Jana Řepková

Identification of resistance genes against powdery mildew in four accessions of Hordeum vulgare ssp. spontaneum

SummaryFour newly detected accessions of wild barley (Hordeumvulgare ssp. spontaneum) resistant to powdery mildew caused by Blumeriagraminis f. sp. hordei were studied with the aim of finding the number of genes/loci conferring the resistance of individual accessions, the type of inheritance of the genes and their relationships to the Mla locus. F2 populations after crosses between the winter variety ‘Tiffany’ and four wild barley accessions and use of microsatellite DNA markers were focused on the identification of individual resistance genes/loci by means of their chromosomal locations. In PI466495, one locus conferring powdery mildew resistance was identified in highly significant linkage with the marker Bmac0213. This location is consistent with the known locus Mla on chromosome 1HS. In the other three accessions the resistance was determined by two independent loci. In PI466197, PI466297 and PI466461, one locus was identified on chromosome 1HS and three new loci were revealed on chromosomes 2HS (highly significant linkage with Bmac0134), 7HS (highly significant linkage with Bmag0021) and 7HL (significant linkage with EBmac0755). Our prospective aim is identification of further linked DNA markers and the exact location of the resistance genes on the barley chromosomes.

Genome assembly and annotation for red clover (Trifolium pratense; Fabaceae)

PREMISE OF THE STUDY Red clover (Trifolium pratense) is an important forage plant from the legume family with great importance in agronomy and livestock nourishment. Nevertheless, assembling its medium-sized genome presents a challenge, given current hardware and software possibilities. Next-generation sequencing technologies enable us to generate large amounts of sequence data at low cost. In this study, the genome assembly and red clover genome features are presented. METHODS First, assembly software was assessed using data sets from a closely related species to find the best possible combination of assembler plus error correction program to assemble the red clover genome. The newly sequenced genome was characterized by repetitive content, number of protein-coding and nonprotein-coding genes, and gene families and functions. Genome features were also compared with those of other sequenced plant species. KEY RESULTS Abyss with Echo correction was used for de novo assembly of the red clover genome. The presented assembly comprises ∼314.6 Mbp. In contrast to leguminous species with comparable genome sizes, the genome of T. pratense contains a larger repetitive portion and more abundant retrotransposons and DNA transposons. Overall, 47 398 protein-coding genes were annotated from 64 761 predicted genes. Comparative analysis revealed several gene families that are characteristic for T. pratense. Resistance genes, leghemoglobins, and nodule-specific cystein-rich peptides were identified and compared with other sequenced species. CONCLUSIONS The presented red clover genomic data constitute a resource for improvement through molecular breeding and for comparison to other sequenced plant species.

New CAPS Marker for Selection of a Barley Powdery Mildew Resistance Gene in the Mla Locus

In PI466495, a powdery mildew resistance source of wild barley (Hordeum vulgare ssp. spontaneum), one gene conferring powdery mildew resistance was identified in the Mla locus. In this paper, the RGH1a gene sequence was used as source for the development of a cleaved amplified polymorphic sequence (CAPS) marker. Co-segregation between this marker and powdery mildew resistance was analysed by specific DNA fragments associated with each allele of the gene using 286 F2 plants derived from a cross between winter barley (H. vulgare L.) variety ‘Tiffany’ and PI466495. For the co-dominant marker RGH1aI1a, three fragments, 370 bp, 82 bp and 59 bp in size, were amplified from F2 plants exhibiting resistance reaction types 0 and 0–1 to powdery mildew; whereas two fragments, 429 bp and 82 bp in size, were amplified in susceptible plants. Simple procedures based on polymerase chain reaction and restriction enzyme digestion allowed for identifying the plants susceptible to powdery mildew (Blumeria graminis f. sp. hordei) and plants homozygous or heterozygous for the resistance allele. The RGH1aI1a marker was positioned 0.85 cM to the resistance gene and the efficiency of marker-assisted selection (MAS), evaluated as the probability of crossing-over between the marker and the targeted gene, was 99%. The CAPS marker RGH1aI1a is a valuable candidate for MAS and gene transfer into barley varieties susceptible to powdery mildew.

Isolation and characterization of a novel semi-lethal Arabidopsis thaliana mutant of gene for pentatricopeptide (PPR) repeat-containing protein.

A novel Arabidopsis thaliana mutant of one member of the pentatricopeptide repeat (PPR) gene family has been identified among T-DNA insertion lines. Tagging of the At1g53330 gene caused the appearance of a semi-lethal mutation with a complex phenotypic expression from embryo lethality associated with the abnormal pattern of cell division during globular to heart transition to fertile plants with just subtle phenotypic changes. The PPR protein At1g53330.1 was predicted to be targeted to mitochondria by TargetP and MitoProt programs. Complementation analysis confirmed that the phenotype is a result of a single T-DNA integration. A thorough functional analysis of this mutant aimed at finding a particular organelle target of At1g53330.1 protein will follow.

Genetic analysis of thirteen accessions of Hordeum vulgare ssp. spontaneum resistant to powdery mildew

Thirteen accessions of wild barley (Hordeum vulgare ssp. spontaneum) resistant to powdery mildew caused by the fungus Blumeria graminis f. sp. hordei were studied with the aim of determining the number of resistance genes and their allelic relationships to the Mla locus on the short arm of chromosome 1H. In five accessions (PI391130, PI466193, PI466200, PI466495 and PI466510), the resistance was caused by one gene, in seven accessions (PI354949, PI391081, PI466158, PI466197, PI466211, PI466297 and PI466461) by two independent genes and in PI301004 by three independent genes. The type of inheritance of all analysed genes except two was dominant or semi-dominant; only one of two genes in PI391081 and PI466297 was recessive. Allelism tests confirmed that in 10 accessions one gene was allelic with the Mla locus, and in three accessions (PI391081, PI466193 and PI466297) the resistance genes were different from the Mla locus.

Characterization and chromosomal location of powdery mildew resistance genes from wild barley PI282605

The objective of this work was to find the identity of three resistance genes against powdery mildew by mapping in an F2 population derived from a cross between winter barley (Hordeum vulgare L.) variety ‘Tiffan’ and the wild barley (H. vulgare ssp. spontaneum) accession PI282605, an effective powdery mildew resistance source.ZusammenfassungDas Ziel dieser Untersuchung bestand in der genetischen Kartierung und Charakterisierung von drei Mehltau-Resistenzgenen in einer F2-Population der Kreuzung der Wintergerstensorte ‘Tiffany’ (Hordeum vulgare L.) und der Akzession PI282605 der Wildgerste (H. vulgare ssp. spontaneum), einer effektiven Mehltau-Resistenzquelle.

New Embryo Lethals in Arabidopsis thaliana: Basic Genetic and Morphological Study

Six different mutations with defects in immature seed development have been identified during screening of a T-DNA collection of Arabidopsis thaliana. The mutations were confirmed to be monogenic and recessive-lethal by genetic analysis. Mutant embryos were blocked in certain steps in the process necessary for embryo viability and development, and therefore they belong to the embryo-lethal class of mutants. The genetic and morphological studies of T-DNA mutations affecting embryo development are presented. The youngest embryos with a defect were observed at the globular stage in the VIII-64 mutation. Externally located cells, precursor of the protoderm, were characterised by abnormal cell division. VIII-41 mutation with a defect at the late globular stage was arrested at the globular-heart stage transition. VIII-111 mutation showed defect at heart stage of embryogenesis with atypical development of cotyledon primordia. The defect was associated with abnormal pattern of cell division constituting the precursor of the shoot apical meristem. In VIII-82 mutation defect in torpedo stage with asymmetric cotyledons was observed. Cotyledon stage of embryos and chlorophyll defect were observed in VIII-75 mutant. Abnormal suspensor consisting of two columns of cells was observed in 280-4-4 mutation. Newly identified embryo-lethals can serve as starting material for more detailed genetic and molecular studies.

Selecting plants with increased total polyphenol oxidases in the genus Trifolium.

One of the aims in red clover (Trifolium pratense) breeding is to increase the polyphenol oxidase (PPO) activity, which may effectively reduce protein breakdown in silage and when cattle are fed by fresh clover. We analysed total PPO activity spectrophotometrically and on the level of gene expression using real-time quantitative PCR in single plants derived from an interspecific T. pratense × T. medium hybrid. Experiments were performed for two years and evaluated according to the general linear model with three factors (family, year, and cut). The analysis revealed considerable variability in total PPO activity between individuals and between families. Four families and two individuals with significantly higher PPO activity were selected. Their PPO activity ranged from 3.411 to 3.547 mkatal/min/g and from 4.041 to 5.731 mkatal/min/g, respectively, in comparison with the control variety Amos (2.370 mkatal/min/g). The majority of PPO transcripts were expressed by the two genes PPO1/5 and PPO2. In some genotypes, the PPO5 gene was expressed. Quantitative PCR confirmed the highest activity of PPO genes in seven hybrid plants with higher DNA contents corresponding to 30 chromosomes with 815 013 copies per plant. Our results indicate the suitability of combining two methods for improved selection: initial expression analysis to assess the PPO transcript level indicating gene activity and subsequent enzymatic assay.

Gene Classification and Mining of Molecular Markers Useful in Red Clover (Trifolium pratense) Breeding

Red clover (Trifolium pratense) is an important forage plant worldwide. This study was directed to broadening current knowledge of red clovers coding regions and enhancing its utilization in practice by specific reanalysis of previously published assembly. A total of 42,996 genes were characterized using Illumina paired-end sequencing after manual revision of Blast2GO annotation. Genes were classified into metabolic and biosynthetic pathways in response to biological processes, with 7,517 genes being assigned to specific pathways. Moreover, 17,727 enzymatic nodes in all pathways were described. We identified 6,749 potential microsatellite loci in red clover coding sequences, and we characterized 4,005 potential simple sequence repeat (SSR) markers as generating polymerase chain reaction products preferentially within 100–350 bp. Marker density of 1 SSR marker per 12.39 kbp was achieved. Aligning reads against predicted coding sequences resulted in the identification of 343,027 single nucleotide polymorphism (SNP) markers, providing marker density of one SNP marker per 144.6 bp. Altogether, 95 SSRs in coding sequences were analyzed for 50 red clover varieties and a collection of 22 highly polymorphic SSRs with pooled polymorphism information content >0.9 was generated, thus obtaining primer pairs for application to diversity studies in T. pratense. A set of 8,623 genome-wide distributed SNPs was developed and used for polymorphism evaluation in individual plants. The polymorphic information content ranged from 0 to 0.375. Temperature switch PCR was successfully used in single-marker SNP genotyping for targeted coding sequences and for heterozygosity or homozygosity confirmation in validated five loci. Predicted large sets of SSRs and SNPs throughout the genome are key to rapidly implementing genome-based breeding approaches, for identifying genes underlying key traits, and for genome-wide association studies. Detailed knowledge of genetic relationships among breeding material can also be useful for breeders in planning crosses or for plant variety protection. Single-marker assays are useful for diagnostic applications.

Identification and Mapping of a T-DNA Induced Flower Mutation in Arabidopsis Thaliana

Collection of the T-DNA tagged lines of Arabidopsis thaliana have been created by Agrobacterium-mediated root transformation. Transgenic lines produced by this method have been screened for morphogenic mutations. A flower mutation with increased number of stamens and carpels (scaf1) was identified. This mutation has similar but weaker phenotype than the known mutant superman. Two mapping procedures, with visible and molecular markers, were used to locate scaf1 flower mutation. Genetic analysis showed that this mutation is located on chromosome 3 near gl1 gene. It is probably one of the SUPERMAN epigenetic alleles.