Fine mapping of the restorer gene <Emphasis Type="Italic">Rfp3</Emphasis> from an Iranian primitive rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Key message

A comparative genetics approach allowed to precisely determine the map position of the restorer gene Rfp3 in rye and revealed that Rfp3 and the restorer gene Rfm1 in barley reside at different positions in a syntenic 4RL/6HS segment.

Abstract

Cytoplasmic male sterility (CMS) is a reliable and striking genetic mechanism for hybrid seed production. Breeding of CMS-based hybrids in cereals requires the use of effective restorer genes as an indispensable pre-requisite. We report on the fine mapping of a restorer gene for the Pampa cytoplasm in winter rye that has been tapped from the Iranian primitive rye population Altevogt 14160. For this purpose, we have mapped 41 gene-derived markers to a 38.8 cM segment in the distal part of the long arm of chromosome 4R, which carries the restorer gene. Male fertility restoration was comprehensively analyzed in progenies of crosses between a male-sterile tester genotype and 21 recombinant as well as six non-recombinant BC₄S₂ lines. This approach allowed us to validate the position of this restorer gene, which we have designated Rfp3, on chromosome 4RL. Rfp3 was mapped within a 2.5 cM interval and cosegregated with the EST-derived marker c28385. The gene-derived conserved ortholog set (COS) markers enabled us to investigate the orthology of restorer genes originating from different genetic resources of rye as well as barley. The observed localization of Rfp3 and Rfm1 in a syntenic 4RL/6HS segment asks for further efforts towards cloning of both restorer genes as an option to study the mechanisms of male sterility and fertility restoration in cereals.

     

Genetic mapping of Dn7, a rye gene conferring resistance to the Russian wheat aphid in wheat

The Russian wheat aphid is a significant pest problem in wheat and barley in North America. Genetic resistance in wheat is the most effective and economical means to control the damage caused by the aphid. Dn7 is a rye gene located on chromosome 1RS that confers resistance to the Russian wheat aphid. The gene was previously transferred from rye into a wheat background via a 1RS/1BL translocation. This study was conducted to genetically map Dn7 and to characterize the type of resistance the gene confers. The resistant line '94M370' was crossed with a susceptible wheat cultivar that also contains a pair of 1RS/1BL translocation chromosomes. The F₂ progeny from this cross segregated for resistance in a ratio of 3 resistant: 1 susceptible, indicating a single dominant gene. One-hundred and eleven RFLP markers previously mapped on wheat chromosomes 1A, 1B and 1D, barley chromosome 1H and rye chromosome 1R, were used to screen the parents for polymorphism. A genetic map containing six markers linked to Dn7, encompassing 28.2 cM, was constructed. The markers flanking Dn7 were Xbcd1434 and XksuD14, which mapped 1.4 cM and 7.4 cM from Dn7, respectively. Dn7 confers antixenosis, and provides a higher level of resistance than that provided by Dn4. The applications of Dn7 and the linked markers in wheat breeding are discussed.Communicated by J. Dvorak     

Development of PCR-based markers linked to dominant genes for male-fertility restoration in Pampa CMS of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Cytoplasmatic male sterility (CMS) is the basis for commercial hybrid seed production of rye. Nuclear restorer genes are indispensable for a complete restoration of fertility of the CMS lines. The drawbacks of current European restorer lines require the utilisation of new genetic resources that have been recently detected in an Iranian primitive rye population (IRAN IX) and an Argentinean landrace (Pico Gentario). The introgression of these effective restorer genes (Rfp1 and Rfp2, respectively) into breeding material can be facilitated by marker-assisted selection. Using two F(2) populations based on crosses between the non-restorer inbred line Lo6 and the restorer IRAN IX, as well as Pico Gentario, RAPDs and AFLPs were screened and led to a closely linked marker set for each of these genes. The conversion of the closest markers into fragment-specific sequence-characterised amplified region (SCAR) markers resulted in flanking ranges of 2.9 cM (Rfp1) and 5.2 cM (Rfp2). The application of these markers in backcross programmes is discussed.     

Evolutionary divergence of the rye <Emphasis Type="Italic">Pm17</Emphasis> and <Emphasis Type="Italic">Pm8</Emphasis> resistance genes reveals ancient diversity

Key message

We have isolated a novel powdery mildew resistance gene in wheat that was originally introgressed from rye. Further analysis revealed evolutionary divergent history of wheat and rye orthologous resistance genes.

Abstract

Wheat production is under constant threat from a number of fungal pathogens, among them is wheat powdery mildew (Blumeria graminis f. sp. tritici). Deployment of resistance genes is the most economical and sustainable method for mildew control. However, domestication and selective breeding have narrowed genetic diversity of modern wheat germplasm, and breeders have relied on wheat relatives for enriching its gene pool through introgression. Translocations where the 1RS chromosome arm was introgressed from rye to wheat have improved yield and resistance against various pathogens. Here, we isolated the Pm17 mildew resistance gene located on the 1RS introgression in wheat cultivar ‘Amigo’ and found that it is an allele or a close paralog of the Pm8 gene isolated earlier from ‘Petkus’ rye. Functional validation using transient and stable transformation confirmed the identity of Pm17. Analysis of Pm17 and Pm8 coding regions revealed an overall identity of 82.9% at the protein level, with the LRR domains being most divergent. Our analysis also showed that the two rye genes are much more diverse compared to the variants encoded by the Pm3 gene in wheat, which is orthologous to Pm17/Pm8 as concluded from highly conserved upstream sequences in all these genes. Thus, the evolutionary history of these orthologous loci differs in the cereal species rye and wheat and demonstrates that orthologous resistance genes can take different routes towards functionally active genes. These findings suggest that the isolation of Pm3/Pm8/Pm17 orthologs from other grass species, additional alleles from the rye germplasm as well as possibly synthetic variants will result in novel resistance genes useful in wheat breeding.

     

Identification and mapping of molecular markers linked to rust resistance genes located on chromosome 1RS of rye using wheat-rye translocation lines

The short arm of rye (Secale cereale) chromosome 1 has been widely used in breeding programs to incorporate new disease resistance genes into wheat. Using wheat-rye translocation and recombinant lines, molecular markers were isolated and mapped within chromosomal regions of 1RS carrying rust resistance genes Lr26, Sr31, Yr9 from 'Petkus' and SrR from 'Imperial' rye. RFLP markers previously mapped to chromosome 1HS of barley - flanking the complex Mla powdery mildew resistance gene locus - and chromosome 1DS of Aegilops tauschii - flanking the Sr33 stem rust resistance gene - were shown to map on either side of rust resistance genes on 1RS. Three non cross-hybridising Resistance Gene Analog markers, one of them being derived from the Mla gene family, were mapped within same region of 1RS. PCR-based markers were developed which were tightly linked to the rust resistance genes in 'Imperial' and 'Petkus' rye and which have potential for use in marker-assisted breeding.     

Genetic mapping of nuclear fertility restorer genes for the ‘Polima’ cytoplasmic male sterility in canola (Brassica napus L.) using DNA markers

 Co-segregation of male fertility with DNA markers selected by targeted mapping approaches as being potentially linked to the Rfp1 restorer gene for the pol cytoplasmic male sterility (CMS) was analyzed using two canola (Brassica napus L.) backcross populations. Eleven DNA markers (10 RFLP markers and one RAPD marker) directly linked to the Rfp1 locus were identified. The linkage group containing the Rfp1 locus was found to correspond to B. napus linkage group 18 of Landry et al. (1991). A similar pattern of co-segregation between DNA markers and male fertility was observed in a backcross population segregating for the pol restorer gene Rfp2 from line ‘UM2383’; one RFLP marker, cRF1b, showed perfect linkage with both Rfp1 and Rfp2 and detected identical polymorphic fragments in both the Rfp1 and Rfp2 restorer lines. Our findings indicate that restoration of pol CMS is controlled by a single nuclear genetic locus on linkage group 18 and that Rfp1 and Rfp2 are likely allelic. Received: 2 October 1996 / Accepted: 20 December 1996     

Characterisation of wheat-rye recombinants with RFLP and PCR probes

Summary The introgression of genetic material from alien species into wheat has become an important tool in modern wheat breeding. Ideally, only the trait of interest and no flanking material should be transferred. Random recombination between the genetic material is therefore of paramount importance. In a model system, we examined 17 recombinants putatively between chromosome 1D of wheat and 1R of rye with 60 random RFLP and three PCR markers. The recombinants had been generated by removing the normal effect of the Ph1 gene in the wheat background. Amongst the nine short-arm recombinants, three breakpoints were identified but no differentiation could be made between the five proximal recombinants. For the eight long-arm recombinants analysed only two breakpoints were identified with 36 markers. However, only a single RFLP marker was able to differentiate between the recombinants. Indeed the long-arm results are consistent with the possibility that only the rye telomeric region had been transferred. These results indicate either a strong clustering of the RFLP markers near the centromere or else imply that recombination induced between wheat and rye in the absence of the normal effect of the Ph1 gene occurs at only restricted sites. The results allow new primary recombinants to be selected for intercrossing to generate secondary recombinants which are expected to have a smaller interstitial rye segment than that present in DR-A1.     

Features of Crossability, Haploidy and Polyembryony in Hybrid Combinations between Cultivated Barley Hordeum vulgare L. (2n = 14) and Wheat-Rye Substitution Lines Triticum aestivum L., Cultivar Saratovskaya 29/Secale cereale L., Cultivar Onokhoiskaya

The role of individual chromosomes of rye in the manifestation of crossability and seedling development in hybrid combinations between cultivated barley Hordeum vulgare L., cultivar Nepolegayushchii (2n = 14) and five wheat-rye substitution lines Triticum aestivum L., cultivar Saratovskaya 29/Secale cereale L., cultivar Onokhoiskaya (2n = 40 wheat + 2 rye chromosomes). Crossability, which was measured by two parameters—frequency of set grains and frequency of grains with embryos—was shown to be significantly affected by each of the five rye chromosomes examined: 1R, 2R, 3R, 5R, and 6R; the development of barley haploids was affected by rye chromosomes 1R, 3R, and 5R. We were the first to demonstrate that polyembryony could be induced by mutual effects of barley cytoplasm and rye chromosome 1R. Possible mechanisms controlling the development of haploids and twins in hybrid combinations H. vulgare × T. aestivum/S. cereale are discussed. The conclusion is drawn that hybrid combinations between cultivated barley and wheat-rye substitution lines can serve as new models for studying incompatibility mechanisms in distant crosses and genetic control of parthenogenesis.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 784–792.Original Russian Text Copyright © 2005 by Pershina, Belova, Devyatkina, Rakovtseva, Kravtsova, Shchapova.     

Features of crossability, haploidy and polyembryony in hybrid combinations between common barley Hordeum vulgare L. (2n = 14) and wheat-rye substitution lines Triticum aestivum L., cultivar Saratovskaya 29/Secale cereale L., cultivar Onokhoiskaya

The role of individual chromosomes of rye in the manifestation of crossability and seedling development in hybrid combinations between common barley Hordeum vulgare L., cultivar Nepolegayushchii (2n = 14) and five wheat-rye substitution lines Triticum aestivum L., cultivar Saratovskaya 29/Secale cereale L., cultivar Onokhoiskaya (2n = 40 wheat + 2 rye chromosomes). Crossability, which was measured by two parameters--frequency of set grains and frequency of grains with embryos--was shown to be significantly affected by each of the five rye chromosomes examined: 1R, 2R, 3R, 5R, and 6R; the development of barley haploids was affected by rye chromosomes 1 R, 3R, and 5R. We were the first to demonstrate that polyembryony could be induced by mutual effects of barley cytoplasm and rye chromosome 1R. Possible mechanisms controlling the development of haploids and twins in hybrid combinations H. vulgare x T. aestivum/S. cereale are discussed. The conclusion is drawn that hybrid combinations between common barley and wheat-rye substitution lines can serve as new models for studying incompatibility mechanisms in distant crosses and genetic control of parthenogenesis.     

Production of wheat-rye substitution lines and identification of chromosome composition of karyotypes using C-banding, GISH, and SSR markers

Based on the cross (Triticum aestivum L. × Secale cereale L.) × T. aestivum L., wheat-rye substitution lines (2n = 42) were produced with karyotypes containing, instead of a pair of homologous wheat chromosomes, a homeologous pair of rye chromosomes. The chromosome composition of these lines was described by GISH and C-banding methods, and SSR analysis. The results of genomic in situ hybridization demonstrated that karyotype of these lines included one pair of rye chromosomes each and lacked wheat-rye translocations. C-banding and SSR markers were used to identify rye chromosomes and determine the wheat chromosomes at which the substitution occurred. The lines were designated 1R(1D), 2R(2D)₂, 2R(2D)₃, 3R(3B), 6R(6A)₂. The chromosome composition of lines 1R(1A), 2R(W)₁, 5R(W), 5R(5A), and 6R(W)₁, which were earlier obtained according to the same scheme for crossing, was characterized using methods of telocentric analysis, GISH, C-banding, and SSR analysis. These lines were identified as 1R(1A), 2R(2D)₁, 5R(5D), 5R(5A), and 6R(6A)₁, C-banding of chromosomes belonging to line 1R(1A) revealed the presence of two translocated chromosomes (3DS.3DL-del. and 4AL.W) during simultaneous amplification of SSR markers located on 3DL and 4AS arms. The “combined” long arm of the newly derived chromosome 4A is assumed to be formed from the long arm of chromosome 4AS itself and a deleted segment 3DL. All examined lines are cytologically stable, except for 3R(3B), which does not affect the stability of rye 3R chromosome transfer. Chromosome identification and classification of the lines will permit them to be models for genetic studies that can be used thereafter as promising “secondary gene pools” for the purpose of plant breeding.     

Resistance genes for rye stem rust (SrR) and barley powdery mildew (Mla) are located in syntenic regions on short arm of chromosome.

Genetic stocks were developed for the localization and eventual cloning of the stem rust resistance gene SrR that occurs in wheat lines carrying the 1RS translocation from Secale cereale 'Imperial' rye. We have used a mutation-based approach for molecular analysis of the SrR region in rye. Forty-one independent mutants resulting in loss of SrR resistance were isolated: many of these were deletions of various sizes that were used to locate SrR with respect to chromosome group 1S markers. The analysis of the mutants showed that markers about 1 Mb apart flanking the barley Mla locus also flank SrR. Additionally, three of the approximately 20 closely related sequences of Mla in rye are deleted in each of six interstitial deletion mutants of SrR. The results indicate that the SrR region in rye is syntenic to the Mla region in barley or that SrR is possibly orthologous to the Mla locus.     

Molecular mapping of wheat. Homoeologous group 3.

A prerequisite for molecular level genetic studies and breeding in wheat is a molecular marker map detailing its similarities with those of other grass species in the Gramineae family. We have constructed restriction fragment length polymorphism maps of the A-, B-, and D-genome chromosomes of homoeologous group 3 of hexaploid wheat (Triticum aestivum L. em. Thell) using 114 F7-8 lines from a synthetic x bread wheat cross. The map consists of 58 markers spanning 230 cM on chromosome 3A, 62 markers spanning 260 cM on 3B, and 40 markers spanning 171 cM on 3D. Thirteen libraries of genomic or cDNA clones from wheat, barley, and T. tauschii, the wheat D genome donor, are represented, facilitating the alignment and comparison of these maps with maps of other grass species. Twenty-four clones reveal homoeoloci on two of the three genomes and the associated linkages are largely comparable across genomes. A consensus sequence of orthologous loci in grass species genomes is assembled from this map and from existing maps of the chromosome-3 homoeologs in barley (Hordeum spp.), T. tauschii, and rice (Oryza spp.). It illustrates the close homoeology among the four species and the partial homoeology of wheat chromosome 3 with oat (Avena spp.) chromosome C. Two orthologous red grain color genes, R3 and R1, are mapped on chromosome arms 3BL and 3DL.     

New wheat-rye 5DS-4RS·4RL and 4RS-5DS·5DL translocation lines with powdery mildew resistance

Powdery mildew is one of the serious diseases of wheat (Triticum aestivum L., 2n = 6 × = 42, genomes AABBDD). Rye (Secale cereale L., 2n = 2 × = 14, genome RR) offers a rich reservoir of powdery mildew resistant genes for wheat breeding program. However, extensive use of these resistant genes may render them susceptible to new pathogen races because of co-evolution of host and pathogen. Therefore, the continuous exploration of new powdery mildew resistant genes is important to wheat breeding program. In the present study, we identified several wheat-rye addition lines from the progeny of T. aestivum L. Mianyang11 × S. cereale L. Kustro, i.e., monosomic addition lines of the rye chromosomes 4R and 6R; a disomic addition line of 6R; and monotelosomic or ditelosomic addition lines of the long arms of rye chromosomes 4R (4RL) and 6R (6RL). All these lines displayed immunity to powdery mildew. Thus, we concluded that both the 4RL and 6RL arms of Kustro contain powdery mildew resistant genes. It is the first time to discover that 4RL arm carries powdery mildew resistant gene. Additionally, wheat lines containing new wheat-rye translocation chromosomes were also obtained: these lines retained a short arm of wheat chromosome 5D (5DS) on which rye chromosome 4R was fused through the short arm 4RS (designated 5DS-4RS·4RL; 4RL stands for the long arm of rye chromosome 4R); or they had an extra short arm of rye chromosome 4R (4RS) that was attached to the short arm of wheat chromosome 5D (5DS) (designated 4RS-5DS·5DL; 5DL stands for the long arm of wheat chromosome 5D). These two translocation chromosomes could be transmitted to next generation stably, and the wheat lines containing 5DS-4RS·4RL chromosome also displayed immunity to powdery mildew. The materials obtained in this study can be used for wheat powdery mildew resistant breeding program.     

A consensus linkage map of rye (Secale cereale L.) including 374 RFLPs, 24 isozymes and 15 gene loci

 Consensus linkage maps were constructed for all seven rye chromosomes using 12 basic RFLP maps. The maps presented contain a total of 413 markers. The number of markers per chromosome varies from 41 (chromosome 3R) to 83 (chromosome 1R). In addition to 374 RFLP and 24 isozyme markers 15 gene loci were incorporated, determining the traits reduced plant height, self fertility, male sterility restoration, vernalization response, resistance against powdery mildew, chlorophyll deficiency, hairy leaf sheath, hairy peduncle, waxy endosperm, waxless plant and absence of ligules. The maps presented allow the selection of markers for the fine mapping of certain regions of the rye genome. In terms of the known chromosomal rearrangements within the Triticeae its utilization can also be extended for mapping in wheat and barley. Received: 13 February 1998 / Accepted: 26 May 1998     

The gibberellic-acid insensitive dwarfing gene<Emphasis Type="Italic"> sdw3</Emphasis> of barley is located on chromosome 2HS in a region that shows high colinearity with rice chromosome 7L

In this study, comparative high resolution genetic mapping of the GA-insensitive dwarfing gene sdw3 of barley revealed highly conserved macrosynteny of the target region on barley chromosome 2HS with rice chromosome 7L. A rice contig covering the sdw3-orthologous region was identified and subsequently exploited for marker saturation of the target interval in barley. This was achieved by (1) mapping of rice markers from the orthologous region of the rice genetic map, (2) mapping of rice ESTs that had been physically localized on the rice contig, or (3) mapping of barley ESTs that show strong sequence similarity to coding sequences present in the rice contig. Finally, the sdw3 gene was mapped to an interval of 0.55 cM in barley, corresponding to a physical distance of about 252 kb in rice, after employing orthologous EST-derived rice markers. Three putative ORFs were identified in this interval in rice, which exhibited significant sequence similarity to known signal regulator genes from different species. These ORFs can serve as starting points for the map-based isolation of the sdw3 gene from barley.Communicated by R. Hagemann     

Fine mapping and candidate gene analysis of the nuclear restorer gene Rfp for pol CMS in rapeseed (Brassica napus L.)

The Polima (pol) system of cytoplasmic male sterility (CMS) in rapeseed is widely used in China for commercial hybrid seed production. Genetic studies have shown that its fertility restorer gene (Rfp) is monogenic dominant. For fine mapping of the Rfp gene, a near isogenic line comprising 3,662 individuals of BC(14)F(1) generation segregating for the Rfp gene was created. Based on the sequences of two SCAR markers, SCAP0612ST and SCAP0612EM2, developed by Zhao et al. (Genes Genom 30(3):191-196, 2008) and the synteny region of Brassica napus and other Brassica species, 13 markers strongly linked with the Rfp gene were identified. By integrating three of these markers to the published linkage map, the Rfp gene was mapped on linkage group N9 of B. napus. Using these markers, the Rfp locus was narrowed down to a 29.2-kb genomic region of Brassica rapa. Seven open reading frames (ORFs) were predicted in the target region, of these, ORF2, encoding a PPR protein, was the most likely candidate gene of Rfp. These results lay a solid foundation for map-based cloning of the Rfp gene and will be helpful for marker-assisted selection of elite CMS restorer lines.     

Comparative RFLP-based genetic maps of barley chromosome 5 (1H) and rye chromosome 1R

Summary A genetic map of barley chromosome 5 (1H) was constructed using DNA markers. Seventeen loci were mapped to 15 locations, and these included the known-function loci (in order from the most distal on the long arm) XAdh (alcohol dehydrogenase), XLec (homologous to wheat germ agglutinin), XHor3 (D-hordein), XPpdk (pyruvate orthophosphate dikinase), centromere, XIcal (chymotrypsin inhibitor), and 6 loci in the B- and C-hordein cluster towards the end of the short arm. The gene order on the barley map agreed closely with that of chromosome 1 of rye. Intervarietal comparisons showed that single-copy cDNA and genomic DNA probes revealed about twice the level of RFLPs found in wheat.     

Molecular and chromosomal organization of DNA sequences coding for the ribosomal RNAs in cereals

The chromosomal locations of ribosomal DNA in wheat, rye and barley have been determined by in situ hybridization using high specific activity ¹²⁵I-rRNA. The 18S-5.8S-26S rRNA gene repeat units in hexaploid wheat (cv. Chinese Spring) are on chromosomes 1B, 6B and 5D. In rye (cv. Imperial) the repeat units occur at a single site on chromosome 1R(E), while in barley (cv. Clipper) they are on both the chromosomes (6 and 7) which show secondary constrictions. In wheat and rye the major 5S RNA gene sites are close to the cytological secondary constrictions where the 18S-5.8S-26S repeating units are found, but in barley the site is on a chromosome not carrying the other rDNA sequences. — Restriction enzyme and R-loop analyses showed the 18S-5.8S-26S repeating units to be approximately 9.5 kb long in wheat, 9.0 kb in rye and barley to have two repeat lengths of 9.5 kb and 10 kb. Electron microscopic and restriction enzyme data suggest that the two barley forms may not be interpersed. Digestion with EcoR1 gave similar patterns in the three species, with a single site in the 26S gene. Bam H1 digestion detected heterogeneity in the spacer regions of the two different repeats in barley, while in rye and wheat heterogeneity was shown within the 26S coding sequence by an absence of an effective Bam H1 site in some repeat units. EcoR1 and Bam H1 restriction sites have been mapped in each species. — The repeat unit of the 5S RNA genes was approximately 0.5 kb in wheat and rye and heterogeneity was evident. The analysis of the 5S RNA genes emphasizes the homoeology between chromosomes 1B of wheat and 1R of rye since both have these genes in the same position relative to the secondary constriction. In barley we did not find a dominant monomer repeat unit for the 5S genes.