Reconstruction of Parental SSR Haplotypes from a Single Grape Seed

Microsatellite (‘single sequence repeats’, SSR) markers were widely used in the last decade for the identification of parents of a given grapevine variety or for pedigree reconstruction as well. By now the pedigree of the majority of the most important varieties is established. At the same time, knowing both of the parents gives information about which one was the mother plant and which one was the pollinator. Analyses of archaeological grapevine seeds can give new opportunities in the research of the evolution of varieties. In most of the angiosperms, the endosperm is triploid with two genome equivalents from the maternal line and one from the paternal line. Our presumption was that this numeral difference in the maternal and paternal alleles causes measureable difference in the amplification of SSR alleles from grapevine seeds. To validate our method, pre-experiments were carried out on 12 ‘Pinot gris’ seeds, which verified our theory

Introgression and pyramiding into common bean market class fabada of genes conferring resistance to anthracnose and potyvirus

Anthracnose and bean common mosaic (BCM) are considered major diseases in common bean crop causing severe yield losses worldwide. This work describes the introgression and pyramiding of genes conferring genetic resistance to BCM and anthracnose local races into line A25, a bean genotype classified as market class fabada. Resistant plants were selected using resistance tests or combining resistance tests and marker-assisted selection. Lines A252, A321, A493, Sanilac BC6-Are, and BRB130 were used as resistance sources. Resistance genes to anthracnose (Co-2 ( C ), Co-2 ( A252 ) and Co-3/9) and/or BCM (I and bc-3) were introgressed in line A25 through six parallel backcrossing programs, and six breeding lines showing a fabada seed phenotype were obtained after six backcross generations: line A1258 from A252; A1231 from A321; A1220 from A493; A1183 and A1878 from Sanilac BC6-Are; and line A2418 from BRB130. Pyramiding of different genes were developed using the pedigree method from a single cross between lines obtained in the introgression step: line A1699 (derived from cross A1258 × A1220), A2438 (A1220 × A1183), A2806 (A1878 × A2418), and A3308 (A1699 × A2806). A characterization based on eight morpho-agronomic traits revealed a limited differentiation among the obtained breeding lines and the recurrent line A25. However, using a set of seven molecular markers linked to the loci used in the breeding programs it was possible to differentiate the 11 fabada lines. Considering the genetic control of the resistance in resistant donor lines, the observed segregations in the last backcrossing generation, the reaction against the pathogens, and the expression of the molecular markers it was also possible to infer the genotype conferring resistance in the ten fabada breeding lines obtained. As a result of these breeding programs, genetic resistance to three anthracnose races controlled by genes included in clusters Co-2 and Co-3/9, and genetic resistance to BCM controlled by genotype I + bc-3 was combined in the fabada line A3308

Root and floral effects of flavonoid transport via an Arabidopsis MATE family transporter

Amongst their numerous roles in plants, flavonoid pigments are hypothesised to affect growth and development via interactions with components of auxin transport networks. Accordingly, our previous work showed that a multidrug and toxin efflux family transporter (FFT) alters root and seed characteristics and was required for full fertility in Arabidopsis. FFT is transcribed in guard cells throughout the plant and inactivation of FFT caused a significant perturbation in flavonoid profile in floral organs. Indeed, SEM and viability staining of mutant flowers reveal reduced anther dehiscence and a proportion of defective pollen, while siliques are smaller with fewer seeds than in WT. Null mutant seedlings grow faster and bolt sooner than WT, and seed size and mucilage are also affected. We are currently quantifying in more detail the flavonoid content of fft plants, and investigating the effect of externally applied auxin, flavonols and an auxin transport inhibitor. Finally, since we see FFT- promoter-GUS induced staining in vegetative tissues, including hydathode guard cells, we are also examining the abiotic stress response in the fft mutant. Co-expressed genes are involved in drought response including salt and osmotic stress

Selección de nuevas variedades de vid resistentes a enfermedades fúngicas, generadas mediante cruzamientos con Monastrell

La viticultura y la vinicultura han sido tradicionales desde la antigüedad en Murcia (España), siendo la vid un cultivo de gran importancia económica en esta zona. Sin embargo, las enfermedades causadas por hongos afectan gravemente al rendimiento, el coste y la calidad de la producción de la vid. Estas enfermedades se controlan en la actualidad mediante tratamientos con fungicidas, repetidos durante cada temporada de cultivo. La obtención de nuevas variedades resistentes de alta calidad reduce tanto el coste de producción de la vid como el riesgo ambiental. Basándonos únicamente en los datos de evaluación fenotípica, es poco factible conseguir la acumulación de genes de resistencia en una nueva línea mejorada. El uso de marcadores moleculares proporciona una nueva herramienta para los mejoradores y puede ayudar a superar este problema. En este trabajo, se presentan las actividades de investigación en el IMIDA orientadas a establecer una selección asistida por marcadores (MAS) para la resistencia a las enfermedades por hongos en la vid, mediante cruzamientos de 'Monastrell', una variedad de vinificación muy bien adaptada a las condiciones secas del clima mediterráneo, con plantas resistentes a enfermedades fúngicas.Los autores desean agradecer el apoyo financiero del Fondo Europeo de Desarrollo Regional y de la Consejería de Agricultura y Agua de la Región de Murcia. Este trabajo está financiado por el proyecto PO07‐37 cofinanciado al 80% por fondos FEDER

Beyond COST Action INTEGRAPE: The Grapevine Genomics Encyclopedia Initiative

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DNA marker identification of Rpv3 downy mildew resistance gene in seedless grape varieties

Table grapes are a valuable dietary product. Seedless grapes are in high demand among consumers. For this reason, the breeding of seedless varieties is one of the popular trends in modern viticulture, along with the production of environmentally friendly products. Downy mildew (Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni) is one of the most common fungal diseases of the grapevine. Most downy mildew resistant grape accessions belong to North American species like Vitis aestivalis Michx., V. berlandieri Planch., V. cinerea (Engelm. ex A. Gray) Engelm. ex Millard, V. riparia Michx., V. rupestris  Scheele, etc. The search for donors of resistance genes is an urgent task. Rpv3 is one of the most significant resistance genes from a number of North American grape varieties. The aim of this work is to identify the downy mildew resistance gene Rpv3 in seedless grape varieties by means of DNA-marker analysis. The grape varieties with rudimentary development of seed in berries and with North American species in the pedigree were chosen as the object of the study. The varieties “Dunavski lazur” and “Seyve Villard 12-375” with reference alleles were used as the positive control, while V. vinifera L. was used as the negative control. UDV305 and UDV737 DNA-markers were used in this study to identify the allelic type of the Rpv3 gene. The work was performed using the polymerase chain reaction. The reaction products were separated by capillary electrophoresis using the ABI Prism 3130 automatic genetic analyzer. Evaluation of the results was done using the GeneMapper and PeakScanner software. Functional alleles of the downy mildew resistance gene Rpv3 were revealed in grape  varieties “Kishmish zaporozhskiy”, “Lady Patricia”, “Remaily seedless”, “Pamyati Smirnova” and “Shayan”. Rpv3299-279, one of the seven known haplotypes, was identified in all the varieties. The pedigree analysis of the studied varieties indicated that the parental forms – “Seyve Villard” and “Seibel” hybrids – are presumably the donors of the gene. Grape accessions with the identified Rpv3 gene can be used in seedless varieties breeding as donors of resistance to downy mildew

Genome assembly of Vitis rotundifolia Michx. using third-generation sequencing (Oxford Nanopore Technologies)

The immune North American grapevine species Vitis rotundifolia Michaux (subgen. Muscadinia Planch.) is regarded as a potential donor of disease resistance genes, withstanding such dangerous diseases of grapes as powdery and downy mildews. The cultivar ‘Dixie’ is the only representative of this species preserved ex situ in Russia: it is maintained by the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) in the orchards of its branch, Krymsk Experiment Breeding Station. Third-generation sequencing on the MinION platform was performed to obtain information on the primary structure of the cultivar’s genomic DNA, employing also the results of Illumina sequencing available in databases. A detailed description of the technique with modifications at various stages is presented, as it was used for grapevine genome sequencing and whole-genome sequence assembly. The modified technique included the main stages of the original protocol recommended by the MinION producer: 1) DNA extraction; 2) preparation of libraries for sequencing; 3) MinION sequencing and bioinformatic data processing; 4) de novo whole-genome sequence assembly using only MinION data or hybrid assembly (MinION+Illumina data); and 5) functional annotation of the whole-genome assembly. Stage 4 included not only de novo sequencing, but also the analysis of the available bioinformatic data, thus minimizing errors and increasing precision during the assembly of the studied genome. The DNA isolated from the leaves of cv. ‘Dixie’ was sequenced using two MinION flow cells (R9.4.1)