂ generation. Therefore, the time required to obtain fixed non-parental type progenies for subsequent performance trials can be drastically shortened. Utilizing this mechanism in plant breeding represents a new approach and requires the modification of conventional plant breeding procedures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance mode of male sterility in bunching onion (<Emphasis Type="Italic">Allium fistulosum</Emphasis> L.) accessions

Cytoplasmic male sterility (CMS) is an indispensable trait for F₁ hybrid seed production in bunching onion (Allium fistulosum L.). Expansion of the cytoplasmic diversity of F₁ hybrid cultivars by introduction of various CMS resources has great potential to eliminate vulnerability to cytoplasm type-specific diseases. This study aimed to evaluate appearance frequency of male sterile plants in several bunching onion accessions and to identify CMS resources. In eight (‘Nogiwa Aigara’, ‘Bansei Hanegi’, ‘Amarume’, ‘Kimnung’, ‘Zhangqiu’, ‘INT/CHN/1990/GOTOU’, ‘Natsunegi’ and ‘Guangzhou’) of 135 accessions collected from Japan, China, Mongolia, Korea and Taiwan, male sterile plants appeared with varied frequencies from 1.7% (‘Nogiwa Aigara’ and ‘Bansei Hanegi’) to 24.5% (‘Zhangqiu’). The inheritance mode of Zhangqiu- and Guangzhou-derived male sterility was confirmed to be CMS by sib-crossings and interbreed crossings. Microscopic examination of microsporogenesis in the CMS plants revealed that microspore protoplasm rapidly degenerated without mitotic division after the release of microspores from tetrads. The CMS germplasm described here would be useful for the development of “A” lines to be used in F₁ hybrid seed production of bunching onion. Male fertility in ‘Nogiwa Aigara’, ‘Bansei Hanegi’, ‘Kimnung’, ‘INT/CHN/1990/GOTOU’ and ‘Natsunegi’ was verified to be controlled by a single fertility restoration locus.     

Identification of molecular markers associated with resistance to squash silverleaf disorder in summer squash (<Emphasis Type="Italic">Cucurbita</Emphasis> <Emphasis Type="Italic">pepo</Emphasis>)

Squash silverleaf (SSL), caused by the silverleaf whitefly [Bemisia argentifolii (formerly known as Bemisia tabaci Gennadius, B strain)], is an important physiological disorder that affects squash (Cucurbita spp.) by reducing yield potential. Breeding squash with resistance to SSL disorder can be facilitated by using marker-assisted selection (MAS). Resistance to SSL disorder, in Cucurbita pepo, is conferred by a single recessive gene (sl). The objective of this study was to identify molecular markers associated with resistance. A zucchini squash, SSL disorder resistant breeding line, ‘Zuc76’ (sl/sl) and a SSL disorder susceptible zucchini cultivar ‘Black Beauty’ (Sl/Sl) were screened with 1,152 randomly amplified polymorphic DNA (RAPD) primers and 432 simple sequence repeat (SSR) markers to identify polymorphisms. Using F₂ and BC₁ progeny segregating for SSL disorder resistance, three RAPD (OPC07, OPL07 and OPBC16) primers and one SSR (M121) marker were found associated with sl. Fragments amplified by RAPD primer OPC07 was linked in coupling phase to sl, whereas RAPD primer OPL07 was linked in repulsion phase. RAPD primer OPBC16 and SSR marker M121 were co-dominant. The allelic order of these loci was found to be M121–sl–OPC07–OPL07–OPBC16. The closest marker to sl is M121 with an estimated genetic distance of 3.3 cM. The markers identified in this study will be useful for breeding summer squash (C. pepo) for SSL disorder resistance derived from zucchini squash breeding line ‘Zuc76’.     

普通小麦DH155抗白粉病基因的分子作图及应用分子标记辅助选择将其转移

普通小麦品系DH155对白粉病菌表现高抗。为明确DH155所携带抗白粉病基因的遗传方式及与抗病基因连锁SSR标记,利用DH155与高感小麦品系SN2890杂交获得的F2和F2:3群体进行接种鉴定和遗传分析,发现DH155对白粉菌菌株E09的抗性受1对显性基因控制,暂命名为Ml DH155。BSA和分子标记分析结果显示,Ml DH155与SSR标记Xcfd81和Xcfd18连锁。利用已发表的中国春和粗山羊草D基因组序列开发新标记,进一步将Ml DH155定位于标记Xsdau K525和Xsdau K527之间,其遗传距离分别为0.2 c M和0.8 c M。将DH155与感白粉病优良品系HB133-4和旱10杂交,在F2~F4代,结合优良农艺性状选择、分子标记辅助选择和抗白粉病鉴定,获得3个高抗白粉病且农艺性状优异的株系(SDAU2100、SDAU2101和SDAU2102)。利用14个白粉菌菌株对DH155进行苗期接种鉴定表明,DH155对13个菌株表现抗病反应型。这些菌株对DH155的毒力谱与已知抗白粉病基因Pm2相似,但DH155对Bg78-3和Bg44-5菌株的反应型与携带Pm2的Ulka/8*Cc不同。结合本试验结果和Pm2基因的相关报道,推测Ml DH155可能是Pm2或其等位基因。     

A new citrus linkage map based on SRAP,SSR, ISSR,POGP, RGA and RAPD markers

Sequence-related amplified polymorphism (SRAP), simple sequence repeats (SSR), inter-simple sequence repeat (ISSR), peroxidase gene polymorphism (POGP), resistant gene analog (RGA), randomly amplified polymorphic DNA (RAPD), and a morphological marker, Alternaria brown spot resistance gene of citrus named as Cabsr caused by (Alternaria alternata f. sp. Citri) were used to establish genetic linkage map of citrus using a population of 164 F₁ individuals derived between ‘Clementine’ mandarin (Citrus reticulata Blanco ‘Clementine) and ‘Orlando’ tangelo’ (C. paradisi Macf. ‘Duncan’ × C. reticulata Blanco ‘Dancy’). A total of 609 markers, including 385 SRAP, 97 RAPD, 95 SSR, 18 ISSR, 12 POGP, and 2 RGA markers were used in linkage analysis. The ‘Clementine’ linkage map has 215 markers, comprising 144 testcross and 71 intercross markers placed in nine linkage groups. The ‘Clementine’ linkage map covered 858 cM with and average map distance of 3.5 cM between adjacent markers. The ‘Orlando’ linkage map has 189 markers, comprising 126 testcross and 61 intercross markers placed in nine linkage groups. The ‘Orlando’ linkage map covered 886 cM with an average map distance of 3.9 cM between adjacent markers. Segregation ratios for Cabsr were not significantly different from 1:1, suggesting that this trait is controlled by a single locus. This locus was placed in ‘Orlando’ linkage group 1. The new map has an improved distribution of markers along the linkage groups with fewer gaps. Combining different marker systems in linkage mapping studies may give better genome coverage due to their chromosomal target site differences, therefore fewer gaps in linkage groups.     

Using molecular markers to pyramid genes for resistance to ascochyta blight and anthracnose in lentil (Lens culinaris Medik)

Ascochyta blight caused by the fungus Ascochyta lentis Vassilievsky and anthracnose caused by Colletotrichum truncatum [(Schwein.) Andrus & W.D. Moore] are the most destructive diseases of lentil in Canada. The diseases reduce both seed yield and seed quality. Previous studies demonstrated that two genes, ral1 and AbR1, confer resistance toA. lentis and a major gene controls the resistance to 95B36 isolate of C. truncatum. Molecular markers linked to each gene have been identified. The current study was conducted to pyramid the two genes for resistance to ascochyta blight and the gene for resistance to anthracnose into lentil breeding lines. A population (F_6:7) consisting of 156 recombinant inbred lines (RILs) was developed from across between ‘CDC Robin’ and a breeding line ‘964a-46’. The RILs were screened for reaction to two isolates (A1 and 3D2) ofA. lentis and one isolate (95B36) ofC. truncatum. χ² analysis of disease reactions demonstrated that the observed segregation ratios of resistant versus susceptible fit the two gene model for resistance to ascochyta blight and a single gene model for resistance to anthracnose. Using markers linked to ral1 (UBC 227₁₂₉₀), to AbR1(RB18₆₈₀) and to the major gene for resistance to anthracnose (OPO6₁₂₅₀),respectively, we confirmed that 11 RILs retained all the three resistance genes. More than 82% of the lines that had either or both RB18₆₈₀ and UBC227₁₂₉₀markers were resistant to 3D2 isolate and had a mean disease score lower than 2.5. By contrast, 80% of the lines that had none of the RAPD markers were susceptible and had a mean disease score of 5.8. For the case of A1 isolate of A. lentis, more than 74% of the lines that carriedUBC227₁₂₉₀ were resistant, whereas more than 79% of the lines that do not have the marker were susceptible. The analysis of the RILs usingOPO6₁₂₅₀ marker demonstrated that 11out of 72 resistant lines carried the marker, whereas 66 out of 84 susceptible lines had the marker present. Therefore, selecting materials with both markers for resistance to ascochyta blight and a marker for resistance to anthracnose can clearly make progress toward resistance in the population. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development and characterization of common black bean lines resistant to anthracnose,rust and angular leaf spot in Brazil

Anthracnose, rust and angular leaf spot caused by Colletotrichum lindemuthianum, Uromyces appendiculatus and Pseudocercospora griseola, respectively, are economically important diseases affecting the common bean production in Brazil. The BIOAGRO/UFV bean breeding program developed Rudá-R, a dry bean line with ‘carioca’ seed type, containing the following disease resistance genes: Co-4, Co-6 and Co-10 (anthracnose); Ur-ON (rust) and Phg-1 (angular leaf spot). To transfer this combination of disease resistance genes present in Rudá-R to a black-seeded bean, a backcrossing program aided by molecular markers was conducted, involving Rudá-R (donor genitor) and Diamante Negro (recurrent genitor). Forty black-seeded BC₃F_3:6 lines were obtained with combinations of at least three markers linked to the indicated disease resistance genes. The lines were evaluated for resistance to the three mentioned pathogens. Eight of the lines were homozygous and resistant to all four evaluated races of C. lindemuthianum, but susceptible to race 2047. Four of the lines were homozygous and resistant to two races of U. appendiculatus. Twenty of the lines were homozygous and resistant to the two races of P. griseola tested. Grain yield of the BC₃F_3:6 lines was evaluated during the ‘winter’ season of 2006 and the ‘dry’ season of 2007. All lines had statistically equal or higher yields than Rudá-R and Diamante Negro. Lines were identified that not only were high yielding but also resistant to the three pathogens tested. These lines are potential genotypes for further testing and for release as new black common bean varieties.     

Identification of Rfd1, a novel restorer-of-fertility locus for cytoplasmic male-sterility caused by DCGMS cytoplasm and development of simple PCR markers linked to the Rfd1 locus in radish (Raphanus sativus L.)

Previously, novel cytoplasmic male-sterility (CMS) caused by DCGMS cytoplasm was discovered in radish (Raphanus sativus L.) introduced from Uzbekistan. We performed extensive progeny tests and identified two fertility restorer lines (‘R171’ and ‘R121’) for this new CMS. Two F₁ hybrid populations were self-pollinated and backcrossed to produce F₂ and BC populations. Inheritance patterns of male-sterility in segregating populations varied depending on paternal lines. Segregation of male-sterility in F₂ populations originating from the cross between MS19 and R121 showed that a single locus was involved in fertility restoration. However, populations originating from the cross between MS15 and R171 showed the involvement of more than one restorer-of-fertility genes. The single fertility restorer locus identified in the cross between MS19 and R121 was designated Rfd1 locus. Bulked segregant analysis was performed using RAPD and AFLP, which identified one marker each. Both RAPD and AFLP markers were converted into simple PCR-based co-dominant markers after their isolated flanking sequences were analyzed. Indels 773-bp and 67-bp in length were identified between two Rfd1 allele-linked flanking sequences of the RAPD and AFLP fragments, respectively, then utilized to develop simple PCR markers. In addition, we prove that the newly identified Rfd1 locus is independent of the Rfo locus, another radish fertility restorer for CMS caused by Ogura cytoplasm.