Larisa Garkava-Gustavsson

Genetic diversity in watermelon (Citrullus lanatus) landraces from Zimbabwe revealed by RAPD and SSR markers.

Low polymorphism in cultivated watermelon has been reported in previous studies, based mainly on US Plant Introductions and watermelon cultivars, most of which were linked to breeding programmes associated with disease resistance. Since germplasm sampled in a putative centre of origin in southern Africa may harbour considerably higher variability, DNA marker-based diversity was estimated among 81 seedlings from eight accessions of watermelon collected in Zimbabwe; five accessions of cow-melons (Citrullus lanatus var. citroides) and three of sweet watermelons (C. lanatus var. lanatus). Two molecular marker methods were used, random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) also known as microsatellite DNA. Ten RAPD primers produced 138 markers of which 122 were polymorphic. Nine SSR primer pairs detected a total of 43 alleles with an average of 4.8 alleles per locus. The polymorphic information content (PIC) ranged from 0.47 to 0.77 for the RAPD primers and from 0.39 to 0.97 for the SSR loci. Similarity matrices obtained with SSR and RAPD, respectively, were highly correlated but only RAPD was able to provide each sample with an individual-specific DNA profile. Dendrograms and multidimensional scaling (MDS) produced two major clusters; one with the five cow-melon accessions and the other with the three sweet watermelon accessions. One of the most variable cow-melon accessions took an intermediate position in the MDS analysis, indicating the occurrence of gene flow between the two subspecies. Analysis of molecular variation (AMOVA) attributed most of the variability to within-accessions, and contrary to previous reports, sweet watermelon accessions apparently contain diversity of the same magnitude as the cow-melons.

RAPD-based analysis of genetic diversity and selection of lingonberry (Vaccinium vitis-idaea L.) material for ex situ conservation

Random amplified polymorphic DNA markers were used to assess relatedness and genetic diversity for 15 lingonberry (Vaccinium vitis-idaea) populations. Seven primers yielding 59 polymorphic bands were used to analyse 13 populations, representing ssp. vitis-idaea from Sweden, Finland, Norway, Estonia and Russia, and two populations, representing ssp. minus from Japan and Canada. A cluster analysis and a multidimensional scaling analysis (MDS) showed similar phenetic patterns among populations, with a pronounced geographic grouping in most cases. Significant correlations were obtained between geographic and genetic distances for the entire set of populations as well as for the 13 ssp. vitis-idaea populations. Mean within-population diversity was 0.206 when estimated with Lynch and Milligans index, and 0.431 when estimated with Shannons index, which is in agreement with the mixed mating system reported for lingonberry. Within-population variability accounted for 68.6% of the total variance when all populations were included, and for 78.8% when only populations of ssp. vitis-idaea were analysed. Two different approaches were applied to the selection of plant material for a potential gene bank: (1) a hierarchical sampling strategy based on a cluster analysis and (2) the Maximum genetic diversity program, developed for the establishment of core collections. Random sampling was undertaken for comparisons with the selected data sets. The most diverse and representative set of lingonberry specimens was obtained when samples were selected with the Maximum diversity program.

Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level.

BackgroundThe amount and structure of genetic diversity in dessert apple germplasm conserved at a European level is mostly unknown, since all diversity studies conducted in Europe until now have been performed on regional or national collections. Here, we applied a common set of 16 SSR markers to genotype more than 2,400 accessions across 14 collections representing three broad European geographic regions (North + East, West and South) with the aim to analyze the extent, distribution and structure of variation in the apple genetic resources in Europe.ResultsA Bayesian model-based clustering approach showed that diversity was organized in three groups, although these were only moderately differentiated (FST = 0.031). A nested Bayesian clustering approach allowed identification of subgroups which revealed internal patterns of substructure within the groups, allowing a finer delineation of the variation into eight subgroups (FST = 0.044). The first level of stratification revealed an asymmetric division of the germplasm among the three groups, and a clear association was found with the geographical regions of origin of the cultivars. The substructure revealed clear partitioning of genetic groups among countries, but also interesting associations between subgroups and breeding purposes of recent cultivars or particular usage such as cider production. Additional parentage analyses allowed us to identify both putative parents of more than 40 old and/or local cultivars giving interesting insights in the pedigree of some emblematic cultivars.ConclusionsThe variation found at group and subgroup levels may reflect a combination of historical processes of migration/selection and adaptive factors to diverse agricultural environments that, together with genetic drift, have resulted in extensive genetic variation but limited population structure. The European dessert apple germplasm represents an important source of genetic diversity with a strong historical and patrimonial value. The present work thus constitutes a decisive step in the field of conservation genetics. Moreover, the obtained data can be used for defining a European apple core collection useful for further identification of genomic regions associated with commercially important horticultural traits in apple through genome-wide association studies.

Genetic diversity among and within watermelon (Citrullus lanatus) landraces in Southern Africa

Summary Genetic diversity in watermelon (Citrullus lanatus) was estimated among 213 seedlings from 22 accessions collected in Botswana, Namibia, South Africa, Zambia, and Zimbabwe. The accessions consisted of two types of watermelon landraces: sweet watermelon (C. lanatus var. lanatus) and cow-melon (C. lanatus var. citroides), also known as citron melon. In addition, three commercial varieties of C. lanatus var. lanatus from the USA were included for comparison. Ten simple sequence repeat (SSR; microsatellite) loci detected a total of 153 alleles. The polymorphic information content (PIC) ranged from 0.833 – 0.963, suggesting sufficient discriminatory power. Both a cluster analysis and a principal co-ordinate analysis produced two major clusters, one with the 13 cow-melon accessions and the other with the 12 sweet watermelon accessions. Within the sweet watermelon cluster, the three US cultivars grouped together with the Botswana accessions. Some of the other accessions also grouped according to their country of origin, but others did not. Within-accession diversity parameters showed that those sweet watermelon accessions found in traditional agrosystems were just as genetically variable as the cow-melon accessions.

A high-density, multi-parental SNP genetic map on apple validates a new mapping approach for outcrossing species

Quantitative trait loci (QTL) mapping approaches rely on the correct ordering of molecular markers along the chromosomes, which can be obtained from genetic linkage maps or a reference genome sequence. For apple (Malus domestica Borkh), the genome sequence v1 and v2 could not meet this need; therefore, a novel approach was devised to develop a dense genetic linkage map, providing the most reliable marker-loci order for the highest possible number of markers. The approach was based on four strategies: (i) the use of multiple full-sib families, (ii) the reduction of missing information through the use of HaploBlocks and alternative calling procedures for single-nucleotide polymorphism (SNP) markers, (iii) the construction of a single backcross-type data set including all families, and (iv) a two-step map generation procedure based on the sequential inclusion of markers. The map comprises 15 417 SNP markers, clustered in 3 K HaploBlock markers spanning 1 267 cM, with an average distance between adjacent markers of 0.37 cM and a maximum distance of 3.29 cM. Moreover, chromosome 5 was oriented according to its homoeologous chromosome 10. This map was useful to improve the apple genome sequence, design the Axiom Apple 480 K SNP array and perform multifamily-based QTL studies. Its collinearity with the genome sequences v1 and v3 are reported. To our knowledge, this is the shortest published SNP map in apple, while including the largest number of markers, families and individuals. This result validates our methodology, proving its value for the construction of integrated linkage maps for any outbreeding species.

Modern apple breeding is associated with a significant change in the allelic ratio of the ethylene production gene Md-ACS1

Summary Inreasing consumer demand for firm apple fruit has raised interest among scientists and breeders to identify heritable traits that affect fruit texture. One such candidate gene is the bi-allelic Md-ACS1 locus, that shows a strong association between genotype and internal ethylene content in ripening fruit. However, associations between genotype and fruit firmness, or between genotype and fruit softening during storage, have been somewhat unclear. In order to determine the potential importance of allelic variation at this locus, we used PCR methods to investigate Md-ACS1 alleles in 137 apple cultivars. In addition, we also compiled data from previously published studies, to achieve an eventual dataset with 270 cultivars. Allele 1, associated with higher ethylene concentrations in ripening apples, was much more common in older cultivars (relative frequency = 0.88 in cultivars introduced before 1800; 0.84 in cultivars introduced between 1800 – 1899; and 0.70 in cultivars introduced in 1900 – 1959). Conversely, allele 2 associated with lower ethylene concentrations, was more common in modern cultivars (relative frequency = 0.57 in cultivars introduced since 1960). The frequencies of the three Md-ACS1 genotypes (1/1, 1/2, 2/2), as well as the relative allele frequencies (1 vs. 2), varied significantly depending on the date of introduction of the investigated cultivars ( i.e., 1800 – 1899 vs. 1900 – 1959; and 1900 – 1959 vs. ≥ 1960). The significant shift in allelic ratios at the Md-ACS1 locus observed during the 20th century was probably associated with the substantial impact that a small number of heterozygous cultivars have had on modern apple breeding. Allelic variation at the Md-ACS1 locus may thus be one of the most influential factors in modern apple breeding.

Marker-aided breeding for resistance to bean common mosaic virus in Kyrgyz bean cultivars

Common bean (Phaseolus vulgaris L.) is an important export crop in Kyrgyzstan. Bean common mosaic virus (BCMV) is a seed-born disease that affects this crop and reduces its yield significantly in the country. The aim of this study was to identify virus strain(s) occurring in Kyrgyzstan and breed host plant resistance to BCMV using DNA markers. Susceptible Kyrgyz cultivars (Ryabaya, Kytayanka and Lopatka) were included in a backcrossing breeding scheme for introducing host plant resistance from resistant cultivars (Vaillant and Flagrano). The virus strains were evaluated according to the symptoms of differential cultivars. The virus strain NL6 was found in northern Kyrgyzstan, where farmers grow most of the common bean produced in this country. Two SCAR markers (SW13 and SBD5) were used successfully in marker-aided backcrossing for pyramiding the I and bc-12 genes, which provide host plant resistance to BCMV. Resistant BC4F2 offspring carrying the I gene showed hypersensitivity reactions to necrosis inducing NL3 strain after detached leaf-assays.

Self-incompatibility alleles of 104 apple cultivars grown in northern Europe

Summary Gametic self-incompatibility in apple is determined by a set of S-locus alleles, which can be identified using PCR with allele-specific DNA primers. Allele composition has previously been determined for a large number of apple cultivars grown in North America, central and southern Europe, and Asia. In the present study, 103 not previously studied apple cultivars that are grown mainly in northern Europe were scored for their S-allele composition in order to provide apple growers and breeders with information on incompatibility among cultivars. In addition, S-allele frequencies were determined in some control cultivars and compared with data reported previously. A different genotype was found for ‘Discovery’, which was therefore added to our list. The most common S-allele in the resulting set of 104 cultivars investigated at Balsgård was S7 (18%) followed by S3 (17%), S5 (14%), and S1 and S2 (both at 11%). Comparisons of allele frequencies obtained from previous compilations showed that the frequency of S9, in particular, deviated considerably; 15–16% was noted in two other studies, but only 1% in the Balsgård study. This discrepancy is most likely due to the importance of ‘Red Delicious’ (S9S28) in almost all areas of the World except northern Europe. A preponderance (30%) of the S3 allele was seen in a collection of 23 scab-resistant cultivars, suggesting that cross-compatibility may become a problem, especially in organic orchards.

Real-time PCR for detection and quantification, and histological characterization of Neonectria ditissima in apple trees

Key messageWe designed a pair of primers from a region of the β-tubulin gene to detect and quantifyNeonectria ditissimain wood of some infected apple cultivars, and optimized light microscopy to study fungal-plant interactions.AbstractNeonectria ditissima, the causal pathogen of fruit tree canker, is a sordariomycete fungus that affects apple orchards, especially in north-western Europe. To prevent serious disease epidemics, an accurate, rapid, and sensitive method for detection of N. ditissima is needed for pathogen identification. A quantitative real-time PCR (qPCR) assay was developed for both detection and quantification of this pathogen in infected apple cultivars. Several primer sets were designed from regions of the β-tubulin gene. One primer set passed several validation tests, and the melting curve confirmed species-specific amplification of the correct product. In addition, the N. ditissima biomass could be detected at variable amounts in samples from the infection sites of six different cultivars, with ‘Aroma’ having the lowest amount of N. ditissima biomass and ‘Elise’ the highest. To complement the qPCR results, tissue from detached shoots and 1-year-old trees of ‘Cox’s Orange Pippin’ (susceptible) and ‘Santana’ (partially resistant) was used in a histopathology study. In both detached shoots and trees, fungal hyphae were found in cells of all tissues. No qualitative differences in the anatomy of the infected samples were observed between the cultivars. In the detached shoot experiment, both cultivars were affected but differences in the rate of disease progression suggest that the partially resistant cultivar could resist the fungus longer. The qPCR assay developed in our study produced reproducible results and can be used for detection of N. ditissima in infected trees.

Elucidating the genetic diversity of farmer cultivars of female date palms (Phoenix dactylifera L.) from Sudan by microsatellite markers

Genetic diversity in female date palms for a total of 89 tree samples, representing 18 cultivar groups, was investigated using 10 loci of microsatellite (SSR) primers. The microsatellites examined showed high polymorphism, a total of 126 for the 10 microsatellite loci, giving an average of 12.6. Low FST values were observed among different fruit classification groups of Sudanese date palms (soft, semi-soft and dry) suggesting that the observed high genetic variability is maintained locally within cultivars. Across all genetic parameters, in comparison among the cultivar groups, the Jaw group was consistently highly variable followed by Korsha, Gondela and Barakawi, respectively. Multivariate analysis of the 89 date palms showed some strong relationships with some mixed grouping, showing a tendency of relate fruit classification and related genotypes. Dry dates almost grouped together while the soft and semi-soft were mixed and somewhat related to genotypes. The effect of genotype was highly supported by the relationship of 14 cultivar groups, in which similar cultivars were associated regardless of the state to which they belong. Further detailed analyses of female date palm populations from different geographic locations represented by a minimum of five plants per cultivar is necessary to provide the basis for understanding of the date palm genomes, revealing the true extent of gene flow between farmer cultivars.