Development and characterization of a new sunflower source of resistance to race G of Orobanche cumana Wallr. derived from Helianthus anomalus.

KEY MESSAGE: A new OrAnom1 gene introgressed in cultivated sunflower from wild Helianthus anomalus confers late post-attachment resistance to Orobanche cumana race G and maps to a target interval in Chromosome 4 where two receptor-like kinases (RLKs) have been identified in the H. anomalus genome as putative candidates. Sunflower broomrape is a parasitic weed that infects sunflower (Helianthus annuus L.) roots causing severe yield losses. Breeding for resistance is the most effective and sustainable control method. In this study, we report the identification, introgression, and genetic and physiological characterization of a new sunflower source of resistance to race G of broomrape developed from the wild annual sunflower H. anomalus (accession PI 468642). Crosses between PI 468642 and the susceptible line P21 were carried out, and the genetic study was conducted in BC1F1, BC1F2, and its derived BC1F3 populations. A BC1F5 germplasm named ANOM1 was developed through selection for race G resistance and resemblance to cultivated sunflower. The resistant trait showed monogenic and dominant inheritance. The gene, named OrAnom1, was mapped to Chromosome 4 within a 1.2 cM interval and co-segregated with 7 SNP markers. This interval corresponds to a 1.32 Mb region in the sunflower reference genome, housing a cluster of receptor-like kinase and receptor-like protein (RLK-RLP) genes. Notably, the analysis of the H. anomalus genome revealed the absence of RLPs in the OrAnom1 target region but featured two RLKs as possible OrAnom1 candidates. Rhizotron and histological studies showed that OrAnom1 determines a late post-attachment resistance mechanism. Broomrape can establish a vascular connection with the host, but parasite growth is stopped before tubercle development, showing phenolic compounds accumulation and tubercle necrosis. ANOM1 will contribute to broadening the genetic basis of broomrape resistance in the cultivated sunflower pool and to a better understanding of the molecular basis of the sunflower-broomrape interaction.

Genetic and physiological characterization of sunflower resistance provided by the wild-derived OrDeb2 gene against highly virulent races of Orobanche cumana Wallr.

KEY MESSAGE: OrDeb2 confers post-attachment resistance to Orobanche cumana and is located in a 1.38 Mbp genomic interval containing a cluster of receptor-like kinase and receptor-like protein genes with nine high-confidence candidates. Sunflower broomrape is a holoparasitic angiosperm that parasitizes on sunflower roots, severely constraining crop yield. Breeding for resistance is the most effective method of control. OrDeb2 is a dominant resistance gene introgressed into cultivated sunflower from a wild-related species that confers resistance to highly virulent broomrape races. The objectives of this study were as follows: (i) locate OrDeb2 into the sunflower genome and determine putative candidate genes and (ii) characterize its underlying resistance mechanism. A segregating population from a cross between the sunflower resistant line DEB2, carrying OrDeb2, and a susceptible line was phenotyped for broomrape resistance in four experiments, including different environments and two broomrape races (FGV and GTK). This population was also densely genotyped with microsatellite and SNP markers, which allowed locating OrDeb2 within a 0.9 cM interval in the upper half of Chromosome 4. This interval corresponded to a 1.38 Mbp genomic region of the sunflower reference genome that contained a cluster of genes encoding LRR (leucine-rich repeat) receptor-like proteins lacking a cytoplasmic kinase domain and receptor-like kinases with one or two kinase domains and lacking an extracellular LRR region, which were valuable candidates for OrDeb2. Rhizotron and histological studies showed that OrDeb2 determines a post-attachment resistance response that blocks O. cumana development mainly at the cortex before the establishment of host-parasite vascular connections. This study will contribute to understand the interaction between crops and parasitic weeds, to establish durable breeding strategies based on genetic resistance and provide useful tools for marker-assisted selection and OrDeb2 map-based cloning.

Genetic Analysis of Reduced γ-Tocopherol Content in Ethiopian Mustard Seeds.

Ethiopian mustard (Brassica carinata A. Braun) line BCT-6, with reduced γ-tocopherol content in the seeds, has been previously developed. The objective of this research was to conduct a genetic analysis of seed tocopherols in this line. BCT-6 was crossed with the conventional line C-101 and the F1, F2, and BC plant generations were analyzed. Generation mean analysis using individual scaling tests indicated that reduced γ-tocopherol content fitted an additive-dominant genetic model with predominance of additive effects and absence of epistatic interactions. This was confirmed through a joint scaling test and additional testing of the goodness of fit of the model. Conversely, epistatic interactions were identified for total tocopherol content. Estimation of the minimum number of genes suggested that both γ- and total tocopherol content may be controlled by two genes. A positive correlation between total tocopherol content and the proportion of γ-tocopherol was identified in the F2 generation. Additional research on the feasibility of developing germplasm with high tocopherol content and reduced concentration of γ-tocopherol is required.

Tocopherols in Sunflower Seedlings under Light and Dark Conditions.

The objective of this study was to evaluate the dynamics of tocopherols in cotyledons and radicles from sunflower seeds with high and low total tocopherol content, mainly in the α-tocopherol form, and from seeds with increased proportions of ß-, γ-, and δ-tocopherol, both under dark and light conditions. Tocopherol content was measured every 24 h from 1 to 12 days after sowing. In all cases, the content of individual tocopherol forms in the cotyledons and radicles was reduced along the sampling period, which was more pronounced under light conditions. The presence of light had a slightly greater effect on α- and γ-tocopherol than on ß- and δ-tocopherol. A marked light effect was also observed on total tocopherol content, with light promoting the reduction of tocopherol content in cotyledons and radicles. The study revealed only slight differences in the patterns of tocopherol losses in lines with different tocopherol profiles, both under dark and light conditions, which suggested that the partial replacement of α-tocopherol by other tocopherol forms had no great impact on the protection against oxidative damage in seedlings.

The genetic structure of wild Orobanche cumana Wallr. (Orobanchaceae) populations in eastern Bulgaria reflects introgressions from weedy populations.

Orobanche cumana is a holoparasitic plant naturally distributed from central Asia to south-eastern Europe, where it parasitizes wild Asteraceae species. It is also an important parasitic weed of sunflower crops. The objective of this research was to investigate genetic diversity, population structure, and virulence on sunflower of O. cumana populations parasitizing wild plants in eastern Bulgaria. Fresh tissue of eight O. cumana populations and mature seeds of four of them were collected in situ on wild hosts. Genetic diversity and population structure were studied with SSR markers and compared to weedy populations. Two main gene pools were identified in Bulgarian populations, with most of the populations having intermediate characteristics. Cross-inoculation experiments revealed that O. cumana populations collected on wild species possessed similar ability to parasitize sunflower to those collected on sunflower. The results were explained on the basis of an effective genetic exchange between populations parasitizing sunflower crops and those parasitizing wild species. The occurrence of bidirectional gene flow may have an impact on wild populations, as new physiological races continuously emerge in weedy populations. Also, genetic variability of wild populations may favour the ability of weedy populations to overcome sunflower resistance mechanisms.

A novel sunflower broomrape race with unusual virulence potentially caused by a mutation.

Introduction: The sunflower broomrape (Orobanche cumana Wallr.) gene pools of the Guadalquivir Valley and Cuenca province in Spain had predominantly race-F virulence. A new race G was observed recently in the Guadalquivir Valley potentially due to the genetic recombination of the avirulence genes of both gene pools. Methods: In this research, we have studied populations with atypical virulence from Cuenca. These populations parasitize on DEB2 sunflower line, resistant to all race-G populations evaluated. Ten populations collected in Cuenca province were evaluated with sunflower differential lines and genotyped with 67 SNP markers. Results: Although genetic recombination with individuals of the Guadalquivir Valley gene pool has been observed in most populations, recombination of avirulence genes was discarded as the cause of the new virulence because the population with the highest degree of attack on DEB2 showed no introgression from an external gene pool. Accordingly, a point mutation is proposed as the putative cause of the new virulence. Discussion: The present study provided a detailed characterization of each population, including the accurate classification of the individuals belonging to each of the classical Spanish gene pools, F1 hybrids, and those that evolved from hybridization between both gene pools. This information is essential to understand how sunflower broomrape populations are evolving in Spain, which in turn may be helpful to understand the dynamics of sunflower broomrape populations in other areas of the world and use this information to develop durable strategies for resistance breeding.

Genetic basis of unstable expression of high gamma-tocopherol content in sunflower seeds.

BACKGROUND: Tocopherols are natural antioxidants with both in vivo (vitamin E) and in vitro activity. Sunflower seeds contain predominantly alpha-tocopherol (>90% of total tocopherols), with maximum vitamin E effect but lower in vitro antioxidant action than other tocopherol forms such as gamma-tocopherol. Sunflower germplasm with stable high levels of gamma-tocopherol (>85%) has been developed. The trait is controlled by recessive alleles at a single locus Tph2 underlying a gamma-tocopherol methyltransferase (gamma-TMT). Additionally, unstable expression of increased gamma-tocopherol content in the range from 5 to 85% has been reported. The objective of this research was to determine the genetic basis of unstable expression of high gamma-tocopherol content in sunflower seeds. RESULTS: Male sterile plants of nuclear male sterile line nmsT2100, with stable high gamma-tocopherol content, were crossed with plants of line IAST-1, with stable high gamma-tocopherol content but derived from a population that exhibited unstable expression of the trait. F2 seeds showed continuous segregation for gamma-tocopherol content from 1.0 to 99.7%. Gamma-tocopherol content in F2 plants (average of 24 individual F3 seeds) segregated from 59.4 to 99.4%. A genetic linkage map comprising 17 linkage groups (LGs) was constructed from this population using 109 SSR and 20 INDEL marker loci, including INDEL markers for tocopherol biosynthesis genes. QTL analysis revealed a major QTL on LG 8 that corresponded to the gamma-TMT Tph2 locus, which suggested that high gamma-tocopherol lines nmsT2100 and IAST-1 possess different alleles at this locus. Modifying genes were identified at LGs 1, 9, 14 and 16, corresponding in most cases with gamma-TMT duplicated loci. CONCLUSIONS: Unstable expression of high gamma-tocopherol content is produced by the effect of modifying genes on tph2a allele at the gamma-TMT Tph2 gene. This allele is present in line IAST-1 and is different to allele tph2 present in line nmsT2100, which is not affected by modifying genes. No sequence differences at the gamma-TMT gene were found associated to allelic unstability. Our results suggested that modifying genes are mostly epistatically interacting gamma-TMT duplicated loci.

Expression of modified tocopherol content and profile in sunflower tissues.

BACKGROUND: Alpha-tocopherol is the predominant tocopherol form in sunflower seeds. Sunflower lines that accumulate increased levels of beta-, gamma- and delta-tocopherol in seeds as well as lines with reduced and increased total seed tocopherol content have been developed. The objective of this research was to evaluate whether the modified tocopherol levels are expressed in plant tissues other than seeds. RESULTS: Lines with increased levels of beta-, gamma- and delta-tocopherol in seeds also possessed increased levels of these tocopherols in leaves, roots and pollen. Correlation coefficients for the proportion of individual tocopherols in different plant tissues were significantly positive in all cases, ranging from 0.68 to 0.97. A line with reduced tocopherol content in seeds also showed reduced content in roots and pollen. CONCLUSION: Genetic modifications producing altered seed tocopherol profiles in sunflower are also expressed in leaves, roots and pollen. Reduced total seed tocopherol content is mainly expressed at the root and pollen level. The expression of tocopherol mutations in other plant tissues will enable further studies on the physiological role of tocopherols and could be of interest for early selection for these traits in breeding programmes.

Association mapping for broomrape resistance in sunflower.

Introduction: Sunflower breeding for resistance to the parasitic plant sunflower broomrape (Orobanche cumana Wallr.) requires the identification of novel resistance genes. In this research, we conducted a genome-wide association study (GWAS) to identify QTLs associated with broomrape resistance. Methods: The marker-trait associations were examined across a germplasm set composed of 104 sunflower accessions. They were genotyped with a 600k AXIOM® genome-wide array and evaluated for resistance to three populations of the parasite with varying levels of virulence (races EFR, FGV, and GTK) in two environments. Results and Discussion: The analysis of the genetic structure of the germplasm set revealed the presence of two main groups. The application of optimized treatments based on the general linear model (GLM) and the mixed linear model (MLM) allowed the detection of 14 SNP markers significantly associated with broomrape resistance. The highest number of marker-trait associations were identified on chromosome 3, clustered in two different genomic regions of this chromosome. Other associations were identified on chromosomes 5, 10, 13, and 16. Candidate genes for the main genomic regions associated with broomrape resistance were studied and discussed. Particularly, two significant SNPs on chromosome 3 associated with races EFR and FGV were found at two tightly linked SWEET sugar transporter genes. The results of this study have confirmed the role of some QTL on resistance to sunflower broomrape and have revealed new ones that may play an important role in the development of durable resistance to this parasitic weed in sunflower.

Wild Helianthus species: A reservoir of resistance genes for sustainable pyramidal resistance to broomrape in sunflower.

Orobanche cumana Wall., sunflower broomrape, is one of the major pests for the sunflower crop. Breeding for resistant varieties in sunflower has been the most efficient method to control this parasitic weed. However, more virulent broomrape populations continuously emerge by overcoming genetic resistance. It is thus essential to identify new broomrape resistances acting at various stages of the interaction and combine them to improve resistance durability. In this study, 71 wild sunflowers and wild relatives accessions from 16 Helianthus species were screened in pots for their resistance to broomrape at the late emergence stage. From this initial screen, 18 accessions from 9 species showing resistance, were phenotyped at early stages of the interaction: the induction of broomrape seed germination by sunflower root exudates, the attachment to the host root and the development of tubercles in rhizotron assays. We showed that wild Helianthus accessions are an important source of resistance to the most virulent broomrape races, affecting various stages of the interaction: the inability to induce broomrape seed germination, the development of incompatible attachments or necrotic tubercles, and the arrest of emerged structure growth. Cytological studies of incompatible attachments showed that several cellular mechanisms were shared among resistant Helianthus species.

An SSR-SNP Linkage Map of the Parasitic Weed Orobanche cumana Wallr. Including a Gene for Plant Pigmentation.

Sunflower broomrape (Orobanche cumana Wallr.) is a holoparasitic plant that causes major yield losses to sunflower crops in the Old World. Efforts to understand how this parasitic weed recognizes and interacts with sunflowers are important for developing long-term genetic resistance strategies. However, such studies are hampered by the lack of genetic tools for O. cumana. The objectives of this research were to construct a genetic linkage map of this species using SSR and SNP markers, and mapping the Pg locus that is involved in plant pigmentation. The genetic map was developed from the progenies of a cross between the O. cumana inbred lines EK-12 and EK-A1, which originated from populations belonging to two distant and geographically separated gene pools identified in Spain. The inbred lines also differed in plant pigmentation, with EK-A1 lacking anthocyanin pigmentation (pgpg genotype). A genetic map comprising 26 SSR and 701 SNP markers was constructed, which displayed 19 linkage groups (LGs), corresponding to the 19 chromosome pairs of O. cumana. The total length of the map was 1795.7 cM, with an average distance between two adjacent positions of 2.5 cM and a maximum map distance of 41.9 cM. The Pg locus mapped to LG19 between the SNP markers OS02468 and OS01653 at 7.5 and 3.4 cM, respectively. This study constitutes the first linkage map and trait mapping study in Orobanche spp., laying a key foundation for further genome characterization and providing a basis for mapping additional traits such as those having a key role in parasitism.

A receptor-like kinase enhances sunflower resistance to Orobanche cumana.

Orobanche cumana (sunflower broomrape) is an obligate parasitic plant that infects sunflower roots, causing yield losses. Here, by using a map-based cloning strategy, we identified HaOr7-a gene that confers resistance to O. cumana race F-which was found to encode a leucine-rich repeat receptor-like kinase. The complete HAOR7 protein is present in resistant lines of sunflower and prevents O. cumana from connecting to the vascular system of sunflower roots, whereas susceptible lines encode a truncated protein that lacks transmembrane and kinase domains.

Increased Virulence in Sunflower Broomrape (Orobanche cumana Wallr.) Populations from Southern Spain Is Associated with Greater Genetic Diversity.

Orobanche cumana Wallr. (sunflower broomrape) is a holoparasitic weed that infects roots of sunflower in large areas of Europe and Asia. Two distant O. cumana gene pools have been identified in Spain, one in Cuenca province in the Center and another one in the Guadalquivir Valley in the South. Race F has been hypothesized to have arisen by separate mutational events in both gene pools. In the Guadalquivir Valley, race F spread in the middle 1990's to become predominant and contained so far with race F hybrids. Recently, enhanced virulent populations of O. cumana have been observed in commercial fields parasitizing race F resistant hybrids. From them, we collected four independent populations and conducted virulence and SSR marker-based genetic diversity analysis. Virulence essays confirmed that the four populations studied can parasitize most of the race F resistant hybrids tested, but they cannot parasitize the differential inbred lines DEB-2, carrying resistance to race F and G, and P-96, resistant to F but susceptible to races G from other countries. Accordingly, the new populations have been classified as race GGV to distinguish them from other races G. Cluster analysis with a set of populations from the two Spanish gene pools and from other areas, mainly Eastern Europe, confirmed that race GGV populations maintain close genetic relatedness with the Guadalquivir Valley gene pool. This suggested that increased virulence was not caused by new introductions from other countries. Genetic diversity parameters revealed that the four populations had much greater genetic diversity than conventional populations of the same area, containing only alleles present in the Guadalquivir Valley and Cuenca gene pools. The results suggested that increased virulence may have resulted from admixture of populations from the Guadalquivir Valley and Cuenca followed by recombination of avirulence genes.

Sunflower Resistance to Broomrape (Orobanche cumana) Is Controlled by Specific QTLs for Different Parasitism Stages.

Orobanche cumana (sunflower broomrape) is an obligatory and non-photosynthetic root parasitic plant that specifically infects the sunflower. It is located in Europe and in Asia, where it can cause yield losses of over 80%. More aggressive races have evolved, mainly around the Black Sea, and broomrape can rapidly spread to new areas. Breeding for resistance seems to be the most efficient and sustainable approach to control broomrape infestation. In our study, we used a population of 101 recombinant inbred lines (RILs), derived from a cross between the two lines HA89 and LR1 (a line derived from an interspecific cross with Helianthus debilis). Rhizotrons, pots and field experiments were used to characterize all RILs for their resistance to O. cumana race F parasitism at three post vascular connection life stages: (i) early attachment of the parasite to the sunflower roots, (ii) young tubercle and (iii) shoot emergence. In addition, RIL resistance to race G at young tubercle development stage was evaluated in pots. The entire population was genotyped, and QTLs were mapped. Different QTLs were identified for each race (F from Spain and G from Turkey) and for the three stages of broomrape development. The results indicate that there are several quantitative resistance mechanisms controlling the infection by O. cumana that can be used in sunflower breeding.