Male Fertility Restoration Research Articles

Evaluation of the cost of restoration of male fertility inBrassica napus

Gynodioecy frequently results from the interplay of mitochondrial cytoplasmic male sterility (CMS) and nuclear fertility-restoration genes. Models suggest that maintaining cytonuclear gynodioecy requires that restorer genes incur a cost to fitness because otherwise they would increase toward fixation. Direct tests of costs of restorer alleles require knowledge of the underlying genetics of sex determination. We use a well characterized CMS system in Brassica napus L. to measure aspects of fitness in four lineages that vary in whether they carry the pol CMS gene or male-fertile cytoplasm (cam), and whether they carry the Rfp restorer of pol or Rfn restorer of the nap CMS gene. As expected, plants with pol CMS and only the Rfn restorer experienced reduced flower size, stamen length, and pollen counts. Plants with pol and the Rfp restorer showed incomplete restoration with shorter stamens than both lines with cam cytoplasm and reduced pollen counts compared with plants with cam cytoplasm and the Rfp restorer. Among plants with cam cytoplasm, pollen counts were higher for those with the Rfp than Rfn restorer, indicating a greater cost of restoration associated with Rfn. These results demonstrate that costs of restoration differ for the Rfn and Rfp alleles in B. napus.

Identification of AFLP and SSR markers linked with the male fertility restorer gene of CMS 06J45 in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

AbstractIn this study, AFLP and SSR techniques were combined with the bulk segregant analysis (BSA) method to map the restorer gene BrRfp using an F2‐segregating population comprising 258 individuals developed by crossing the polima (pol)‐like cytoplasmic male sterility (CMS) line 06J45 and the restorer line 01S325 of heading Chinese cabbage. A survey of 2048 AFLP primer pairs identified 21 polymorphic fragments, approximately half of which exhibited high similarity with the A09 chromosome sequence of Brassica rapa in the Brassica database (BRAD). Based on the genome sequence, three specific AFLP fragments linked with BrRfp were successfully converted into sequence‐characterized amplified region (SCAR) markers, named SC1233, SC2673 and SC2141. Subsequently, 178 pairs of SSR primers were redesigned for further screening, with five producing polymorphic amplification patterns. Linkage analysis showed that these markers were distributed along both sides of the BrRfp gene, with two markers, SSR03 and SSR2528, co‐segregating with the BrRfp locus in the F2 population. These results may be valuable for marker‐assisted selection and map‐based cloning in heading Chinese cabbage.

Transgenic pearl millet male fertility restorer line (ICMP451) and hybrid (ICMH451) expressing Brassica juncea Nonexpressor of pathogenesis related genes 1 (BjNPR1) exhibit resistance to downy mildew disease.

Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into pearl millet male fertility restorer line ICMP451 by Agrobacterium tumefaciens-mediated genetic transformation. Transgenic pearl millet plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with A. tumefaciens strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in transgenic pearl millet lines. Transgenes BjNPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Transgenic pearl millet hybrid ICMH451-BjNPR1 was developed by crossing male-sterile line 81A X homozygous transgenic line ICMP451-BjNPR1. T3 and T4 homozygous lines of ICMP451-BjNPR1 and hybrid ICMH451-BjNPR1 exhibited resistance to three strains of downy mildew pathogen, while the untransformed ICMP451 and the isogenic hybrid ICMH451 plants were found susceptible. Following infection with S. graminicola, differential expression of systemic acquired resistance pathway genes, UDP-glucose salicylic acid glucosyl transferase and pathogenesis related gene 1 was observed in transgenic ICMP451-BjNPR1 and untransformed plants indicating the activation of systemic acquired resistance pathway contributing to the transgene-mediated resistance against downy mildew. The transgenic pearl millet expressing BjNPR1 showed resistance to multiple strains of S. graminicola and, as such, seems promising for the development of durable downy mildew resistant hybrids.

Molecular cloning and expression of the male sterility-related CtYABBY1 gene in flowering Chinese cabbage (Brassica campestris L. ssp chinensis var. parachinensis).

Expression of the YABBY gene family in the abaxial surface of lateral plant organs determines abaxial destiny of cells, enhances growth and expansion of lateral organs, and plays an important role in polar establishment of lateral organs. However, the YABBY gene has not been studied in male sterility and fertility restoration. We homologously cloned the CtYABBY1 gene of male-sterile TC1 in Brassica campestris L. ssp chinensis var. parachinensis; its expression was analyzed by real-time PCR. A 937-bp sequence was cloned from TC1 and named CtYABBY1. The ORF of this gene has 702 bp, contains a "C2C2 zinc finger" motif at the N-terminal end, and a "YABBY" structural domain at the C-terminal end. This gene had the highest homology with DBC43-3-2 gene in B. campetris ssp pekinensis. Expression of CtYABBY1 gene has a wide range of functions. It is involved in growth and development of lateral organs, such as leaves and flowers, enhancing expansion of the area and volume of young organs. CtYABBY1 is a gene that promotes thermo-sensitive fertility restoration. At room temperature, expression level of this gene was found to be lower in the stamens of sterile flowers. After treating TC1 at a low temperature of 2°-6°C for 20 days, expression of this gene was upregulated in the stamen of fertile flowers. We conclude that male sterility in TC1 is negatively regulated by this gene, which facilitates transition from male sterility to fertility.

Identification of sequence-related amplified polymorphism and insertion-deletion markers linked to the male fertility restorer gene of pol-like CMS06J45 in heading Chinese cabbage (Brassica rapa subsp pekinensis).

In order to map the restorer gene BrRfp of the polima (pol)-like cytoplasmic male sterility (CMS) 06J45 line in heading Chinese cabbage, an F2 segregating population with 258 individuals of CMS06J45 and the restorer line 01S325 were tested by sequence-related amplified polymorphism (SRAP) and insertion-deletion (InDel) technologies combined with the bulked segregant analysis method. As a result, two SRAP markers, me3em3.366 and pm88bg5.263, that were linked with the BrRfp gene were identified from 463 SRAP primer pairs. By cloning, sequencing, and basic local alignment search tool analysis, the two markers were targeted to the BGIScaffold000053 of Brassica rapa in the Brassica database. Using the BGIScaffold000053 sequence, four InDel primer pairs were designed and identified to be linked with the BrRfp gene in this population. Linkage analysis showed that these markers were distributed on both sides of the BrRfp gene, the linkage distances of two nearest markers InDel878.1125 and InDel920.713 were 0.82 and 0.46 cM, respectively, and the BrRfp gene was restricted to a 243-kb genomic region of B. rapa. These specific markers provided basic information for map-based cloning of the BrRfp gene and will be very valuable for the marker-assisted selection of a new restorer line in heading Chinese cabbage.

Male Sterility and Fertility Restoration in Crops

In plants, male sterility can be caused either by mitochondrial genes with coupled nuclear genes or by nuclear genes alone; the resulting conditions are known as cytoplasmic male sterility (CMS) and genic male sterility (GMS), respectively. CMS and GMS facilitate hybrid seed production for many crops and thus allow breeders to harness yield gains associated with hybrid vigor (heterosis). In CMS, layers of interaction between mitochondrial and nuclear genes control its male specificity, occurrence, and restoration of fertility. Environment-sensitive GMS (EGMS) mutants may involve epigenetic control by noncoding RNAs and can revert to fertility under different growth conditions, making them useful breeding materials in the hybrid seed industry. Here, we review recent research on CMS and EGMS systems in crops, summarize general models of male sterility and fertility restoration, and discuss the evolutionary significance of these reproductive systems.

Effect of alien cytoplasm and fertility restorer genes on agronomic and physiological traits of Brassica juncea (L.) Czern

AbstractTo assess the effect of alien cytoplasm and fertility restorer genes on agronomic and physiological traits in Brassica juncea, cytoplasmic male sterile (CMS) and fertility restorer lines involving five alloplasms in three nuclear backgrounds were constituted through repeated backcrossing. These lines were evaluated along with euplasmic lines for agronomic traits in field experiments. Respiration efficiency of in vitro cultured hypocotyls, and chlorophyll content in leaves were also estimated. Significant adverse effects of trachystoma and catholica cytoplasms on yield‐contributing traits and yield were noticed. The restorer gene corrected the floral defects in CMS trachystoma and restored yield to the level of euplasmic line, while it did so only partly in CMS catholica. Restorer genes had no adverse effect on traits in any of the systems. On the contrary, the Rf gene of moricandia CMS system exerted a positive effect on leaf chlorophyll content. Likewise, in vitro assay indicated the moricandia CMS system as vigorous. The moricandia CMS plants gave higher seed yield perhaps due to overall better vigour and higher leaf chlorophyll content.

Registration of a Male Fertility Restorer Oilseed Sunflower Germplasm, HA‐R9, Resistant to Sunflower Rust

The sunflower (Helianthus annuus L.) germplasm HA‐R9 (Reg. No. GP‐326, PI 667595) was developed by the USDA‐ARS, Sunflower and Plant Biology Research Unit in collaboration with the Agricultural Experiment Station of North Dakota State University and released in January 2013. Sunflower rust (caused by Puccinia helianthi Schwein.) is a worldwide disease of sunflower that is an increasing threat to North American production due to newly emerged virulent races. HA‐R9 was selected from an oilseed male fertility restorer line, Rf ANN‐1742 (PI 596476), which was released by the USDA‐ARS and the Agricultural Experiment Station of North Dakota State University in 1997. The rust resistance in HA‐R9 originated from wild H. annuus PI 613748. Rust evaluation was conducted at the seedling stage under greenhouse conditions. HA‐R9 has resistance to all rust races tested so far, including the most predominant and the most virulent races currently identified in the United States. The rust resistance in HA‐R9 is conditioned by a single dominant gene R11 which has been mapped to linkage group 13 of sunflower and is closely linked to the Rf5 male fertility restorer gene present in HA‐R9 in the coupling phase. Germplasm HA‐R9 will fill an urgent need for resistance to new virulent rust races and diversify the pool of fertility restoration genes in sunflower.

Single Nucleotide Polymorphisms in Linkage Disequilibrium with the Male-fertility Restoration (Ms) Locus in Open-pollinated and Inbred Populations of Onion

Maintainer lines are used to seed propagate male-sterile lines for the development of hybrid onion (Allium cepa L.) cultivars. The identification of maintainer lines would be more efficient with molecular markers distinguishing genotypes at the nuclear male-fertility restoration (Ms) locus. Ms has been mapped to chromosome 2 of onion and linked genetic markers identified. However, linkages between these markers and Ms were detected using F2 or BC1 families at maximum linkage disequilibrium and, for many markers, their efficacy to predict genotypes at Ms in onion populations at or near linkage equilibrium remains unknown. In this research, near isogenic lines homozygous-dominant and -recessive at Ms were developed and screened for 930 single nucleotide polymorphisms (SNPs). Three SNPs tightly linked on chromosome 2 remained in linkage disequilibrium with genotypes at Ms among randomly selected plants from three open-pollinated populations of onion as well as among a collection of inbred lines. These SNPs should be useful for selection of the recessive ms allele to aid in the development of maintainer lines for hybrid onion development.

Precocious flowering of Citrus seedlings and its use for determination of cultivars generating male sterile progenies

Precocious flowering in several-month-old to one-year-old seedlings is known in many Citrus cultivars with very long juvenile phase of around ten years. The Citrus accessions generating precocious flowering seedlings are interesting materials for rapid genetic analysis. Precocious flowering was observed in the zygotic seedlings obtained from the most pummelo and pummelo-relative accessions used, and many of yuzu, mandarin and their relative accessions with the range between less than 1% and about 20%. The rates indicated that some pummelo and orange cultivars have high ability to generate precocious-flowering seedlings. Male sterility in the seedlings showed stability in the flowers of first, second, and third years after seed germination. Male sterile seedlings did not segregate in the progenies of pummelo and pummelo-relative accessions used as pistillate parents, whereas they segregated in those of accessions related to ‘Yuzu’ (C. junos) and mandarin, indicating pummelo cytoplasm is fertile for male fertility, whereas ‘Yuzu’ and mandarin cytoplasm was sterile. The segregation of sterility in the progenies also indicated that most Citrus accessions are heterozygous for male fertility restoration genes.

Transcriptome sequencing and de novo analysis of a cytoplasmic male sterile line and its near-isogenic restorer line in chili pepper (Capsicum annuum L.).

BackgroundThe use of cytoplasmic male sterility (CMS) in F1 hybrid seed production of chili pepper is increasingly popular. However, the molecular mechanisms of cytoplasmic male sterility and fertility restoration remain poorly understood due to limited transcriptomic and genomic data. Therefore, we analyzed the difference between a CMS line 121A and its near-isogenic restorer line 121C in transcriptome level using next generation sequencing technology (NGS), aiming to find out critical genes and pathways associated with the male sterility.ResultsWe generated approximately 53 million sequencing reads and assembled de novo, yielding 85,144 high quality unigenes with an average length of 643 bp. Among these unigenes, 27,191 were identified as putative homologs of annotated sequences in the public protein databases, 4,326 and 7,061 unigenes were found to be highly abundant in lines 121A and 121C, respectively. Many of the differentially expressed unigenes represent a set of potential candidate genes associated with the formation or abortion of pollen.ConclusionsOur study profiled anther transcriptomes of a chili pepper CMS line and its restorer line. The results shed the lights on the occurrence and recovery of the disturbances in nuclear-mitochondrial interaction and provide clues for further investigations.

Effect of different cryoprotectant agents on spermatogenesis efficiency in cryopreserved and grafted neonatal mouse testicular tissue

Restoration of male fertility associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The purpose of this study was to test the effects of four different cryoprotectant agents (CPA) on spermatogenesis and steroidogenesis in cryopreserved and allotransplanted neonatal mouse testicular tissue. Hank’s balanced salt solution (HBSS) with 5% fetal bovine serum including either 0.7M dimethyl sulfoxide (DMSO), 0.7M propylene glycol (PrOH), 0.7M ethylene glycol (EG), or glycerol was used as the cryoprotectant solution. Donor testes were collected and dissected from neonatal pups of CD-1 mice (one day old). Freezing and seeding of the testicular whole tissues was performed using an automated controlled-rate freezer. Four fresh (non-frozen) or frozen–thawed pieces of testes were subcutaneously grafted onto the hind flank of each castrated male NCr nude recipient mouse and harvested after 3months. Fresh neonatal testes grafts recovered from transplant sites had the most advanced rate of spermatogenesis with elongated spermatid and spermatozoa in 46.6% of seminiferous tubules and had higher levels of serum testosterone compared to all other frozen–thawed-graft groups (p<0.05). Fresh grafts and frozen–thawed grafts in the DMSO group had the highest rate of tissue survival compared to PrOH, EG, and glycerol after harvesting (p>0.05). The most effective CPA for the freezing and thawing of neonatal mouse testes was DMSO in comparison with EG (p<0.05) in both pre-grafted and post-grafted tissues based on histopathological evaluation. Likewise, the highest level of serum testosterone was obtained from the DMSO CPA group compared to all other cryoprotectants evaluated (p<0.05). The typical damage observed in the frozen–thawed grafts included disruption of the interstitial stroma, intercellular connection ruptures, and detachment of spermatogonia from the basement membrane. These findings indicate that neonatal mouse testes were most effectively preserved when frozen with HBSS medium with DMSO and that the type of CPA is a significant factor to obtain the most advanced stages of spermatogenesis and steroidogenesis after cryopreservation, thawing, and transplantation of neonatal mouse testes.

The importance of chromosomes from the sixth homeologic group in the restoration of male fertility in winter triticale with Triticum timopheevii cytoplasm

The sterilising cytoplasm from Triticum timopheevii is presently considered to be the most promising as regards to the seed production of triticale hybrid cultivars. This study was aimed at the utilisation of Diversity Arrays Technology (DArT) for the preliminary identification of genomic regions with loci controlling male sterility/fertility in the field-grown F2 generation of the interline hybrid between male sterile line CMS-Salvo 15/1 and restorer line Stan I. The fertility of plants was examined by visual scoring as well as by the assessment of seed setting within bagged spikes. For DNA analyses, 92 individuals representing opposite phenotypes (male sterile vs. fully male fertile) were chosen from the whole F2 population, which consisted of 414 plants. The constructed genetic map consists of 759 DArT markers distributed in 24 linkage groups that cover a distance of 974.4 cM. Application of the interval mapping method and the Kruskal–Wallis test enabled the identification of six genomic regions engaged in the restoration of male fertility within the mapping population. The most effective restorer genes were found on chromosomes of the sixth homeologic group, i.e. on 6R (the most efficient), 6A and 6B. Additionally, linkage groups assigned to chromosomes 1BS, 3A and 3A/3B were important for the determination of male fertility.

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 of a codominant CAPS marker linked to the Ms locus controlling fertility restoration in onion (Allium cepa L.)

A molecular marker associated with male fertility restoration was newly identified in onion in this study using random amplification of polymorphic DNA (RAPD) and bulk segregant analysis. A polymorphic amplicon from RAPD reaction was observed in DNA from fertile plants. The amplicon was designated as the RAPD marker OBC14.1000 and a 980-base pair (bp) nucleotide sequence was obtained. Genome walking was performed to obtain the flanking sequences of the OBC14.1000 marker to develop a co-dominant marker. The acquired sequences were a 2049-bp fragment linked to a male-fertile allele and a 1621-bp fragment linked to a male-sterile allele. Approximately 95% similarity was shown when the nucleotide sequences between fertile and sterile alleles were aligned. Using a polymorphic region containing a restriction site of AvaII in a fertile allele, the RAPD marker OBC14.1000 was successfully converted into a co-dominant CAPS marker and it was designated as ACms.1100. It appeared to be positioned much closer to the restorer-of-fertility locus, Ms, based on genotyping analysis as compared to other currently available markers. No putative homology was found to the newly identified ACms.1100 marker sequences through a Blast database search and the identification of sequences is still unknown.

Innovative basic and applied researches in cytoplasmic male sterility and fertility restoration

Innovative basic and applied researches in cytoplasmic male sterility and fertility restoration

Genetic Analysis and Mapping of an Enclosed Panicle Mutant Locus esp1 in Rice (Oryza sativa L.)

A mutant was isolated from the M2 of 60Co-γ ray mutagenized male-fertility restorer line Zao-R974 in rice. The mutant showed pleiotropic phenotypes including dwarfism, delayed heading time, short and partially enclosed panicles, short uppermost internode, decreased grain and secondary branch numbers per panicle, and partially degenerated spikelets. The mutant was named as esp1 (enclosed shorter panicle 1). Genetic analysis indicated that the mutant phenotype was controlled by a recessive locus. Spraying exogenous GA3 did not rescue the panicle enclosure. Using an F2 and a BC1 population of the cross between esp1 and a japonica cultivar Nipponbare, we mapped the ESP1 locus to a region of ∼260 kb on chromosome 11. This result provides a basis for further map-based cloning of the ESP1 locus.

TESTING MODELS OF SEX RATIO EVOLUTION IN A GYNODIOECIOUS PLANT: FEMALE FREQUENCY COVARIES WITH THE COST OF MALE FERTILITY RESTORATION

In many gynodioecious species, cytoplasmic male sterility genes (CMS) and nuclear male fertility restorers (Rf) jointly determine whether a plant is female or hermaphrodite. Equilibrium models of cytonuclear gynodioecy, which describe the effect of natural selection within populations on the sex ratio, predict that the frequency of females in a population will primarily depend on the cost of male fertility restoration, a negative pleiotropic effect of Rf alleles on hermaphrodite fitness. Specifically, when the cost of restoration is higher, the frequency of females at equilibrium is predicted to be higher. To test this prediction, we estimated variation in the cost of restoration across 26 populations of Lobelia siphilitica, a species in which Rf alleles can have negative pleiotropic effects on pollen viability. We found that L. siphilitica populations with many females were more likely to contain hermaphrodites with low pollen viability. This is consistent with the prediction that the cost of restoration is a key determinant of variation in female frequency. Our results suggest that equilibrium models can explain variation in sex ratio among natural populations of gynodioecious species.

Molecular mapping of a male fertility restorer locus of Brassica oleracea using expressed sequence tag-based single nucleotide polymorphism markers and analysis of a syntenic region in Arabidopsis thaliana for identification of genes encoding pentatricopeptide repeat proteins

An F2 population was developed from a cross between a mur-cytoplasmic male sterile broccoli line and a restorer Chinese kale line. Phenotypic analysis of F2 plants indicated that the pollen fertility is controlled by two genes and segregated in a duplicate gene interaction mode with a ratio of 15:1. A total of 236 single nucleotide polymorphism (SNP) markers were developed utilizing 1,448 primers designed for production of expressed sequence tag (EST)-SNP markers of Raphanus sativus and analyzed by the dot-blot technique in 205 F2 individuals. A linkage map was constructed with a total of 142 markers and these markers were assigned to nine linkage groups together with simple sequence repeat markers mapped previously on the published linkage maps of Brassica oleracea. The linkage map spanned 909 cM with an average marker distance of 6.4 cM. A fertility restorer locus (Rfm1) was mapped on LG1, corresponding to chromosome 3, along with a flower color locus at a distance of 25 cM. SNP markers flanking the Rfm1 locus were BoCL2642s at a distance of 2.5 cM on one side and BoCL2901s at a distance of 7.5 cM on the other side. All the SNP markers showed homology with Arabidopsis thaliana and Brassica rapa genome sequences. Three pentatricopeptide repeat genes of the P-subfamily, particularly expressed in buds of the restorer line, were identified and these genes could be potential candidate fertility restorer genes.

Molecular tagging of a novel rust resistance gene R 12 in sunflower (Helianthus annuus L.)

Sunflower production in North America has recently suffered economic losses in yield and seed quality from sunflower rust (Puccinia helianthi Schwein.) because of the increasing incidence and lack of resistance to new rust races. RHA 464, a newly released sunflower male fertility restorer line, is resistant to both of the most predominant and most virulent rust races identified in the Northern Great Plains of the USA. The gene conditioning rust resistance in RHA 464 originated from wild Helianthus annuus L., but has not been molecularly marked or determined to be independent from other rust loci. The objectives of this study are to identify molecular markers linked to the rust resistance gene and to investigate the allelism of this gene with the unmapped rust resistance genes present in HA-R6, HA-R8 and RHA 397. Virulence phenotypes of seedlings for the F(2) population and F(2:3) families suggested that a single dominant gene confers rust resistance in RHA 464, and this gene was designated as R(12). Bulked segregant analysis identified ten markers polymorphic between resistant and susceptible bulks. In subsequent genetic mapping, the ten markers covered 33.4 cM of genetic distance on linkage group 11 of sunflower. A co-dominant marker CRT275-11 is the closest marker distal to R(12) with a genetic distance of 1.0 cM, while ZVG53, a dominant marker linked in the repulsion phase, is proximal to R(12) with a genetic distance of 9.6 cM. The allelism test demonstrated that R(12) is not allelic to the rust resistance genes in HA-R6, HA-R8 and RHA 397, and it is also not linked to any previously mapped rust resistance genes. Discovery of the R(12) novel rust resistance locus in sunflower and associated markers will potentially support the molecular marker-assisted introgression and pyramiding of R(12) into sunflower breeding lines.