Development and evaluation of robust molecular markers linked to disease resistance in tomato for distinctness, uniformity and stability testing

Molecular markers linked to phenotypically important traits are of great interest especially when traits are difficult and/or costly to be observed. In tomato where a strong focus on resistance breeding has led to the introgression of several resistance genes, resistance traits have become important characteristics in distinctness, uniformity and stability (DUS) testing for Plant Breeders Rights (PBR) applications. Evaluation of disease traits in biological assays is not always straightforward because assays are often influenced by environmental factors, and difficulties in scoring exist. In this study, we describe the development and/or evaluation of molecular marker assays for the Verticillium genes Ve1 and Ve2, the tomato mosaic virusTm1 (linked marker), the tomato mosaic virus Tm2 and Tm22 genes, the Meloidogyne incognita Mi1-2 gene, the Fusarium I (linked marker) and I2 loci, which are obligatory traits in PBR testing. The marker assays were evaluated for their robustness in a ring test and then evaluated in a set of varieties. Although in general, results between biological assays and marker assays gave highly correlated results, marker assays showed an advantage over biological tests in that the results were clearer, i.e., homozygote/heterozygote presence of the resistance gene can be detected and heterogeneity in seed lots can be identified readily. Within the UPOV framework for granting of PBR, the markers have the potential to fulfil the requirements needed for implementation in DUS testing of candidate varieties and could complement or may be an alternative to the pathogenesis tests that are carried out at present

Variance of allele balance calculated from low coverage sequencing data infers departure from a diploid state

Abstract Background Polyploidy and heterokaryosis are common and consequential genetic phenomena that increase the number of haplotypes in an organism and complicate whole-genome sequence analysis. Allele balance has been used to infer polyploidy and heterokaryosis in diverse organisms using read sets sequenced to greater than 50× whole-genome coverage. However, sequencing to adequate depth is costly if applied to multiple individuals or large genomes. Results We developed VCFvariance.pl to utilize the variance of allele balance to infer polyploidy and/or heterokaryosis at low sequence coverage. This analysis requires as little as 10× whole-genome coverage and reduces the allele balance profile down to a single value, which can be used to determine if an individual has two or more haplotypes. This approach was validated using simulated, synthetic, and authentic read sets from the oomycete species Bremia lactucae and Phytophthora infestans, the fungal species Saccharomyces cerevisiae, and the plant species Arabidopsis arenosa. This approach was deployed to determine that nine of 21 genotyped European race-type isolates of Bremia lactucae were inconsistent with diploidy and therefore likely heterokaryotic. Conclusions Variance of allele balance is a reliable metric to detect departures from a diploid state, including polyploidy, heterokaryosis, a mixed sample, or chromosomal copy number variation. Deploying this strategy is computationally inexpensive, can reduce the cost of sequencing by up to 80%, and used to test any organism. </jats:sec

Variance of allele balance calculated from low coverage sequencing data infers departure from a diploid state

BackgroundPolyploidy and heterokaryosis are common and consequential genetic phenomena that increase the number of haplotypes in an organism and complicate whole-genome sequence analysis. Allele balance has been used to infer polyploidy and heterokaryosis in diverse organisms using read sets sequenced to greater than 50× whole-genome coverage. However, sequencing to adequate depth is costly if applied to multiple individuals or large genomes.ResultsWe developed VCFvariance.pl to utilize the variance of allele balance to infer polyploidy and/or heterokaryosis at low sequence coverage. This analysis requires as little as 10× whole-genome coverage and reduces the allele balance profile down to a single value, which can be used to determine if an individual has two or more haplotypes. This approach was validated using simulated, synthetic, and authentic read sets from the oomycete species Bremia lactucae and Phytophthora infestans, the fungal species Saccharomyces cerevisiae, and the plant species Arabidopsis arenosa. This approach was deployed to determine that nine of 21 genotyped European race-type isolates of Bremia lactucae were inconsistent with diploidy and therefore likely heterokaryotic.ConclusionsVariance of allele balance is a reliable metric to detect departures from a diploid state, including polyploidy, heterokaryosis, a mixed sample, or chromosomal copy number variation. Deploying this strategy is computationally inexpensive, can reduce the cost of sequencing by up to 80%, and used to test any organism

Variance of allele balance calculated from low coverage sequencing data infers departure from a diploid state

AbstractMotivationPolyploidy and heterokaryosis are common and consequential genetic phenomena that increase the number of haplotypes in an organism and complicate whole-genome sequence analysis. Allele balance has been used to infer polyploidy and heterokaryosis in diverse organisms using read sets sequenced to greater than 50x whole-genome coverage. However, Sequencing to adequate depth is costly if applied to multiple individuals or large genomes.ResultsWe developed VCFvariance.pl to utilize the variance of allele balance to infer polyploidy and/or heterokaryosis at low sequence coverage. This analysis requires as little as 10x whole-genome coverage and reduces the allele balance profile down to a single value, which can be used to determine if an individual has two or more haplotypes. This approach was validated on simulated, synthetic, and authentic read sets from an oomycete, fungus, and plant. The approach was deployed to ascertain the genome status of multiple isolates of Bremia lactucae and Phytophthora infestans.Availability and implementationVCFvariance.pl is a Perl script available at https://github.com/kfletcher88/VCFvariance.</jats:sec

New evidence for the synteny of rice chromosome 1 and barley chromosome 3H from rice expressed sequence tags

To provide improved access to the wealth of resources and genomic information that is presently being developed for rice a set of 88 rice expressed sequence tags (ESTs) previously mapped on rice chromosome 1 in the cross 'Nipponbare' × 'Kasalath' was used for comparative mapping in a cross of the barley cultivars 'Igri' and 'Franka'. As expected, most (89%) of the clones gave distinct banding patterns in barley of which about one-third was polymorphic between 'Igri' and 'Franka'. These polymorphisms were mapped, and most of these (56%) confirmed that rice chromosome 1 and barley chromosome 3H are syntenous. All single-copy markers identified conserved collinear positions, while markers with multiple copies did so in a few cases only. The markers that were not fitting in the collinear order were distributed randomly across the barley genome. The comparative maps of barley chromosome 3H and rice chromosome 1 comprise in total 26 common markers covering more than 95% of the genetic length of both chromosomes. A 30-fold reduction of recombination is seen around the barley centromere, and synteny may be interrupted in this region. However, the good overall synteny on a mesoscale (1–10 cM) justifies the use of rice as a platform for map-based cloning in barley.Key words: Oryza sativa, Hordeum vulgare, RFLP, synteny, comparative mapping.</jats:p

Additional file 1 of Variance of allele balance calculated from low coverage sequencing data infers departure from a diploid state

Additional file 1 Figures S1 Histograms of allele balance generated from simulated data; Fig. S2 Histograms of allele balance generated from synthetic E. coli data, Fig. S3 Polymorphisms identified for 30 isolates of B. lactucae, downsampled to different coverages, used to generate Fig. 3; Fig. S4 Results of running six isolates of B. lactucae through nQuire; Fig. S5 Polymorphisms identified for 24 isolates of S. cerevisiae, downsampled to different coverages, used to generate Fig. 5; and Fig. S6 Polymorphisms analyzed for 24 A. arenosa individuals used to generate Fig. 6 (PDF 1167 kb)

Additional file 2 of Variance of allele balance calculated from low coverage sequencing data infers departure from a diploid state

Additional file 2 Tables S1 Summary results for 1,000 tests simulating different ploidies and different levels of coverage; Table S2 SRA information and values calculated for downsampled data of Bremia lactucae isolates used to validate the variance of allele balance approach; Table S3 SRA information and values calculated for Phytophthora infestans isolates used in this study; Table S4 SRA information and values calculated for downsampled Saccharomyces cerevisiae isolates used in this study; Table S5 SRA information and values calculated for Arabidopsis arenosa individuals used in this study; Table S6 Variance of allele balance values calculated for European race type isolates of Bremia lactucae in this study; and Table S7 Number of mutations introduced into each synthetic haplotype. Lower numbers are a subset of the larger preceding number. Each table is on a separate tab of the excel workbook (XLSX 35 kb)

Distribution of races and virulence factors of Bremia lactucae in the main lettuce production area in Brazil

Lettuce downy mildew, caused by Bremia lactucae, occurs throughout São Paulo, the main lettuce production state in Brazil, during winter due to low temperatures and high humidity. Thus, the aim of this work was to monitor the races and virulence factors in São Paulo’s B. lactucae population from 2003 to 2015, and to compare the results with similar studies in other countries. Data from 582 isolates collected from Lactuca sativa cultivars in 33 municipalities of São Paulo were analyzed. Seventeen virulence patterns (races) were identified. Only few races have been stable over the years. The virulence factors v1, v2, v3, v4, v5/8, v7, v10, v11, v12 and v13 have always been present since the first screening in 2003 while the factors v6, v14, v15, v16, v36 and v37 were found in varying frequencies. The population diversity in São Paulo increased over time with five new races emerging in 2014 and 2015. However, the virulence factors identified are the same as previously described in other areas. The races of São Paulo state are similar to the new races from Australia, California, Europe and Japan, grouping in the same clade by analysis the virulence patterns. The genes Dm17, Dm18, R38 and the resistance factors of the cultivars Balesta and Bellisimo can be recommended as source of resistance to the pathotypes characterized to date in São Paulo.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Gulf Coast Research and Education Center University of Florida, 14625 CR 672, 33598Department of Crop Production School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP), Jaboticabal, Via de Acesso Prof. Paulo Donato Castellane - s/n – CEP: 14Naktuinbouw, Sotaweg 22, 2371 GDDepartment of Plant Pathology School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP), Jaboticabal, Via de Acesso Prof. Paulo Donato Castellane - s/n – CEP: 14Department of Crop Production School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP), Jaboticabal, Via de Acesso Prof. Paulo Donato Castellane - s/n – CEP: 14Department of Plant Pathology School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP), Jaboticabal, Via de Acesso Prof. Paulo Donato Castellane - s/n – CEP: 14FAPESP: 2002/13968-9FAPESP: 2012/09705-4FAPESP: 2014/10923-1FAPESP: and 2014/11242-

Harmorescoll: a harmonized collection of reference isolates, controls and differentials to facilitate disease resistance testing

Plant breeding for resistance to pests is a commercial activity that benefits from a regulatory framework that promotes steady progress. In Europe, a significant part of this framework is applied by national variety examination offices and provided by the Community Plant Variety Office, CPVO. In the vegetable sector, disease resistances are commonly used as characteristics for establishing distinctness, uniformity and stability (DUS) of new cultivars. CPVO has published official protocols for resistance testing, of which many have been harmonized between European countries and seed companies over the last 15 years. As a next step, we now propose to improve information on the availability and quality of the most essential reference materials (controls, differentials, and isolates) for relevant vegetable crops. The Harmorescoll project involves examination offices, accredited examination centres, seed companies and trade associations. The inventory of the reference materials described in CPVO protocols and guidelines of UPOV, the International Union for the Protection of New Varieties of Plants, contains materials already available through existing initiatives (i.e., IBEB, IWGP, MATREF, Plantum), and some missing materials. In this new initiative, a series of experimental validations will be performed on prioritized pathosystems, respecting some criteria to consider the materials as a reference. The aim of the manuscript is to present the project, next steps, and its benefits. Harmorescoll will build a new network by using the experience obtained in existing initiatives that serve the same purpose in different European countries. Harmorescoll wishes to become a user-friendly platform providing reliable information on reference materials for a given resistance test, and how to obtain it. The aim is to improve efficiency and trust in DUS resistance tests. Having a harmonized system ensuring access to high quality reference material will be an asset for breeders worldwide to characterize vegetable genetic resources for disease resistance.We thank all people involved in the project as representatives of their company and/or initiatives, among them Ton Allersma (Bayer/Euroseeds), Cécile Collonnier (CPVO), Carole Constant (Sakata/UFS), Pascal Coquin (GEVES), Perrine David (HM.CLAUSE), Michel de Lange (Syngenta/IBEB/IWGP), Raphaël Fernandez (GEVES), Eelco Gilijamse (Rijk Zwaan/Plantum), Aurélia Gouleau (GEVES), Monique Hoogenboom (Naktuinbouw), Catherine Langat (Euroseeds), Christelle Lavaud (GEVES), Claudius Marondedze (Euroseeds), Lesley McCarthy (SASA), Bertrand Monsimier (Vilmorin Mikado), Céline Morineau (CPVO), Raquel Piqueras (INIA-CSIC), Rose Souza Richards (ISF), Monique van Vegchel (Plantum). We thank the providers of reference materials in the initiatives, partners that store back-up of the isolates and the initiatives’ members for daily care of the reference material. The Harmorescoll project has received co-funding from the Community Plant Variety Office (CPVO) under grant agreement N°7515996.Peer reviewe