Genetic Diversity and Gene Flow of Four South African Venturia inaequalis (Apple Scab) Populations.

Venturia inaequalis isolates were collected during the 2012/13 and 2013/14 seasons from the four principal apple growing regions of South Africa, Elgin (n = 114), Koue Bokkeveld (n = 126), Lower Langkloof (n = 92), and Upper Langkloof (n = 103). Sequence analysis of the ribosomal internal transcribed spacer (ITS) gene regions and genotyping with six (2012/13) and seven (2013/14) microsatellite (SSR) markers was conducted. A subset of 12 isolates from the individual ITS haplotype groups were sequenced for the translation elongation factor-1 alpha (TEF1) and the large subunit of the RNA polymerases II (RPB1) gene regions. Four haplotypes were found for ITS, whereas all isolates were identical for the TEF1 and RPB1 gene regions. The SSR markers revealed considerable variation with an average gene diversity (H) of 0.675. Multivariate analysis (discriminant analysis of principal components [DAPC]) revealed that the two Langkloof populations clustered together with the Koue Bokkeveld population. The population from the warmer winter region, Elgin, clustered separately from the rest of the populations (ΦPT = 0.076 to 0.116; P ≤ 0.05). Estimates of gene flow showed the highest migration rate from the Koue Bokkeveld, toward the Lower Langkloof (M = 151.1), and the least migration to and from the Elgin region (average M = 42.75). Occasionally, identical genotypes (clones) were detected across seasons in the Koue Bokkeveld and Elgin area, which might contribute to overwintering conidia. From this study, it is evident that South Africa most likely has V. inaequalis subpopulations linked to diverse climatic conditions of the coastal Elgin region compared with the mountainous inland regions of the Koue Bokkeveld and the Langkloof.

The development of a bin mapping population and the selective mapping of 103 markers in the diploid Fragaria reference map.

We have identified a set of plants (the bin set) to permit "selective" or "bin" mapping using the diploid strawberry mapping population FV x FN, derived from the F2 cross F. vesca 815 x F. nubicola 601, which has been used to develop the Fragaria reference map. The bin set consists of 8 plants: the F. vesca 815 parent, the F1 hybrid individual, and 6 seedlings of the F2 population. This bin set divides the 578 cM of the diploid Fragaria genome into 46 bins, the largest mapping bin being 26 cM in length and the average bin size being 12.6 cM. To validate the FV x FN bin set, we used it to locate 103 loci into bins on the FV x FN map. These loci comprised 61 previously described SSRs, 38 new SSRs developed in this investigation from Fragaria x ananassa genomic DNA, EST and gene sequences, and 4 ripening-related genes developed for Prunus. The 103 markers were located to bins on all 7 linkage groups of the Fragaria map and a new mapping bin was identified with the novel markers, demonstrating that the map covers the majority of the diploid Fragaria genome and that the 6 bin-set seedlings selected were appropriate for bin mapping using this progeny.

The development and mapping of functional markers in Fragaria and their transferability and potential for mapping in other genera.

We have developed 46 primer pairs from exon sequences flanking polymorphic introns of 23 Fragaria gene sequences and one Malus sequence deposited in the EMBL database. Sequencing of a set of the PCR products amplified with the novel primer pairs in diploid Fragaria showed the products to be homologous to the sequences from which the primers were originally designed. By scoring the segregation of the 24 genes in two diploid Fragaria progenies FV x FN (F. vesca x F. nubicola F(2)) and 815 x 903BC (F. vesca x F. viridis BC(1)) 29 genetic loci at discrete positions on the seven linkage groups previously characterised could be mapped, bringing to 35 the total number of known function genes mapped in Fragaria. Twenty primer pairs, representing 14 genes, amplified a product of the expected size in both Malus and Prunus. To demonstrate the applicability of these gene-specific loci to comparative mapping in Rosaceae, five markers that displayed clear polymorphism between the parents of a Malus and a Prunus mapping population were selected. The markers were then scored and mapped in at least one of the two additional progenies.

An enhanced microsatellite map of diploid Fragaria.

A total of 45 microsatellites (SSRs) were developed for mapping in Fragaria. They included 31 newly isolated codominant genomic SSRs from F. nubicola and a further 14 SSRs, derived from an expressed sequence tagged library (EST-SSRs) of the cultivated strawberry, F. x ananassa. These, and an additional 64 previously characterised but unmapped SSRs and EST-SSRs, were scored in the diploid Fragaria interspecific F2 mapping population (FVxFN) derived from a cross between F. vesca 815 and F. nubicola 601. The cosegregation data of these 109 SSRs, and of 73 previously mapped molecular markers, were used to elaborate an enhanced linkage map. The map is composed of 182 molecular markers (175 microsatellites, six gene specific markers and one sequence-characterised amplified region) and spans 424 cM over seven linkage groups. The average marker spacing is 2.3 cM/marker and the map now contains just eight gaps longer than 10 cM. The transferability of the new SSR markers to the cultivated strawberry was demonstrated using eight cultivars. Because of the transferable nature of these markers, the map produced will provide a useful reference framework for the development of linkage maps of the cultivated strawberry and for the development of other key resources for Fragaria such as a physical map. In addition, the map now provides a framework upon which to place transferable markers, such as genes of known function, for comparative mapping purposes within Rosaceae.

Isolation of S-locus F-box alleles in Prunus avium and their application in a novel method to determine self-incompatibility genotype.

This study characterises a series of 12 S-locus haplotype-specific F-box protein genes (SFB) in cherry (Prunus avium) that are likely candidates for the pollen component of gametophytic self-incompatibility in this species. Primers were designed to amplify 12 SFB alleles,including the introns present in the 50 untranslated region;sequences representing the S-alleles S1, S2, S3, S4, S40, S5,S6, S7, S10, S12, S13 and S16 were cloned and characterized. [The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank database under the following accession numbers: PaSFB1(AY805048), PaSFB2 (AY805049), PaSFB3 (AY805057),PaSFB4 (AY649872), PaSFB40 (AY649873), PaSFB5(AY805050), PaSFB6 (AY805051), PaSFB7 (AY805052),PaSFB10 (AY805053), PaSFB12 (AY805054), PaSFB13(AY805055), PaSFB16 (AY805056).] Though the coding regions of six of these alleles have been reported previously,the intron sequence has previously been reported only for S6. Analysis of the introns revealed sequence and length polymorphisms. A novel, PCR-based method to genotype cultivars and wild accessions was developed which combines fluorescently labelled primers amplifying the intron of SFB with similar primers for the first intron of S-RNase alleles. Intron length polymorphisms were then ascertained using a semi-automated sequencer. The convenience and reliability of this method for the determination of the self-incompatibility (SI) genotype was demonstrated both in sweet cherry cultivars representing alleles S1 to S16 and in individuals from a wild population encompassing S-alleles S17 to S22. This method will greatly expedite SI characterisation in sweet cherry and also facilitate large-scale studies of self-incompatibility in wild cherry and other Prunus populations.

Inheritance and interactions of incompatibility alleles in the tetraploid sour cherry.

Three progenies of sour cherry (Prunus cerasus) were analysed to correlate self-(in)compatibility status with S-RNase phenotype in this allotetraploid hybrid of sweet and ground cherry. Self-(in)compatibility was assessed in the field and by monitoring pollen tube growth after selfing. The S-RNase phenotypes were determined by isoelectric focusing of stylar proteins and staining for RNase activity and, for the parents, confirmed by PCR. Seedling phenotypes were generally consistent with disomic segregation of S-RNase alleles. The genetic arrangements of the parents were deduced to be 'Köröser' (self-incompatible) S1S4.S(B) S(D), 'Schattenmorelle' (self-compatible) S6S13.S(B)S(B), and clone 43.87 (self-compatible) S4S13.S(B)S(B), where "." separates the two homologous genomes. The presence of S4 and S6 alleles at the same locus led to self-incompatibility, whereas S13 and S(B) at homologous loci led to self-compatibility. The failure of certain heteroallelic genotypes in the three crosses or in the self-incompatible seedlings indicates that S4 and S6 are dominant to S(B). However, the success of S13S(B) pollen on styles expressing corresponding S-RNases indicates competitive interaction or lack of pollen-S components. In general, the universal compatibility of S13S(B) pollen may explain the frequent occurrence of S13 and S(B) together in sour cherry cultivars. Alleles S(B) and S(D), that are presumed to derive from ground cherry, and S13, presumably from sweet cherry, were sequenced. Our findings contribute to an understanding of inheritance of self-(in)compatibility, facilitate screening of progenies for self-compatibility and provide a basis for studying molecular interactions in heteroallelic pollen.

Single-locus gametophytic incompatibility in autotetraploids.

It is known that a single-locus gametophytic self-incompatibility (GSI) system can persist with just two distinct alleles in an autotetraploid population, in contrast to diploid GSI systems, assuming "competitive interaction" in which heteroallelic pollen is universally compatible. The steady-state population structure of a GSI system in autotetraploids was investigated in an undivided population assuming "competitive interaction." A deterministic model was developed to predict the frequencies of genotypes with two, three, or four distinct S alleles, assuming no mutation or population subdivision. The model showed that unlike in diploid GSI systems, the limiting values of the frequencies of genotype classes do not minimize pollen wastage.

Determining self-incompatibility genotypes in Belgian wild cherries.

ABSTRACT The self-incompatibility (S) genotypes of a collection of 65 Belgian accessions of wild cherry, selected within two populations and planted in a seed orchard, were determined using polymerase chain reaction (PCR) methods. Initially, DNA extracts were amplified with consensus primers that amplify across the second intron of the S-ribonuclease gene which shows considerable length polymorphism. The provisional genotypes deduced were checked with the appropriate allele-specific primers for the known alleles S(1) to S(16). Putative new alleles were subjected to PCR with consensus primers amplifying across the first intron. Six new alleles, S(17) to S(22), were thus indicated on the basis of the estimated lengths of the first and second intron PCR products. Examples of these alleles were partially sequenced and were indeed mutually distinct and different from the known alleles. The incompatibility genotypes of all 65 accessions were determined and one triploid individual was found. Seventeen alleles were detected in all. Allele frequencies differed between samples and the expected total number of alleles in the underlying populations was estimated. The wild cherry populations differed significantly with respect to allelic frequencies from sweet cherry cultivars; alleles S(4) and S(5), which are moderately frequent in sweet cherry, were absent from the wild cherry accessions. The knowledge of the S genotypes will be useful for studying the gene flow within the seed orchard and these approaches should also be informative in wild populations.

A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria.

Diploid Fragaria provide a potential model for genomic studies in the Rosaceae. To develop a genetic linkage map of diploid Fragaria, we scored 78 markers (68 microsatellites, one sequence-characterised amplified region, six gene-specific markers and three morphological traits) in an interspecific F2 population of 94 plants generated from a cross of F.vesca f. semperflorens x F. nubicola. Co-segregation analysis arranged 76 markers into seven discrete linkage groups covering 448 cM, with linkage group sizes ranging from 100.3 cM to 22.9 cM. Marker coverage was generally good; however some clustering of markers was observed on six of the seven linkage groups. Segregation distortion was observed at a high proportion of loci (54%), which could reflect the interspecific nature of the progeny and, in some cases, the self-incompatibility of F. nubicola. Such distortion may also account for some of the marker clustering observed in the map. One of the morphological markers, pale-green leaf (pg) has not previously been mapped in Fragaria and was located to the mid-point of linkage group VI. The transferable nature of the markers used in this study means that the map will be ideal for use as a framework for additional marker incorporation aimed at enhancing and resolving map coverage of the diploid Fragaria genome. The map also provides a sound basis for linkage map transfer to the cultivated octoploid strawberry.

Identification of incompatibility alleles in the tetraploid species sour cherry.

The incompatibility genetics of sour cherry ( Prunus cerasus), an allotetraploid species thought to be derived from sweet cherry (diploid) and ground cherry (tetraploid), were investigated by test crossing and by analysis of stylar ribonucleases which are known to be the products of incompatibility alleles in sweet cherry. Stylar extracts of 36 accessions of sour cherry were separated electrophoretically and stained for ribonuclease activity. The zymograms of most accessions showed three bands, some two or four. Of the ten bands seen, six co-migrated with bands that in sweet cherry are attributed to the incompatibility alleles S(1), S(3), S(4), S(6, ) S(9) and S(13). 'Cacanski Rubin', 'Erdi Botermo B', 'Koros' and 'Ujfehertoi Furtos', which showed bands apparently corresponding to S(1) and S(4), were test pollinated with the sweet cherry 'Merton Late' ( S(1) S(4)). Monitoring pollen tube growth, and, in one case, fruit set, showed that these crosses were incompatible and that the four sour cherries indeed have the alleles S(1) and S(4). Likewise, test pollination of 'Marasca Piemonte', 'Marasca Savena' and 'Morello, Dutch' with 'Noble' ( S(6) S(13)) showed that these three sour cherries have the alleles S(6) and S(13). S(13) was very frequent in sour cherry cultivars, but is rare in sweet cherry cultivars, whereas with S(3) the situation is reversed. It was suggested that the other four bands are derived from ground cherry and one of these, provisionally attributed to S(B), occurred frequently in a small set of ground cherry accessions surveyed. Analysing some progenies from sour by sweet crosses by S allele-specific PCR and monitoring the success of some sweet by sour crosses were informative. They indicated mostly disomic inheritance, with sweet cherry S alleles belonging to one locus and, presumably, the ground cherry alleles to the other, and helped clarify the genomic arrangement of the alleles and the interactions in heteroallelic pollen.

Allele-specific PCR detection of sweet cherry self-incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers.

PCR-based identification of all 13 known self-incompatibility (S) alleles of sweet cherry is reported. Two pairs of consensus primers were designed from our previously published cDNA sequences of S(1) to S(6) S-RNases, the stylar components of self-incompatibility, to reveal length variation of the first and the second introns. With the exception of the first intron of S(13), these also amplified S(7) to S(14) and an allele previously referred to as S(x), which we now label S(16). The genomic PCR products were cloned and sequenced. The partial sequence of S(11) matched that of S(7) and the alleles were shown to have the same functional specificity. Allele-specific primers were designed for S(7) to S(16), so that allele-specific primers are now available for all 13 S alleles of cherry (S(8), S(11) and S(15) are duplicates). These can be used to distinguish between S alleles with introns of similar size and to confirm genotypes determined with consensus primers. The reliability of the PCR with allele-specific primers was improved by the inclusion of an internal control. The use of the consensus and allele-specific primers was demonstrated by resolving conflicting genotypes that have been published recently and by determining genotypes of 18 new cherry cultivars. Two new groups are proposed, Group XXIII (S(3) S(16)), comprising 'Rodmersham Seedling' and 'Strawberry Heart', and Group XXIV (S(6) S(12)), comprising 'Aida' and 'Flamentiner'. Four new self-compatibility genotypes, S(3) S(3)', S(4)' S(6), S(4)' S(9) and S(4)' S(13), were found. The potential use of the consensus primers to reveal incompatibility alleles in other cherry species is also demonstrated.

Polylink: to support two-point linkage analysis in autotetraploids.

SUMMARY: Polylink runs under Microsoft Windows (95 or later). It performs various calculations that are useful for investigating two-point linkage analysis for autopolyploids, based on the random chromosome pairing model. These include calculation of offspring phenotypic probabilities as functions of the recombination fraction, calculation of theoretical standard errors for the maximum likelihood estimator of and numerical computation of maximum likelihood estimates. It also includes simulation facilities. AVAILABILITY: Polylink is free and available from Xiangming Xu via email