Non-linear transformation of enzyme-linked immunosorbent assay (ELISA) measurements allows usage of linear models for data analysis.

BACKGROUND: In research questions such as in resistance breeding against the Beet necrotic yellow vein virus it is of interest to compare the virus concentrations of samples from different groups. The enzyme-linked immunosorbent assay (ELISA) counts as the standard tool to measure virus concentrations. Simple methods for data analysis such as analysis of variance (ANOVA), however, are impaired due to non-normality of the resulting optical density (OD) values as well as unequal variances in different groups. METHODS: To understand the relationship between the OD values from an ELISA test and the virus concentration per sample, we used a large serial dilution and modelled its non-linear form using a five parameter logistic regression model. Furthermore, we examined if the quality of the model can be increased if one or several of the model parameters are defined beforehand. Subsequently, we used the inverse of the best model to estimate the virus concentration for every measured OD value. RESULTS: We show that the transformed data are essentially normally distributed but provide unequal variances per group. Thus, we propose a generalised least squares model which allows for unequal variances of the groups to analyse the transformed data. CONCLUSIONS: ANOVA requires normally distributed data as well as equal variances. Both requirements are not met with raw OD values from an ELISA test. A transformation with an inverse logistic function, however, gives the possibility to use linear models for data analysis of virus concentrations. We conclude that this method can be applied in every trial where virus concentrations of samples from different groups are to be compared via OD values from an ELISA test. To encourage researchers to use this method in their studies, we provide an R script for data transformation as well as the data from our trial.

Deciphering the complex nature of bolting time regulation in Beta vulgaris.

KEY MESSAGE: Only few genetic loci are sufficient to increase the variation of bolting time in Beta vulgaris dramatically, regarding vernalization requirement, seasonal bolting time and reproduction type. Beta species show a wide variation of bolting time regarding the year of first reproduction, seasonal bolting time and the number of reproduction cycles. To elucidate the genetics of bolting time control, we used three F3 mapping populations that were produced by crossing a semelparous, annual sugar beet with iteroparous, vernalization-requiring wild beet genotypes. The semelparous plants died after reproduction, whereas iteroparous plants reproduced at least twice. All populations segregated for vernalization requirement, seasonal bolting time and the number of reproduction cycles. We found that vernalization requirement co-segregated with the bolting locus B on chromosome 2 and was inherited independently from semel- or iteroparous reproduction. Furthermore, we found that seasonal bolting time is a highly heritable trait (h 2 > 0.84), which is primarily controlled by two major QTL located on chromosome 4 and 9. Late bolting alleles of both loci act in a partially recessive manner and were identified in both iteroparous pollinators. We observed an additive interaction of both loci for bolting delay. The QTL region on chromosome 4 encompasses the floral promoter gene BvFT2, whereas the QTL on chromosome 9 co-localizes with the BR 1 locus, which controls post-winter bolting resistance. Our findings are applicable for marker-assisted sugar beet breeding regarding early bolting to accelerate generation cycles and late bolting to develop bolting-resistant spring and winter beets. Unexpectedly, one population segregated also for dwarf growth that was found to be controlled by a single locus on chromosome 9.

Genetic analysis of bolting after winter in sugar beet (Beta vulgaris L.).

KEY MESSAGE: This study reveals for the first time a major QTL for post-winter bolting resistance in sugar beet ( Beta vulgaris L.). The knowledge of this QTL is a major contribution towards the development of a winter sugar beet with controlled bolting behavior. In cool temperate climates, sugar beets are currently grown as a spring crop. They are sown in spring and harvested in autumn. Growing sugar beet as a winter crop with an extended vegetation period fails due to bolting after winter. Bolting after winter might be controlled by accumulating genes for post-winter bolting resistance. Previously, we had observed in field experiments a low post-winter bolting rate of 0.5 for sugar beet accession BETA 1773. This accession was crossed with a biennial sugar beet with regular bolting behavior to develop a F3 mapping population. The population was grown in the greenhouse, exposed to artificial cold treatment for 16 weeks and transplanted to the field. Bolting was recorded twice a week from May until October. Post-winter bolting behavior was assessed by two different factors, bolting delay (determined as days to bolt after cold treatment) and post-winter bolting resistance (bolting rate after winter). For days to bolt, means of F3 families ranged from 25 to 164 days while for bolting rate F3 families ranged from 0 to 1. For each factor one QTL explaining about 65% of the phenotypic variation was mapped to the same region on linkage group 9 with a partially recessive allele increasing bolting delay and post-winter bolting resistance. The results are discussed in relation to the potential use of marker-assisted breeding of winter sugar beets with controlled bolting.

Comparative QTL analysis of root lesion nematode resistance in barley.

KEY MESSAGE: This study demonstrates for the first time that resistance to different root lesion nematodes ( P. neglectus and P. penetrans ) is controlled by a common QTL. A major resistance QTL ( Rlnnp6H ) has been mapped to chromosome 6H using two independent barley populations. Root lesion nematodes (Pratylenchus spp.) are important pests in cereal production worldwide. We selected two doubled haploid populations of barley (Igri × Franka and Uschi × HHOR 3073) and infected them with Pratylenchus penetrans and Pratylenchus neglectus. Nematode multiplication rates were measured 7 or 10 weeks after infection. In both populations, continuous phenotypic variations for nematode multiplication rates were detected indicating a quantitative inheritance of resistance. In the Igri × Franka population, four P. penetrans resistance QTLs were mapped with 857 molecular markers on four linkage groups (2H, 5H, 6H and 7H). In the Uschi × HHOR 3073 population, eleven resistance QTLs (P. penetrans and P. neglectus) were mapped with 646 molecular markers on linkage groups 1H, 3H, 4H, 5H, 6H and 7H. A major resistance QTL named Rlnnp6H (LOD score 6.42-11.19) with a large phenotypic effect (27.5-36.6 %) for both pests was mapped in both populations to chromosome 6H. Another resistance QTL for both pests was mapped on linkage group 5H (Igri × Franka population). These data provide first evidence for common resistance mechanisms against different root lesion nematode species. The molecular markers are a powerful tool for the selection of resistant barley lines among segregating populations because resistance tests are time consuming and laborious.

GWAS reveals a rapidly evolving candidate avirulence effector in the Cercospora leaf spot pathogen.

The major resistance gene BvCR4 recently bred into sugar beet hybrids provides a high level of resistance to Cercospora leaf spot caused by the fungal pathogen Cercospora beticola. The occurrence of pathogen strains that overcome BvCR4 was studied using field trials in Switzerland conducted under natural disease pressure. Virulence of a subset of these strains was evaluated in a field trial conducted under elevated artificial disease pressure. We created a new C. beticola reference genome and mapped whole genome sequences of 256 isolates collected in Switzerland and Germany. These were combined with virulence phenotypes to conduct three separate genome-wide association studies (GWAS) to identify candidate avirulence genes. We identified a locus associated with avirulence containing a putative avirulence effector gene named AvrCR4. All virulent isolates either lacked AvrCR4 or had nonsynonymous mutations within the gene. AvrCR4 was present in all 74 isolates from non-BvCR4 hybrids, whereas 33 of 89 isolates from BvCR4 hybrids carried a deletion. We also mapped genomic data from 190 publicly available US isolates to our new reference genome. The AvrCR4 deletion was found in only one of 95 unique isolates from non-BvCR4 hybrids in the United States. AvrCR4 presents a unique example of an avirulence effector in which virulent alleles have only recently emerged. Most likely these were selected out of standing genetic variation after deployment of BvCR4. Identification of AvrCR4 will enable real-time screening of C. beticola populations for the emergence and spread of virulent isolates.

EcoTILLING in Beta vulgaris reveals polymorphisms in the FLC-like gene BvFL1 that are associated with annuality and winter hardiness.

BACKGROUND: Sugar beet (Beta vulgaris ssp. vulgaris L.) is an important crop for sugar and biomass production in temperate climate regions. Currently sugar beets are sown in spring and harvested in autumn. Autumn-sown sugar beets that are grown for a full year have been regarded as a cropping system to increase the productivity of sugar beet cultivation. However, for the development of these "winter beets" sufficient winter hardiness and a system for bolting control is needed. Both require a thorough understanding of the underlying genetics and its natural variation. RESULTS: We screened a diversity panel of 268 B. vulgaris accessions for three flowering time genes via EcoTILLING. This panel had been tested in the field for bolting behaviour and winter hardiness. EcoTILLING identified 20 silent SNPs and one non-synonymous SNP within the genes BTC1, BvFL1 and BvFT1, resulting in 55 haplotypes. Further, we detected associations of nucleotide polymorphisms in BvFL1 with bolting before winter as well as winter hardiness. CONCLUSIONS: These data provide the first genetic indication for the function of the FLC homolog BvFL1 in beet. Further, it demonstrates for the first time that EcoTILLING is a powerful method for exploring genetic diversity and allele mining in B. vulgaris.

QTL analysis of root-lesion nematode resistance in barley: 1. Pratylenchus neglectus.

The root-lesion nematode Pratylenchus neglectus can cause severe losses in barley cultivation. Multiplication rates had been found to vary greatly between different barley accessions. Two winter barley cultivars, Igri and Franka, had been found to differ in their ability to resist this parasite. An existing Igri × Franka doubled haploid population was chosen to genetically map resistance genes after artificial inoculation with P. neglectus in the greenhouse and climate chamber. A continuous phenotypic variation was found indicating a quantitative inheritance of P. neglectus resistance. An existing map was enriched by 527 newly developed Diversity Array Technology markers (DArTs). The new genetic linkage map was comprised of 857 molecular markers that cover 1,157 cM on seven linkage groups. Using phenotypic data collected from four different experiments in 3 years, five quantitative trait loci were mapped by composite interval mapping on four (3H, 5H, 6H and 7H) linkage groups. A quantitative trait locus with a large phenotypic effect of 16% and likelihood of odds (LOD) score of 6.35 was mapped on linkage group 3H. The remaining four QTLs were classified as minor or moderate with LOD scores ranging from 2.71 to 3.55 and R (2) values ranging from 8 to 10%. The DNA markers linked to the resistance QTLs should be quite useful for marker-assisted selection in barley breeding because phenotypic selection is limited due to time constraints and labor costs.

Effect of crop improvement on genetic diversity in oilseed Brassica rapa (turnip-rape) cultivars, detected by SSR markers.

With the improvement of seed quality, Brassica rapa oilseed germplasm went through 2 major breeding bottlenecks during the introgression of genes for zero erucic acid content and low glucosinolate content, respectively. This study investigates the impact of these bottlenecks on the genetic diversity in European winter B. rapa by comparing 3 open-pollinated cultivars, each representing a different breeding period. Diversity was estimated on 32 plants per cultivar, with 16 simple sequence repeat (SSR) markers covering each of the B. rapa linkage groups. There was no significant loss of genetic diversity over the 3 cultivars as indicated by allele number (ranging from 59 to 55), mean allele number (from 3.68 to 3.50), Shannon information index (from 0.94 to 0.87) and expected heterozygosity (from 0.53 to 0.48). About 83% of the total variation was attributed to within-cultivar variation, and the remaining 17% to between-cultivar variation by analysis of molecular variance (AMOVA). Individual plants were separated into the 3 cultivars by principal coordinate analysis (PCoA). In conclusion, genetic diversity within cultivars was high and quality breeding in B. rapa did not significantly reduce the genetic diversity of B. rapa winter cultivars, so there is no risk of decline in performance due to quality improvement.

A Detailed Analysis of the BR1 Locus Suggests a New Mechanism for Bolting after Winter in Sugar Beet (Beta vulgaris L.).

Sugar beet (Beta vulgaris ssp. vulgaris) is a biennial, sucrose-storing plant, which is mainly cultivated as a spring crop and harvested in the vegetative stage before winter. For increasing beet yield, over-winter cultivation would be advantageous. However, bolting is induced after winter and drastically reduces yield. Thus, post-winter bolting control is essential for winter beet cultivation. To identify genetic factors controlling bolting after winter, a F2 population was previously developed by crossing the sugar beet accessions BETA 1773 with reduced bolting tendency and 93161P with complete bolting after winter. For a mapping-by-sequencing analysis, pools of 26 bolting-resistant and 297 bolting F2 plants were used. Thereby, a single continuous homozygous region of 103 kb was co-localized to the previously published BR1 QTL for post-winter bolting resistance (Pfeiffer et al., 2014). The BR1 locus was narrowed down to 11 candidate genes from which a homolog of the Arabidopsis CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR 73-I (CPSF73-I) was identified as the most promising candidate. A 2 bp deletion within the BETA 1773 allele of BvCPSF73-Ia results in a truncated protein. However, the null allele of BvCPSF73-Ia might partially be compensated by a second BvCPSF73-Ib gene. This gene is located 954 bp upstream of BvCPSF73-Ia and could be responsible for the incomplete penetrance of the post-winter bolting resistance allele of BETA 1773. This result is an important milestone for breeding winter beets with complete bolting resistance after winter.

A candidate gene-based association study of tocopherol content and composition in rapeseed (Brassica napus).

Rapeseed (Brassica napus L.) is the most important oil crop of temperate climates. Rapeseed oil contains tocopherols, also known as vitamin E, which is an indispensable nutrient for humans and animals due to its antioxidant and radical scavenging abilities. Moreover, tocopherols are also important for the oxidative stability of vegetable oils. Therefore, seed oil with increased tocopherol content or altered tocopherol composition is a target for breeding. We investigated the role of nucleotide variations within candidate genes from the tocopherol biosynthesis pathway. Field trials were carried out with 229 accessions from a worldwide B. napus collection which was divided into two panels of 96 and 133 accessions. Seed tocopherol content and composition were measured by HPLC. High heritabilities were found for both traits, ranging from 0.62 to 0.94. We identified polymorphisms by sequencing selected regions of the tocopherol genes from the 96 accession panel. Subsequently, we determined the population structure (Q) and relative kinship (K) as detected by genotyping with genome-wide distributed SSR markers. Association studies were performed using two models, the structure-based GLM + Q and the PK-mixed model. Between 26 and 12 polymorphisms within two genes (BnaX.VTE3.a, BnaA.PDS1.c) were significantly associated with tocopherol traits. The SNPs explained up to 16.93% of the genetic variance for tocopherol composition and up to 10.48% for total tocopherol content. Based on the sequence information we designed CAPS markers for genotyping the 133 accessions from the second panel. Significant associations with various tocopherol traits confirmed the results from the first experiment. We demonstrate that the polymorphisms within the tocopherol genes clearly impact tocopherol content and composition in B. napus seeds. We suggest that these nucleotide variations may be used as selectable markers for breeding rapeseed with enhanced tocopherol quality.