A linkage of gene of resistance to a broomrape race G with microsatellite loci of a sunflower linedonor RGP1 of VNIIMK’s breeding

Broomrape (Orobanche cumana Wallr.) is one of the main biotic factors limiting high sunflower yield formation. The most effective and environmentally safe method of protection is cultivation of resistant varieties and hybrids of sunflower. Development of resistant sunflower genotypes includes search and usage of sources of resistance in breeding process as well as accurate and productive procedures of material assessment. The purpose of the research is to analyze a linkage of a gene Or7 with microsatellite loci of the line-donor of resistance to broomrape race G from the VNIIMK’s collection. The objects of the research are the line RGP1 – a donor of resistance to broomrape race G and a susceptible to this race line VR 678 from the VNIIMK’s collection. Sunflower plants were crossed in field to produce F1. Also we conducted self-pollination of F1 plants to obtain F2 progeny. Plants were tested in a greenhouse in soil infected with seeds of broomrape race G using a method of early diagnostic. Sunflower DNA was extracted from the top leaves of the young sprouts of the vegetative plants. For PCR-analysis we used three SSR-primers demonstrated polymorphism in parental lines: ORS 683, ORS 1040, and ORS 1112. We tested joint inheritance of the gene Or7 and these loci, and inheritance between SSR-loci. An independent inheritance of the gene Or7 with DNA-loci ORS 683, ORS 1040, and ORS 1112, as well as SSR-loci between ORS 1040 and ORS 1112, ORS 1040 and ORS 683 was showed. Loci ORS683 – ORS 1112 are linked with a frequency of recombination of 0.27 ± 0.41 (27 cM). As a result of our research location of the gene Or7 in the nearest area to microsatellite loci ORS 683, ORS 1040, and ORS 1112 was excluded. Basing on studied literary sources and a representative sunflower genome HanXRQr2.0-SUNRISE we made a partial physical map LG3 for determination of an area for the further search of a localization of the Or7 and DNAmarkers co-segregating with this gene.

Genomic and Transcriptomic Survey Provides New Insight into the Organization and Transposition Activity of Highly Expressed LTR Retrotransposons of Sunflower (Helianthus annuus L.)

LTR retrotransposons (RTEs) play a crucial role in plant genome evolution and adaptation. Although RTEs are generally silenced in somatic plant tissues under non-stressed conditions, some expressed RTEs (exRTEs) escape genome defense mechanisms. As our understanding of exRTE organization in plants is rudimentary, we systematically surveyed the genomic and transcriptomic organization and mobilome (transposition) activity of sunflower (Helianthus annuus L.) exRTEs. We identified 44 transcribed RTEs in the sunflower genome and demonstrated their distinct genomic features: more recent insertion time, longer open reading frame (ORF) length, and smaller distance to neighboring genes. We showed that GAG-encoding ORFs are present at significantly higher frequencies in exRTEs, compared with non-expressed RTEs. Most exRTEs exhibit variation in copy number among sunflower cultivars and one exRTE Gagarin produces extrachromosomal circular DNA in seedling, demonstrating recent and ongoing transposition activity. Nanopore direct RNA sequencing of full-length RTE RNA revealed complex patterns of alternative splicing in RTE RNAs, resulting in isoforms that carry ORFs for distinct RTE proteins. Together, our study demonstrates that tens of expressed sunflower RTEs with specific genomic organization shape the hidden layer of the transcriptome, pointing to the evolution of specific strategies that circumvent existing genome defense mechanisms.

Search and analysis of nuclear sequences in sunflower genome associated with genes of resistance to bromrape (Orobanche cumana Wallr.) races

Studying of sunflower lines resistant to broomrape race G, bred in V.S. Pustovoit All-Russian Research Institute of Oil Crops (VNIIMK), Krasnodar showed the genetic control of resistance is monogenic with incomplete dominance. The purpose of the research was to seek and analyze molecular markers for the genes of resistance to broomrape race G using PCRmethods. In our research we used six developed in VNIIMK lines-donors resistant to broomrape race G: RGP1, RGP2, RGB, RGL1, RGL2, RGA, and susceptible lines VK 101, VK 678, VK 680. We extracted DNA from the leaves of young sunflower plants using STAB-buffer. For PCR-analysis we used 17 pairs of primers of two types: SCAR (sequence characterized amplified region) and SSR (simple sequence repeat). Conditions of amplification are as recommended by authors of markers with experimental selection of a temperature for primers hybridization. Within the research we balanced involved into work SSR- and 12 SCAR-loci using BLAST and a set of HanXRQr1.0. Due to these data we composed a physical map of loci location. After assessment of six resistant and three susceptible parental lines, seven markers demonstrated polymorphism by a length of locus DNA and two ones – by presence/absence of amplified fragment. For markers ORS 683 and ORS 1112 we observed a relation between loci polymorphism and susceptibility of parental lines to broomrape. This was certified with a presence of an allele of 364 n.p. length in ORS 683 and an absence of an allele of 375 n.p. length in ORS 1112. These markers were noted as primary ones for hybridological analysis in F1 и F2 with selected pairs for crossing. Additional markers will be selected individually using obtained data on loci polymorphism. Thus, as a result of the research we created a physical map for the further markers selection, found hybrid combinations and markers from the studied ones for hybridological analysis.

On the Trail of Tetu1: Genome-Wide Discovery of CACTA Transposable Elements in Sunflower Genome

Much has been said about sunflower (Helianthus annuus L.) retrotransposons, representing the majority of the sunflower’s repetitive component. By contrast, class II transposons remained poorly described within this species, as they present low sequence conservation and are mostly lacking coding domains, making the identification and characterization of these transposable elements difficult. The transposable element Tetu1, is a non-autonomous CACTA-like element that has been detected in the coding region of a CYCLOIDEA (CYC) gene of a sunflower mutant, tubular ray flower (turf). Based on our knowledge of Tetu1, the publicly available genome of sunflower was fully scanned. A combination of bioinformatics analyses led to the discovery of 707 putative CACTA sequences: 84 elements with complete ends and 623 truncated elements. A detailed characterization of the identified elements allowed further classification into three subgroups of 347 elements on the base of their terminal repeat sequences. Only 39 encode a protein similar to known transposases (TPase), with 10 TPase sequences showing signals of activation. Finally, an analysis of the proximity of CACTA transposons to sunflower genes showed that the majority of CACTA elements are close to the nearest gene, whereas a relevant fraction resides within gene-encoding sequences, likely interfering with sunflower genome functionality and organization.

Characterization of CYP71AX36 from Sunflower (Helianthus annuus L., Asteraceae)

Sesquiterpene lactones (STL) are a subclass of isoprenoids with many known bioactivities frequently found in the Asteraceae family. In recent years, remarkable progress has been made regarding the biochemistry of STL, and today the biosynthetic pathway of the core backbones of many STLs has been elucidated. Consequently, the focus has shifted to the discovery of the decorating enzymes that can modify the core skeleton with functional hydroxy groups. Using in vivo pathway reconstruction assays in heterologous organisms such as Saccharomyces cerevisiae and Nicotiana benthamiana, we have analyzed several cytochrome P450 enzyme genes of the CYP71AX subfamily from Helianthus annuus clustered in close proximity to one another on the sunflower genome. We show that one member of this subfamily, CYP71AX36, can catalyze the conversion of costunolide to 14-hydroxycostunolide. The catalytic activity of CYP71AX36 may be of use for the chemoenzymatic production of antileukemic 14-hydroxycostunolide derivatives and other STLs of pharmaceutical interest. We also describe the full 2D-NMR assignment of 14-hydroxycostunolide and provide all 13C chemical shifts of the carbon skeleton for the first time.

Composite plants for a composite plant: An efficient protocol for root studies in the sunflower using composite plants approach

Sunflower (Helianthus annuus L.) is important oilseed crop in the world and the sunflower oil is prized for its’ exceptional quality and flavor. The recent availability of the sunflower genome can allow genome-wide characterization of genes and gene families. With plant transformation usually being the rate limiting step for gene functional studies of sunflower, composite plants can alleviate this bottleneck. Composite plants, produced using Agrobacterium rhizogenes, are plants with transgenic roots and wild type shoots. Composite plants offer benefits over creating fully transgenic plants, namely time and cost. Here we outlined the critical steps and parameters for a protocol for the sunflower composite plants production. We use more than a dozen genotypes and three constitutive promoters to validates the utility and efficiency of this protocol. Moreover, functional gene characterization by overexpression and RNAi silencing of a root related transcription factor HaLBD16 further emphasize the value of the system in the sunflower studies. With the protocol developed here an experiment can be carried out with efficiency and in only two months. This procedure adds to arsenal of approaches for the functional genetics/genomics in sunflower for characterization candidate genes involved in root development and stress adaptation.Key messageComposite plants technique described here is fast and efficient approach for roots functional studies in sunflower.

Assessment of RAPD Markers to Analyse the Genetic Diversity among Sunflower (Helianthus annuus L.) Genotypes

Genetic diversity estimation among different species is an important tool for genetic improvement to maximize the yield, desirable quality, wider adaptation, pest and insect resistance that ultimately boosting traditional plant breeding methods. The most efficient way of diversity estimation is application of molecular markers. In this study, twenty random amplified polymorphic DNA (RAPD) primers were utilized to estimate the genetic diversity between ten sunflower genotypes. Overall 227 bands were amplified by 20 primers with an average of 11.35 bands per primer. RAPD data showed 86.34% polymorophic bands and 13.65% of monomorophic bands. Genetic similarity was ranged from 50.22% to 87.22%. The lowest similarity (50.22%) was observed between FH-352 and FH-359 and the maximum similarity 87.22% was observed between A-23 and G-46. Polymorphic information content (PIC) values were varying from 0.05 to 0.12 with a mean of 0.09. Cluster analysis based on RAPD results displayed two major distinct groups 1 and 2. Group-2 contains FH-352 which was the most diverse genotype, while group-1 consists of few sub groups with all other genotypes. Ample diversity was found in all the genotypes. Present study reveals novel information about sunflower genome which can be used in future studies for sunflower improvement.