Abstract:The entire chloroplast and mitochondrial genomes of domesticated and wild type sunflower were sequenced. The comparative analysis of chloroplast genomes revealed 43 variant sites, including 21 polymorphic SSR loci and 22 SNPs. About 14 variant sites were found by collation of mitochondrial DNA (mtDNA), among them 4 SSRs, 8 SNPs and 2 deletions. About 9 SNPs were located in coding region of chloroplast DNA (cpDNA) and single SNP was mapped in mitochondrial gene. Only three SNPs caused amino acid changes: Two SNPs in cpDNA and one mtDNA SNP. Despite the fact that sunflower mitochondrial genome sequence is twice as long as chloroplast genome sequence, mtDNA has one third as much variant sites than cpDNA.

BackgroundCytoplasmic male sterility (CMS) is a common phenotype in higher plants, that is often associated with rearrangements in mitochondrial DNA (mtDNA), and is widely used to produce hybrid seeds in a variety of valuable crop species. Investigation of the CMS phenomenon promotes understanding of fundamental issues of nuclear-cytoplasmic interactions in the ontogeny of higher plants. In the present study, we analyzed the structural changes in mitochondrial genomes of three alloplasmic lines of sunflower (Helianthus annuus L.). The investigation was focused on CMS line PET2, as there are very few reports about its mtDNA organization.MethodsThe NGS sequencing, de novo assembly, and annotation of sunflower mitochondrial genomes were performed. The comparative analysis of mtDNA of HA89 fertile line and two HA89 CMS lines (PET1, PET2) occurred.ResultsThe mtDNA of the HA89 fertile line was almost identical to the HA412 line (). The comparative analysis of HA89 fertile and CMS (PET1) analog mitochondrial genomes revealed 11,852 bp inversion, 4,732 bp insertion, 451 bp deletion and 18 variant sites. In the mtDNA of HA89 (PET2) CMS line we determined 27.5 kb and 106.5 kb translocations, 711 bp and 3,780 bp deletions, as well as, 5,050 bp and 15,885 bp insertions. There are also 83 polymorphic sites in the PET2 mitochondrial genome, as compared with the fertile line.DiscussionThe observed mitochondrial reorganizations in PET1 resulted in only one new open reading frame formation (orfH522), and PET2 mtDNA rearrangements led to the elimination of orf777, duplication of atp6 gene and appearance of four new ORFs with transcription activity specific for the HA89 (PET2) CMS line—orf645, orf2565, orf228 and orf285. Orf228 and orf285 are the atp9 chimeric ORFs, containing transmembrane domains and possibly may impact on mitochondrial membrane potential. So orf228 and orf285 may be the cause for the appearance of the PET2 CMS phenotype, while the contribution of other mtDNA reorganizations in CMS formation is negligible.

One of the areas of biotechnology sunflower is the development and testing of DNA markers of important agronomic traits and in particular markers of resistance to downy mildew. Resistance of 16 Rf-lines of sunflower to the races 330 and 710 of Plasmopara halstedii has been studied. Genotyping of these lines was carried out using 9 STS-markers of three Pl-loci, Pl 5 , Pl 6 and Pl 8 , associated with the resistance of sunflower to downy mildew. Only two out of nine STS-markers, НаР2 and НаР3 (locus Pl 6), allowed us to identify the lines, which demonstrated resistance to the downy mildew under the conditions of artificial infection.

In this work we developed and exploited a spray-induced gene silencing (SIGS)-based approach to deliver double-stranded RNA (dsRNA), which was found to protect potato against potato virus Y (PVY) infection. Given that dsRNA can act as a defence-inducing signal that can trigger sequence-specific RNA interference (RNAi) and non-specific pattern-triggered immunity (PTI), we suspected that these two pathways may be invoked via exogeneous application of dsRNA, which may account for the alterations in PVY susceptibility in dsRNA-treated potato plants. Therefore, we tested the impact of exogenously applied PVY-derived dsRNA on both these layers of defence (RNAi and PTI) and explored its effect on accumulation of a homologous virus (PVY) and an unrelated virus (potato virus X, PVX). Here, we show that application of PVY dsRNA in potato plants induced accumulation of both small interfering RNAs (siRNAs), a hallmark of RNAi, and some PTI-related gene transcripts such as WRKY29 (WRKY transcription factor 29; molecular marker of PTI), RbohD (respiratory burst oxidase homolog D), EDS5 (enhanced disease susceptibility 5), SERK3 (somatic embryogenesis receptor kinase 3) encoding brassinosteroid-insensitive 1-associated receptor kinase 1 (BAK1), and PR-1b (pathogenesis-related gene 1b). With respect to virus infections, PVY dsRNA suppressed only PVY replication but did not exhibit any effect on PVX infection in spite of the induction of PTI-like effects in the presence of PVX. Given that RNAi-mediated antiviral immunity acts as the major virus resistance mechanism in plants, it can be suggested that dsRNA-based PTI alone may not be strong enough to suppress virus infection. In addition to RNAi- and PTI-inducing activities, we also showed that PVY-specific dsRNA is able to upregulate production of a key enzyme involved in poly(ADP-ribose) metabolism, namely poly(ADP-ribose) glycohydrolase (PARG), which is regarded as a positive regulator of biotic stress responses. These findings offer insights for future development of innovative approaches which could integrate dsRNA-induced RNAi, PTI and modulation of poly(ADP-ribose) metabolism in a co-ordinated manner, to ensure a high level of crop protection.

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Markin N., Usatov A., Makarenko M., Azarin K., Gorbachenko O., Kolokolova N., Usatenko T., Markina O., Gavrilova V. (2017): Study of informative DNA markers of the Rf1 gene in sunflower for breeding practice. Czech J. Genet. Plant Breed., 53: 69−75.The investigation of DNA markers associated with the pollen fertility restoration gene (Rf1) was conducted in Helianthus. Two sequence-characterized amplified region (SCAR) markers -HRG01 and HRG02 were informative for the identification of Rf1 gene in selections of sunflower plants. The codominant character of the HRG01 marker and HRG01 amplicon polymorphism has been determined. Five annual and twenty-six perennial species of sunflower were tested for the presence of Rf genotypes. HRG02 proved to be a more appropriate marker for Rf1 determination in perennial species, and HRG01 was more informative for annual species. We have also developed the multiplex RT-PCR test system, which allows simultaneously detecting the dominant allele of Rf1 and CMS-PET1 associated mitotype.

A comparative analysis of full-genome sequences of chloroplast DNA (cpDNA) of the original inbred line 3629 and three extranuclear mutants, which were obtained by the method of mutagenesis induced with N-nitroso-N-methylurea (NMU) and characterized by different level of chlorophyll insufficiency (en:chlorina-7-yellow-green leaves; chlorophyll content (a + b)-67.8% with respect to the line 3629, variegated-10-leaves with white zones; chlorophyll content (a + b)-2.9% with respect to the line 3629 and variegated-13-leaves with yellow zones; chlorophyll content (a + b)-6.1% with respect to the line 3629), has been carried out. Single-parent maternal inheritance of chlorophyll defects was confirmed by analysis of progeny obtained from reciprocal crossbreedings between the original line 3629 and mutants. Chlorophyll mutants carried modified cpDNA unique for each mutant. We anticipate that chlorophyll defect of en:chlorina-7 may control the observed non-synonymous mutations (transitions) in the genes rpoB, psaA and psbB, which encode β-subunit of RNA-polymerase, the A1 apoprotein of chlorophyll a of the photosystem I, P700 and 47 kDa protein of the photosystem II respectively. In variegated-10, it may control mutations in the genes rpoA and rpoC2, which encode α and β" subunits of RNA-polymerase and in variegated-13-two mutations in the ycf3 gene that encodes photosystem I assembly factor.

Marker-assisted selection (MAS) was adopted for introgression of broad-spectrum blast resistance genes Pi1, Pi2 and Pi33 into elite Russian rice varieties. It was shown that microsatellite markers RM 224, RM 527 and RM 310 may be effectively used to transfer Pi1, Pi2 and Pi33 genes into selected Russian genotypes of rice. Based on the highly productive variety Kuboyar, we obtained the lines, 200-5668 (Pi 1+2+33 IL14 × Kuboyar), 207-5671 (Pi 1+2+33 IL28 × Kuboyar) and 208-5674 (Pi 1+2+33 IL28 × Kuboyar) carrying three pyramided genes Pi1, Pi2 and Pi33 in homozygous state. The lines 200-5668, 207-5671 and 208-5674 are used as an improved resistance donor source to obtain hybrids and pyramiding additional Pi genes in order to provide stable long-term resistance for rice blast disease.