Locating the petunia Rf gene on a 650-kb DNA fragment

 A bulked segregant analysis was conducted in order to find RAPD and AFLP markers linked to the restorer of fertility (Rf ) gene in petunia. One RAPD marker, OP704, and one AFLP marker, ECCA/ MACT, were found to be closely linked to Rf (<1 cM) in our mapping population produced from an intraspecific Petunia hybrida cross. These two single-copy markers bracketing Rf were then mapped as RFLPs on the tomato map. Despite some rearrangement between the petunia and the tomato genomes, this synteny survey revealed two tomato markers, TG250 and CT24, closely linked to Rf. Physical mapping indicates that CT24, OP704 and ECCA/MACT lie on the same 650-kb MluI fragment. A physical to genetic distance ratio of 400 kb/cM around the Rf gene should make it feasible to identify markers physically very close to Rf. Received: 20 August 1997 / Accepted: 21 October 1997     

Development of SNP markers linked to the <Emphasis Type="Italic">L</Emphasis> locus in <Emphasis Type="Italic">Capsicum</Emphasis> spp. by a comparative genetic analysis

In pepper, the TMV resistance locus L is syntenic to the tomato I2 and the potato R3 loci on chromosome 11. In this report, we identified pepper bacterial artificial chromosome (BAC) clones corresponding to the I2 and R3 loci and developed L-linked markers using the BAC sequence information. A BAC library was screened using the tomato I2C-1 gene as a probe. The resulting clones were sorted further by PCR screening, sequencing, and genetic mapping. A linkage analysis revealed that BAC clone 082F03 could be anchored to the target region near TG36 on chromosome 11. Using the 082F03 sequence, more BAC clones were identified and a BAC contig spanning 224 kb was constructed. Gene prediction analysis showed that there were at least three I2/R3 R gene analogs (RGAs) in the BAC contig. Three DNA markers closely linked (about 1.2 cM) to the L ⁴ gene were developed by using the BAC contig sequence. The single nucleotide polymorphism marker 087H3T7 developed in this study was subjected to linkage analysis in L ⁴ - and L ³ -segregating populations together with previously developed markers. The 189D23M marker, which is known to co-segregate with L ³ , was located on the opposite side of 087H3T7, about 0.7 cM away from L ⁴ . This supports the idea that L ³ and L ⁴ may be different genes closely linked within the region instead of different alleles at the same locus. Finally, use of flanking markers in molecular breeding program for introgression of L ⁴ to elite germplasm against most aggressive tobamoviruses pathotype P_1,2,3 is discussed.     

Physical mapping of the <Emphasis Type="Italic">Rf</Emphasis><Subscript><Emphasis Type="Italic">1</Emphasis></Subscript> fertility-restoring gene to a 100 kb region in cotton

Cytoplasmic male sterility (CMS) plays an important role in crop heterosis exploitation. Determining one or more nuclear genes that can restore male fertility to CMS is essential for developing hybrid cultivars. Genetic and physical mapping is the standard technique required for isolating these restoration genes. By screening 2,250 simple sequence repeat (SSR) primer pairs in cotton (Gossypium hirsutum L.), we identified five new SSR markers that are closely linked to the Rf ₁ gene, a fertility restorer gene of cotton for CMS-D2. Based on our previous fine mapping of the Rf ₁ gene and assemblage of three published STS markers, we constructed a high-resolution genetic map of Rf ₁ containing 13 markers in a genetic distance of 0.9 cM. The 13 molecular markers were used to screen a bacterial artificial chromosome (BAC) library from a restorer line 0-613-2R containing Rf ₁ gene, which yielded 50 single positive clones. There was an average of 3.8 clones ranging from 1 to 12 BAC clones per PCR marker. These 50 clones produced an average insert size of 120 kb (ranging between 80 and 225 kb). Thirty-five primer pairs were designed based on 38 sequences of BAC ends, and two new STS markers tightly linked to Rf ₁ gene have been tagged and integrated into this map. The physical map for the Rf ₁ gene was constructed by fingerprinting the positive clones digested with the HindIII enzyme. We were able to delimit the possible location of the Rf ₁ gene to a minimum of two BAC clones spanning an interval of approximately 100 kb between two clones designated 081-05K and 052-01N. Further work using these two BAC clones will lead to isolation of the Rf ₁ gene in cotton.     

Molecular markers from a BAC contig spanning the Rdr1 locus: a tool for marker-assisted selection in roses

We constructed a BAC contig of about 300 kb spanning the Rdr1 locus for black spot resistance in Rosa multiflora hybrids, using a new BIBAC library from DNA of this species. From this contig, we developed broadly applicable simple sequence repeat (SSR) markers tightly linked to Rdr1, which are suitable for genetic analyses and marker-assisted selection in roses. As a source for the high molecular weight DNA, we chose the homozygous resistant R. multiflora hybrid 88/124-46. For the assembly of the BAC contig, we made use of molecular markers derived from a previously established R. rugosa contig. In order to increase the resolution for fine mapping, the size of the population was increased to 974 plants. The genomic region spanning Rdr1 is now genetically restricted to 0.2 cM, corresponding to a physical distance of about 300 kb. One single-stranded conformational polymorphism (SSCP) and one SSR marker cosegregate with the Rdr1-mediated black spot resistance, while one SSR and several cleaved amplified polymorphic sequence or SSCP markers are very tightly linked with one to three recombinants among the 974 plants. The benefits of the molecular markers developed from the R. multiflora contig for the genetic analysis of roses and the integration of rose genetic maps are discussed.     

Identification of AFLP markers linked to the male fertility restorer gene of CMS (Moricandia arvensis) Brassica juncea and conversion to SCAR marker

We have developed a cytoplasmic male sterile (CMS) line of Brassica juncea through somatic hybridization with Moricandia arvensis and introgressed the fertility restorer gene into B. juncea. This fertility restorer locus is unique in that it is capable of restoring male fertility to two other alloplasmic CMS systems of B. juncea. As a first step toward cloning of this restorer gene we attempted molecular tagging of the Rf locus using the amplified fragment length polymorphism (AFLP) technique. A BC₁F₁ population segregating for male sterility/fertility was used for tagging using the bulk segregant analysis method. Out of 64 primer combinations tested in the bulks, 5 combinations gave polymorphic amplification patterns. Further testing of these primers in individual plants showed four amplicons associated with the male fertility trait. Polymorphic amplicons were cloned and used for designing SCAR primers. One of the SCAR primers generated amplicons mostly in the fertile plants. Linkage analysis using MAPMAKER showed two AFLP and one SCAR markers linked to the male fertility gene with a map distance ranging from 0.6 to 2.9 cM. All the markers are located on one side of the Rf locus.     

Construction of a BAC library and a physical map of a major QTL for CBB resistance of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

A major quantitative trait loci (QTL) conditioning common bacterial blight (CBB) resistance in common bean (Phaseolus vulgaris L.) lines HR45 and HR67 was derived from XAN159, a resistant line obtained from an interspecific cross between common bean lines and the tepary bean (P. acutifolius L.) line PI319443. This source of CBB resistance is widely used in bean breeding. Several other CBB resistance QTL have been identified but none of them have been physically mapped. Four molecular markers tightly linked to this QTL have been identified suitable for marker assisted selection and physical mapping of the resistance gene. A bacterial artificial chromosome (BAC) library was constructed from high molecular weight DNA of HR45 and is composed of 33,024 clones. The size of individual BAC clone inserts ranges from 30 kb to 280 kb with an average size of 107 kb. The library is estimated to represent approximately sixfold genome coverage. The BAC library was screened as BAC pools using four PCR-based molecular markers. Two to seven BAC clones were identified by each marker. Two clones were found to have both markers PV-tttc001 and STS183. One preliminary contig was assembled based on DNA finger printing of those positive BAC clones. The minimum tiling path of the contig contains 6 BAC clones spanning an estimated size of 750 kb covering the QTL region.     

A CAPS marker associated with the partial restoration of cytoplasmic male sterility in chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS), an economically important trait for hybrid seed production in many crops, is a maternally inherited trait in which a plant fails to produce functional anthers, pollen grains, or male gametes. It has long been reported that the restoration of CMS in chili pepper is controlled by a major nuclear gene termed restorer-of-fertility (Rf), along with several modifiers and some environmental factors. In this study, we identified the partial restoration (pr) locus related to the fertility restoration of CMS, demonstrated the inheritance of the trait, and developed a CAPS marker closely linked to the locus. The partially restored plant had normal anthers that produced a mix of normal and aborted pollen grains that stuck tightly to the anther wall, even after dehiscence. This trait was expressed only when the pepper plant had the sterile (S) cytoplasm and homozygous recessive pr alleles. A total of 768 AFLP primer combinations were screened, and bulked segregant analysis (BSA) was performed by preparing two pools of eight Pr/Pr (fully fertile) and eight pr/pr (partially fertile) plants, respectively, selected from the 87 individuals of the F₂ segregating population. Of the eight Pr-linked AFLP markers that were identified, E-AGC/M-GCA₁₂₂ and E-TCT/M-CCG₁₁₆ were the closest to the locus, estimated at about 1.8 cM in genetic distance. E-AGC/M-GCA₁₂₂ was converted into a CAPS marker, PR-CAPS, based on the sequences of the internal and flanking regions of the AFLP fragment. This PR-CAPS marker could be useful in selecting fully fertile lines (Pr/Pr) and eliminating partially fertile (pr/pr) and potential (Pr/pr) lines in segregant populations during the development of new inbred restorer lines.     

Development of a candidate gene marker for Rf 1 based on a PPR gene in cytoplasmic male sterile CMS-D2 Upland cotton

The cytoplasmic male sterility (CMS) system is convenient and efficient for hybrid seed production in Upland cotton (Gossypium hirsutum L.). However, it has not been widely used because of limited restorer lines carrying the restorer gene Rf ₁ in the CMS-D2 system. In this study, the fertility segregation in a backcross (BC8F1) population of 409 individuals and an F2 population of 695 plants confirmed that the fertility restoration was determined by one dominant restorer gene (Rf ₁ ). A sequence alignment showed that 13 Rf ₁ -linked simple sequence repeat marker sequences were distributed on nine scaffolds of chromosome 9 in the sequenced D5 genome of G. raimondii Ulbrich. Ten pentotricopeptide repeat (PPR)-like genes were identified on two scaffolds, including Scaffold 333 where nine PPR-like genes were clustered in a region of about 160 kb. Among them, PPR-like gene Cotton_D_gene_10013437 was identified as the candidate for the Rf ₁ gene through a comparative sequence analysis of the homologous gene among sterile (A), maintainer (B) and restorer (R) lines, and co-segregation analysis. Compared with the non-restoring lines, the restorer had a 9-nucleotide (nt) insertion and a single nucleotide polymorphism (SNP) 8 nt upstream of the insertion at the 3′ untranslated regions (3′ UTRs) in this gene. A cleaved amplified polymorphic sequence (CAPS) marker named CAPS-R was developed from the SNP site using the restriction enzyme DraI, and was further used to track the restorer gene and its homozygous or heterozygous status in molecular breeding for restorer lines. A marker-assisted selection system using the Rf ₁ -specific CAPS-R marker and a CMS-D2 cytoplasm-specific SCAR marker was established to distinguish the three-line hybrids from other genotypes.     

Molecular mapping of the Rf 3 fertility restoration gene to facilitate its utilization in breeding confection sunflower

The inheritance of a previously identified dominant Rf gene in the confection sunflower line RHA 280 has been determined and designated as Rf ₃ . This study reports the mapping of the Rf ₃ locus using an F2 population of 227 individuals derived from CMS HA 89-3149 × RHA 280. Bulked segregant analysis with 624 pairs of simple sequence repeat (SSR) primers and sequence tagged site (STS) primers identified two polymorphic SSR markers each of linkage groups (LGs) 7 and 11 from a previous map. Results on 90 F2 individuals with 42 polymorphic markers of LGs 7 and 11 indicated that the Rf ₃ gene was linked with eight markers on LG 7, including five SSR markers (ORS328, ORS331, ORS928, ORS966, and ORS1092) and three expressed sequence tag (EST)-SSR markers (HT619-1, HT619-2, and HT1013). Further analysis of the total F2 population of 227 individuals identified a co-dominant marker, ORS328, linked to Rf ₃ at a genetic distance of 0.7 cM on one side, and a female-dominant marker HT1013 at 12.6 cM proximal to Rf ₃ on the other side; a genetic distance of 47.1 cM for LG 7 was covered. This is the first report of an Rf gene from the confection sunflower. The closely linked marker to Rf ₃ will facilitate marker-assisted selection, and provide a basis for cloning of this gene.     

Genetic analysis and molecular mapping of a new fertility restorer gene Rf8 for Triticum timopheevi cytoplasm in wheat (Triticum aestivum L.) using SSR markers

A study on mode of inheritance and mapping of fertility restorer (Rf) gene(s) using simple sequence repeat (SSR) markers was conducted in a cross of male sterile line 2041A having Triticum timopheevi cytoplasm and a restorer line PWR4099 of common wheat (Triticum aestivum L.). The F₁ hybrid was completely fertile indicating that fertility restoration is a dominant trait. Based on the pollen fertility and seed set of bagged spikes in F₂ generation, the individual plants were classified into fertile and sterile groups. Out of 120 F₂ plants, 97 were fertile and 23 sterile (based on pollen fertility) while 98 plants set ≥5 seeds/spike and 22 produced ≤4 or no seed. The observed frequency fits well into Mendelian ratio of 3 fertile: 1 sterile with χ² value of 2.84 for pollen fertility and 2.17 for seed setting indicating that the fertility restoration is governed by a single dominant gene in PWR4099. The three linked SSR markers, Xwmc503, Xgwm296 and Xwmc112 located on the chromosome 2DS were placed at a distance of 3.3, 5.8 and 6.7 cM, respectively, from the Rf gene. Since, no known Rf gene is located on the chromosome arm 2DS, the Rf gene in PWR4099 is a new gene and proposed as Rf8. The closest SSR marker, Xwmc503, linked to the Rf8 was validated in a set of Rf, maintainer and cytoplasmic male sterile lines. The closely linked SSR marker Xwmc503 may be used in marker-assisted backcross breeding facilitating the transfer of fertility restoration gene Rf8 into elite backgrounds with ease.     

An approach to cloning of Pi-b rice blast resistance gene

A contig of clones from BAC rice genomic library encompassing blast resistance gene Pi-b was constructed. On an average eight clones (8 ± 2.6) were picked up by each marker, which was expected basing on the BAC library size (Nakamura et al. 1997). The 2.4 cM distance between flanking RFLP markers G 1234 and RZ 213 (Miyamoto et al. 1996) was spanned with 4 steps of contig including 25 clones. The physical distance of 370 kb between flanking markers corresponds to a small ratio of physical and genetical distances (155 kb/cM) due to a probable structure of the gene locus near the telomeric end of the chromosome. Markers cosegregating with blast resistance against Magnoporthe grisea were localized in a 2 kb restriction fragment. A new border marker was found on the telomeric side of the Pi-b gene, less than 10 kb from cosegregating markers. No clear marker for the centromeric side of the gene was found but the position of Pi-b rice blast resistant gene was narrowed to within at least 50 kb, which is to our knowledge the most precised estimation of the position of this gene.     

Molecular mapping of<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis> f. sp.<Emphasis Type="Italic"> ciceris</Emphasis> race 3 resistance gene in chickpea

Sequence-tagged microsatellite site (STMS) and sequence-tagged site (STS) markers linked closely to Fusarium oxysporum f. sp. ciceris race 3 resistance gene in chickpea were identified, and linkage between three wilt resistance genes was elucidated. The resistance to race 3 in chickpea germplasm accession WR-315 was inherited as a single gene, designated foc-3, in 100 F₇ recombinant inbred lines derived from the cross of WR-315 (resistant) × C-104 (susceptible). The foc-3 gene was mapped 0.6 cM from STMS markers TA96 and TA27 and STS marker CS27A. Another STMS marker, TA194, at 14.3 cM, flanked the gene on the other side. Linkage between foc-3 and two other chickpea wilt resistance genes, foc-1 (syn. h ₁ ) and foc-4, was established. foc-3 was mapped 9.8 cM from foc-1 and 8.7 cM from foc-4, whereas foc-1 and foc-4 are closely linked at 1.1 cM. The identification of closely linked markers to resistance genes will facilitate marker-assisted selection for introgression of the race 3 resistance gene to susceptible chickpea lines.Communicated by H.C. Becker     

Inheritance and molecular mapping of two fertility-restoring loci for Honglian gametophytic cytoplasmic male sterility in rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.)

The Honglian cytoplasmic male sterility (cms-HL) system, a novel type of gametophytic CMS in indica rice, is being used for the large-scale commercial production of hybrid rice in China. However, the genetic basis of fertility restoration (Rf) in cms-HL remains unknown. Previous studies have shown that fertility restoration is controlled by a single locus located on chromosome 10, close to the loci Rf1 and Rf4, which respond to cms-BT and cms-WA, respectively. To determine if the Rf locus for cms-HL is different from these Rf loci and to establish fine-scale genetic and physical maps for map-based cloning of the Rf gene, high-resolution mapping of the Rf gene was carried out using RAPD and microsatellite markers in three BCF₁ populations. The results of the genetic linkage analysis indicated that two Rf loci respond to cms-HL, and that these are located in different regions of chromosome 10. One of these loci, Rf5 , co-segregates with the SSR marker RM3150, and is flanked by RM1108 and RM5373, which are 0.9 cM and 1.3 cM away, respectively. Another Rf locus, designated as Rf6(t), co-segregates with RM5373, and is flanked by RM6737 and SBD07 at genetic distances of 0.4 cM. The results also demonstrated these loci are distinct from Rf1 and Rf4. A 105-kb BAC clone covering the Rf6(t) locus was obtained from a rice BAC library. The sequence of a 66-kb segment spanning the Rf6(t) locus was determined by a BLASTX search in the genomic sequence database established for the cultivar 93-11.Communicated by R. Hagemann     

Molecular mapping and inheritance of restoration of fertility (<Emphasis Type="Italic">Rf</Emphasis>) in A4 hybrid system in pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp.)

Key message

We report molecular mapping and inheritance of restoration of fertility (Rf) in A4 hybrid system in pigeonpea. We have also developed PCR-based markers amenable to low-cost genotyping to identify fertility restorer lines.

Abstract

Commercial hybrids in pigeonpea are based on A4 cytoplasmic male sterility (CMS) system, and their fertility restoration is one of the key prerequisites for breeding. In this context, an effort has been made to understand the genetics and identify quantitative trait loci (QTL) associated with restoration of fertility (Rf). One F₂ population was developed by crossing CMS line (ICPA 2039) with fertility restorer line (ICPL 87119). Genetic analysis has shown involvement of two dominant genes in regulation of restoration of fertility. In parallel, the genotyping-by-sequencing (GBS) approach has generated ~?33 Gb data on the F₂ population. GBS data have provided 2457 single nucleotide polymorphism (SNPs) segregating across the mapping population. Based on these genotyping data, a genetic map has been developed with 306 SNPs covering a total length 981.9 cM. Further QTL analysis has provided the region flanked by S8_7664779 and S8_6474381 on CcLG08 harboured major QTL explained up to 28.5% phenotypic variation. Subsequently, sequence information within the major QTLs was compared between the maintainer and the restorer lines. From this sequence information, we have developed two PCR-based markers for identification of restorer lines from non-restorer lines and validated them on parental lines of hybrids as well as on another F₂ mapping population. The results obtained in this study are expected to enhance the efficiency of selection for the identification of restorer lines in hybrid breeding and may reduce traditional time-consuming phenotyping activities.

     

Genomic analysis and marker development for the Tsn1 locus in wheat using bin-mapped ESTs and flanking BAC contigs

The wheat Tsn1 gene confers sensitivity to the host-selective toxin Ptr ToxA produced by the tan spot fungus (Pyrenophora tritici-repentis). The long-term goal of this research is to isolate Tsn1 using a positional cloning approach. Here, we evaluated 54 ESTs (expressed sequence tags) physically mapped to deletion bin 5BL 0.75–0.76, which is a gene-rich region containing Tsn1. Twenty-three EST loci were mapped as either PCR-based single-stranded conformational polymorphism or RFLP markers in a low-resolution wheat population. The genetic map corresponding to the 5BL 0.75–0.76 deletion bin spans 18.5 cM and contains 37 markers for a density of 2 markers/cM. The EST-based genetic map will be useful for tagging other genes, establishing colinearity with rice, and anchoring sequence ready BAC contigs of the 5BL 0.75–0.76 deletion bin. High-resolution mapping showed that EST-derived markers together with previously developed AFLP-derived markers delineated Tsn1 to a 0.8 cM interval. Flanking markers were used to screen the Langdon durum BAC library and contigs of 205 and 228 kb flanking Tsn1 were assembled, sequenced, and anchored to the genetic map. Recombination frequency averaged 760 kb/cM across the 228 kb contig, but no recombination was observed across the 205 kb contig resulting in an expected recombination frequency of more than 10 Mb/cM. Therefore, chromosome walking within the Tsn1 region may be difficult. However, the sequenced BACs allowed the identification of one microsatellite in each contig for which markers were developed and shown to be highly suitable for marker-assisted selection of Tsn1.     

Localization of<Emphasis Type="Italic"> jointless-2</Emphasis> gene in the centromeric region of tomato chromosome 12 based on high resolution genetic and physical mapping

Abstract Abscission is a universal process whereby plants shed their organs, such as flowers, fruit and leaves. In tomato, the non-allelic mutations jointless and jointless-2 have been discovered as recessive mutations that completely suppress the formation of pedicel abscission zones. A high resolution genetic map of jointless-2 was constructed using 1,122 jointless F₂ plants. Restriction fragment length polymorphism (RFLP) marker RPD140 completely co-segregated with the jointless-2 locus and mapped in a 2.4 cM interval between RFLP markers CD22 and TG618. To chromosome walk to jointless-2, all three markers were used to screen a bacterial artificial chromosome (BAC) library and contigs were developed. Intensive efforts to expand and merge the BAC contigs were unsuccessful because of the highly repetitive sequence content on the distal ends of each contig. To determine the physical distance between and the orientation of the three contigs, we used high resolution pachytene fluorescence in situ hybridization (FISH) mapping. The RPD140 contig was positioned in the centromeric region of chromosome 12 between two large pericentric heterochromatin blocks, about 50 Mb from the TG618 contig on the short arm and 10 Mb from the CD22 contig on the long arm, respectively. Based on high resolution genetic and physical mapping, we conclude that the jointless-2 gene is located within or near the chromosome 12 centromere where 1 cM is approximately 25 Mb in length.Communicated by Q. ZhangM.A. Budiman, S-B. Chang and S. Lee contributed equally to the work.     

Genetic mapping of a new semi-dwarf gene, <Emphasis Type="Italic">sd-t(t)</Emphasis>, in indica rice and estimating of the physical distance of the mapping region

Application and functional study of dwarf and semi-dwarf genes are of great importance to both crop breeding and molecular biology. A new semi-dwarf gene, sd-t(t), non-allelic to sd-1, had been identified in an indica rice variety, Aitaiyin 2. In this study the gene was genetically mapped by using an F₂ population, which consisted of 474 individuals developed from a cross between Aitaiyin 2 and B30. The sd-t(t) gene was located between the RFLP markers R514 and R1408B with a distance of 1.1 cM to R514, and 4.5 cM to R1408B on chromosome 4. A physical contig covering the sd-t(t) mapping region was further constructed by screening a BAC library with R514 and R1408B as probes, and the physical distance between R514 and R1408B was estimated at approximately 147 kb. This result will facilitate map-based cloning of the sd-t(t) gene.     

Diversifying Sunflower Germplasm by Integration and Mapping of a Novel Male Fertility Restoration Gene

The combination of a single cytoplasmic male-sterile (CMS) PET-1 and the corresponding fertility restoration (Rf) gene Rf₁ is used for commercial hybrid sunflower (Helianthus annuus L., 2n = 34) seed production worldwide. A new CMS line 514A was recently developed with H. tuberosus cytoplasm. However, 33 maintainers and restorers for CMS PET-1 and 20 additional tester lines failed to restore the fertility of CMS 514A. Here, we report the discovery, characterization, and molecular mapping of a novel Rf gene for CMS 514A derived from an amphiploid (Amp H. angustifolius/P 21, 2n = 68). Progeny analysis of the male-fertile (MF) plants (2n = 35) suggested that this gene, designated Rf₆, was located on a single alien chromosome. Genomic in situ hybridization (GISH) indicated that Rf₆ was on a chromosome with a small segment translocation on the long arm in the MF progenies (2n = 34). Rf₆ was mapped to linkage group (LG) 3 of the sunflower SSR map. Eight markers were identified to be linked to this gene, covering a distance of 10.8 cM. Two markers, ORS13 and ORS1114, were only 1.6 cM away from the gene. Severe segregation distortions were observed for both the fertility trait and the linked marker loci, suggesting the possibility of a low frequency of recombination or gamete selection in this region. This study discovered a new CMS/Rf gene system derived from wild species and provided significant insight into the genetic basis of this system. This will diversify the germplasm for sunflower breeding and facilitate understanding of the interaction between the cytoplasm and nuclear genes.     

Development and mapping of AFLP markers linked to the sorghum fertility restorer gene <Emphasis Type="Italic">rf4</Emphasis>

The restoration of male fertility in the sorghum IS1112 C (A3) male-sterile cytoplasm is through a two-gene gametophytic system involving complementary action of the restoring alleles Rf3 and Rf4. To develop markers suitable for mapping rf4, AFLP technology was applied to bulks of sterile and fertile individuals from a segregating BC₃F₁ population. Three AFLP markers linked to rf4 were identified and subsequently converted to STS/CAPS markers, two of which are co-dominant. Based on a population of 378 BC₁F₁ individuals, two STS/CAPS markers, LW7 and LW8, mapped to within 5.31 and 3.18 cM, respectively, of rf4, while an STS marker, LW9, was positioned 0.79 cM on the flanking side of rf4. Markers LW8 and LW9 were used to screen sorghum BAC libraries to identify the genomic region encoding rf4. A series of BAC clones shown to represent a genomic region of linkage group E were identified by the rf4-linked markers. A contig of BAC clones flanking the LW9 marker represent seed clones on linkage group E, from which fine mapping of the rf4 locus and chromosome walking can be initiated. Received: 20 June 2001 / Accepted: 3 August 2001