The myrobalan (<Emphasis Type="Italic">Prunus cerasifera</Emphasis> L.): a useful diploid model for studying the molecular genetics of self-incompatibility in plums

A series of PCR methods were used to detect S-RNase alleles and SFB alleles and to determine S-genotypes in 25 accessions of myrobalan (Prunus cerasifera L.). Firstly, primers flanking the polymorphic second intron were used to identify S-RNases in agarose gels. These primers amplified one or two bands per accession in 25 accessions. Then consensus primers were designed for amplifying the polymorphic first intron, unique to Prunus S-RNases, for automated fluorescent detection. Each accession produced one or two peaks. New primers were then developed to amplify the intron in the SFB gene, for detection by fluorescence. Cross-referencing PCR bands and peaks indicated 15 S-alleles were present in the 25 accessions. Cloning, sequencing and comparison with published data indicated that the amplified products were S-RNase alleles. Sequence information was used to design primers specific for each S-RNase. Full and consistent S-genotypes were obtained by cross-comparing PCR data for 23 of the 25 accessions, and two accessions appeared to have a single allele. Pollen-tube microscopy indicated function of some but not all of the S-alleles sequenced.     

Genetic diversity evaluation of a loquat (<Emphasis Type="Italic">Eriobotrya japonica</Emphasis> (Thunb) Lindl) germplasm collection by SSRs and <Emphasis Type="Italic">S</Emphasis>-allele fragments

Loquat (Eriobotrya japonica (Thunb.) Lindl.) is a minor Rosaceae fruit of growing interest as an alternative to the main fruit crops. In this context, the selection of new cultivars to satisfy the market demand will request the suitable characterization of the available germplasm. In this work, genetic relationships among 83 loquat accessions from different countries belonging to the European loquat germplasm collection, held at the Instituto Valenciano de Investigaciones Agrarias (IVIA) in Moncada (Spain) were evaluated using microsatellites and S-allele fragments. A total of nine single sequence repeats (SSRs) from Malus and Eriobotrya genera revealed 53 informative alleles and the S-RNases consensus primers detected 11 self-incompatibility putative alleles. The combined data allow to distinguish unambiguously 80 out of the 83 accessions studied. Unweighted pair-group method (UPGMA) cluster and principal coordinates analysis (PCoA), based on Dice’s genetic distance, generally grouped genotypes according to their geographic origins and pedigrees. Discrepancies and similarities of the results obtained with other variability analysis, based on pomological traits or molecular markers, on the same loquat collection are discussed.     

Identification of self-incompatibility (S) alleles in Latvian and Swedish sweet cherry genetic resources collections by PCR based typing

The Latvian and the Swedish sweet cherry (Prunus avium L.) genetic resources collections comprise valuable material for breeding. The collections represent local Latvian and Scandinavian genetic resources: semi-wild samples, landraces, and cultivars developed in local breeding programmes, as well as diverse germplasm from the northern temperate zone. The objective of this investigation was to determine which S ₁ –S ₆ alleles are most important in the sweet cherry genetic resources collections and to compare the identified allelic and genotypic frequencies in material of different origin. Accessions in the two collections were screened for the presence of the self-incompatibility (S) S ₁ to S ₆ alleles, using PCR based typing. Significant differences (P < 0.05) between screened collections were found in frequencies of S ₄ and S ₅ alleles. Analysis of allele combinations identified the high occurrence of selections with the S-genotype S ₃ S ₆ in both collections. Compared to the S-allele frequencies published for over 250 sweet cherry cultivars from Western and Southern Europe, the Latvian and Swedish germplasm appeared to have a high frequency of the S ₆ allele in both collections, and a relatively high frequency of the S ₅ allele in Latvian germplasm. This study represents the first comprehensive S-allele screening for the sweet cherry genetic resources collections in Latvia and Sweden. Both sweet cherry collections contain high proportion of accessions adapted to north central European growing conditions, not typical for the majority of the documented sweet cherry genetic resources, which explains differences in certain S-allele occurrence.     

Primers amplifying a range of Prunus S-alleles

Although various consensus polymerase chain reaction (PCR) primers have been reported for identifying Prunus S‐alleles, they have been developed from and optimized on a limited set of alleles, which may limit their applicability to a broader allele range. To develop a primer set for use across the genus, degenerate consensus primers were designed from conserved regions of 27 S‐RNase sequences available from five Prunus species. The primers were tested in 15 previously genotyped cultivars of cherry, almond and apricot, representing alleles S₁ to S₆ in each crop and also S_c in apricot. Comparisons were made with previously published primers tested in the same 15 cultivars under reported reaction conditions. The new primers generated an amplification product for each of the 19 S‐alleles whereas those previously available amplified no more than 14. The primers will be useful for genotyping and genetic studies in cultivars and wild populations.     

Molecular markers linked to the <Emphasis Type="Italic">fin</Emphasis> gene controlling determinate growth habit in common bean

The S-genotypes of 16 apricot (Prunus armeniaca L.) cultivars native to China were determined by the S-allele PCR approach and the results were confirmed by cross-pollination tests among these cultivars. Primer combination EM-PC2consFD + EM-PC3consR, based on the conserved regions C2 and C3 of Rosaceous S-RNase genes, was the most useful primer combination for identifying Chinese apricot S-alleles. Twelve S-RNase alleles were identified using this primer combination, and they were defined as follows: S ₉ was 657 bp, S ₁₀ was 266 bp, S ₁₁ was 464 bp, S ₁₂ was 360 bp, S ₁₃ was 401 bp, S ₁₄ was 492 bp, S ₁₅ was 469 bp, S ₁₆ was 481 bp, S ₁₇ was 487 bp, S ₁₈ was 1337 bp, S ₁₉ was 546 bp and S ₂₀ was 1934 bp. S ₁₁–S ₂₀ were new S-RNase genes deposited in GenBank under accession numbers DQ868316, DQ870628-DQ870634, EF133689 and EF160078, respectively. Our findings contribute to a more efficient breeding program of Chinese apricot and further studies on the S-RNase genes.     

Identification of S-alleles in almond using multiplex PCR

The S-genotypes of eight almond (Prunus dulcis Miller (D.A. Webb)) cultivars from different geographical origins and of nine new selections from the CEBAS-CSIC (Murcia, Spain) breeding program were determined using single and multiplex PCR with different sets of specific oligonucleotide primers. The results of PCR using the AS1II- and AmyC5R-specific primers showed amplification in a single reaction of 10 different self-incompatibility alleles and of the self-compatibility allele S _f. However, the amplified fragments of the S _f allele were of similar sizes to those amplified from the S ₃ self-incompatibility allele. For this reason, a specific PCR primer CEBASf was designed from the intron sequence of S _f. A multiplex-PCR reaction using the AS1II, CEBASf and AmyC5R primers permitted unequivocal identification of the 10 self-incompatibility alleles and of the self-compatibility allele. Multiplex PCR opens the possibility to identify new S-alleles using different sets of primers. The applications of these PCR markers in the almond-breeding programs are discussed.     

Development of PCR-based markers for the identification of the <Emphasis Type="Italic">S</Emphasis>-<Emphasis Type="Italic">RNase</Emphasis> alleles in wild potato species

Sexual self-incompatibility in wild diploid potato species is controlled by a single multiallelic S-locus encoding a polymorphic stylar ribonuclease (S-RNase) that is responsible for the female function in pollen–pistil recognition. In this study, an approach using PCR-based markers were originally developed to amplify the S-RNase alleles in Solanum chacoense. Subsequently, to investigate their general applicability in Solanum, this molecular approach was successfully tested on S. spegazzinii and S. kurtzianum. Application of PCR-SSCP approach revealed potentially new S-RNase alleles in the three species, demonstrating for the first time the existence of S-RNase genetic variability within and between populations of wild diploid potato species. Species-specific SSCP markers that may be successfully used in gene flow studies was also detected in this investigation.     

Identification of self-incompatibility alleles and pollen incompatibility groups in sweet cherry by PCR based s-allele typing and controlled pollination

A total of 17 pollen incompatibility groups in sweet cherry (Prunusavium L.) were identified among 46 accessions by PCR based S-alleletyping analysis and by controlled test pollinations. Two putativeS-alleles different from S ₁ to S ₆,S _z and S _y were identified. Five S-genotypes, S ₁ S ₅, S ₁ S ₆,S ₂ S ₆, S ₄ S ₆, andS ₅ S ₆, combinations of S ₁ toS ₆ alleles that had not previously been identified from cultivars in NYSAES, were positively confirmed by PCR based S-genotyping analysis. Also, the S-genotypes of cultivars in some pollen incompatibility groups that had previously been incorrectly reported have been clarified. Several popular cultivars, which were previously used as testers for S-allele typing analysis, were found to have been inaccurately genotyped. In addition, the S-genotypes and self-incompatibility groups of some relatively recentlyintroduced cultivars were identified. The molecular typing system ofS-genotypes based on PCR is a useful and rapid method for identifying newS-alleles and incompatibility groups in sweet cherry.     

Diallel analyses reveal the genetic control of resistance to ascochyta blight in diverse chickpea and wild Cicer species

Ascochyta blight is a major fungal disease affecting chickpea production worldwide. The genetics of ascochyta blight resistance was studied in five 5 × 5 half-diallel cross sets involving seven genotypes of chickpea (ICC 3996, Almaz, Lasseter, Kaniva, 24B-Isoline, IG 9337 and Kimberley Large), three accessions of Cicer reticulatum (ILWC 118, ILWC 139 and ILWC 184) and one accession of C. echinospermum (ILWC 181) under field conditions. Both F₁ and F₂ generations were used in the diallel analysis. The disease was rated in the field using a 1–9 scale. Almaz, ICC 3996 and ILWC 118 were the most resistant (rated 3–4) and all other genotypes were susceptible (rated 6–9) to ascochyta blight. Estimates of genetic parameters, following Hayman’s method, showed significant additive and dominant gene actions. The analysis also revealed the involvement of both major and minor genes. Susceptibility was dominant over resistance to ascochyta blight. The recessive alleles were concentrated in the two resistant chickpea parents ICC 3996 and Almaz, and one C. reticulatum genotype ILWC 118. The wild Cicer accessions may have different major or minor resistant genes compared to the cultivated chickpea. High narrow-sense heritability (ranging from 82% to 86% for F₁ generations, and 43% to 63% for F₂ generations) indicates that additive gene effects were more important than non-additive gene effects in the inheritance of the trait and greater genetic gain can be achieved in the breeding of resistant chickpea cultivars by using carefully selected parental genotypes.     

Genetics of self-compatibility in dihaploids of Solanum tuberosum L. 5. The S-genotype of tetraploid potato cultivar gineke

Summary Investigations of the genetics of self-compatibility and self-incompatibility in dihaploids and diploid derivatives from cv. Gineke revealed the presence of S ₁, S₂ and S ₃ at the S-locus of Gineke and in addition an S ₁-allele on a translocation. By means of a complete tester set involving the S-alleles S ₁, S₂ and S ₃ (all from Gineke) and S ₄ (from Black 4495) it was demonstrated that some Gineke dihaploids were compatible with all six testers. This indicated a fourth S-allele in Gineke, which differs from those in the tester series and was therefore assigned S ₅. Additional evidence was obtained from an analysis of F₁'s from crosses of two S ₅-bearing dihaploids and one of the testers. So the S-genotype of cv. Gineke was identified as S ₁S₂S₃S₅/S₁, the second S ₁ being the S-allele on a translocated fragment.     

S-genotype identification based on allele-specific PCR in Japanese pear

Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear. We designed S allele-specific PCR primer pairs for ten previously reported S-RNase alleles (S¹–S⁹ and S^k) as simple and reliable method. Specific nucleotide sequences were chosen to design the primers to amplify fragments of only the corresponding S alleles. The developed primer pairs were evaluated by using homozygous S-genotypes (S¹/S¹–S⁹/S⁹ and S^4sm/S^4sm) and 14 major Japanese pear cultivars, and found that S allele-specific primer pairs can identify S-genotypes effectively. The S allele-specific primer pairs developed in this study will be useful for efficient S-genotyping and for marker-assisted selection in Japanese pear breeding programs.     

Inheritance of fruit colour in a wild Russian red raspberry seedling

Summary A new gene, i, from a self-incompatible wild Russian Rubus idaeus seedling is described. The gene i interacts with the fruit colour gene T, Tii plants having apricot instead of red fruits, and spines, leaves and stems pigmented to varying degrees. A deficit of apricot-fruited seedlings in progenies segregating for i could be explained by linkage between i and the self-incompatibility S locus with a crossover value of approximately 26.7%.Segregations for spine colour in crosses and backcrosses of the Russian seedling with two green-spined (ttII) raspberries selected for their yellow fruit colour, showed that both the latter carried the spine colour intensifier gene P. This suggests that earlier authors were incorrect in attributing the apricot fruit colour developed by some tt plants to a pleiotropic effect of P.     

Description of a new trans-generic <Emphasis Type="Italic">S</Emphasis>kb-RNase allele in apple

Polish apple cvs: ‘Ligol’, ‘Odra’ and ‘Primula’ served for studies of self-incompatibility. Basing on available sequence data, a new set of primers upstream and downstream of the hypervariable (HV) region of apple S-RNases were designed. Using the RT-PCR method, cDNA was amplified on RNA isolated from styles. PCR products were cloned and sequenced. A new trans-generic S-RNase allele, designated as Skb (GenBank accession no. EU443101), was discovered in cvs ‘Odra’ and ‘Primula’. Nucleotide sequence alignment revealed that Skb-RNase shows 98% identity to SaucS19-RNase from Sorbus aucuparia and 97% identity to CmonS17-RNase from Crataegus monogyna. The occurrence of extensive intergeneric hybridization among extant Pyrinae is considered since the deduced amino acid sequence of Skb-RNase from M. × domestica showed higher similarity to CmonS17 from C. monogyna, SaucS19-RNase from S. aucuparia, St from Malus transitoria, S5-RNase and S3-RNase from Pyrus pyrifolia, and S40-RNase from P. ussuriensis than to S-alleles from Malus × domestica and all of them are grouped in the same cluster of phylogenetic tree. In respect to extremely high similarities between aforementioned S-RNases it could be possible that these alleles existed before the separation of Malus, Pyrus, Sorbus and Crataegus genera. Within Malus, the Skb-RNase from M. × domestica and St-RNase from M. transitoria show 100% identity of the HV region at the deduced amino acid level, suggesting that these S-RNases diverged more recently than the other Malus S-RNases. In ‘Ligol’, the agronomically most important cultivar in Poland, the S2 and S9 were identified.     

Genetics of self-compatibility in dihaploids of Solanum tuberosum L. 2. Detection and identification of all possible incompatibility and compatibility genotypes in six F1'S from interdihaploid crosses

Summary Two self-compatible (sc) dihaploids, G254 and B16, and one self-incompatible (si) dihaploid, G609, from Solanum tuberosum L. were intercrossed reciprocally. Segregation ratios sc : si : pc (pseudo-compatible) were determined in all 6 F₁'s in three successive years and critically tested and discussed. Genotypes at the S-locus could be assigned to the dihaploid parents and the S-allele on the translocation in sc G254 identified as S ₁. Using these genotypes all sc and si genotypes were derived which could be expected in the F₁'s.Incompatibility groups were detected in each F₁ from the results of complete diallels involving si plants. The genotype of each group was identified by test crosses. Compatibility groups could be both detected and identified by crossing in each F₁ the sc plants as females with the already identified si sibs. In this way a complete series of 6 si testers and corresponding sc genotypes was obtained involving four alleles at the S-locus and S ₁ and S _x on the translocation.Certative disadvantage of pollen carrying the translocation could be ruled out as a possible cause of unexpected ratios. The hypothesis of an S-bearing translocation as the cause of self-compatibility could account for all results on the assumption that translocation homozygotes are lethal and the S-allele on the translocation is active in the pollen only.The following bachelor students have contributed to the experimental data used in this article: Janny Olsder, J. Marelis, H. v.d. Brink, J. Sonneveld, D. Vreugdenhil, Digna van Ballegooijen and Els Staas-Ebregt.     

Determination of incompatibility genotypes in almond using first and second intron consensus primers: detection of new S alleles and correction of reported S genotypes

The work aimed to develop a reliable and convenient PCR approach for determining incompatibility S genotypes in almond. Initially, genomic DNAs of 24 accessions of known S genotype were amplified with novel consensus primers flanking the first and second introns of the S‐RNase gene. The PCR products separated on agarose showed length polymorphisms and correlated well with the reference alleles S1‐S₂3 and Sf. In addition, to improve discrimination between alleles of similar sizes, the same sets of primers but fluorescently labelled were used, and the products sized on an automated sequencer. These fluorescent primers were particularly informative in the case of the first intron, variation in the length of which has not been used previously for S genotyping in almond. Some reference alleles showed the same patterns with first and second intron primers, and others showed a microsatellite‐like trace. Subsequently, the S genotypes of 26 cultivars not genotyped previously and of four of uncertain genotype were determined. An allele described in Australian work as putative S₁₀ was shown to be a ‘new’ allele and ascribed to S₂₄ and evidence of five more ‘new’S alleles was found, for which the labels S₂₅‐S₂₉ are proposed. This PCR approach should be useful for genotyping in other Prunus crops.     

Isolation of a new repetitive DNA sequence from Secale africanum enables targeting of Secale chromatin in wheat background

A genome specific DNA sequence that detects Secale africanum chromatin incorporated into wheat was developed in this study. Random amplified polymorphic DNA (RAPD) analysis was used to search for genome specific DNA sequences of S. africanum in lines, R111, “mianyang11” (MY11) and wheat-rye 1RS/1BL translocations R25 and R57. A high copy rye-specific DNA segment pSaD15₉₄₀ of the S. africanum genome was obtained. The sequence of pSaD15 did not show any significant homology to other reported sequences in databases and it is therefore a new repetitive sequence of Secale. PCR primers were designed for pSaD15₉₄₀, which amplify a clear 887 bp fragment in S. africanum but not in any wheat. The primers also amplified an 887 bp fragment in other accessions of rye, Chinese Spring-Imperial rye chromosome additions and a diverse range of material carrying different rye chromosomes or chromosomal segments. In situ hybridization showed that probe pSaD15₉₄₀ was specifically hybridized throughout all rye chromosomes arms except for the terminal regions. The advantage of the rye-specific probe developed herein compared to those of previous reports is that it has been shown to be widely applicable to other Secale species. The probe will be useful as a molecular marker for the introgression of S. africanum and other rye chromosome segments into the wheat genome.     

Diversity analysis and genotyping in Pisum with sequence tagged microsatellite site (STMS) primers

Pisum sativum specific sequence tagged microsatellite site primers were used to amplify genomic profiles from 15accessions of P. sativum L. that represented the genetic base of the Australian field pea-breeding program and five accessions of the wild related species P. fulvum. The STMS primers were used to assess genetic relationships among the Pisum accessions in two ways. Firstly, to produce RAPD-like multiple banding marker profiles using an adapted RAMS method, for intra- and interspecific diversity analysis. From the 14 flanking primer pairs assessed, 133 markers were obtained. Conservation and reproducibility of markers among individuals within accessions was demonstrated. The largest distance observed among P. sativumaccessions was 22% and among P.fulvum accessions was 40%, similar to that revealed with other PCR-based methods. The maximum distance between P.sativum and P. fulvum accessions was 46%. Phylogenetic clustering of P. sativum accessions, using the neighbour joining method and based on simple matching distances, was distinct and distant to P. fulvum. Secondly, PCR with a higher annealing temperature and fluorescent labeling identified simple and allelic loci markers useful for creating agenotype/fingerprint database for P. sativum cultivars. This is the first report to demonstrate the use of Pisum specific STMS sequences for both diversity analysis and genotype identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sequence of the S 9-RNase cDNA and PCR-RFLP system for discriminating S 1- to S 9-allele in Japanese pear

A new S ₉-allele was discovered in 6 Japanese pear cultivars, ‘Shinkou’, ‘Shinsei’, ‘Niitaka’, ‘Amanogawa’, ‘Nangetsu’ and ‘Nansui’. cDNA encoding S ₉-RNase, a stylar product of S ₉-allele, was cloned from pistils of ‘Shinkou’ and ‘Shinsei’ by 3' and 5' RACE. The S ₉-RNase gene had an open reading frame of 684 nucleotides encoding 228 amino acid residues. S ₉-RNase had a hypervariable (HV) region different from S ₁- to S ₈-RNase and shared higher similarity (95.2%) with apple S ₃-RNase than with 8 Japanese pear S-RNases (from 61.0% to 70.7%). Genomic PCR with primers ‘FTQQYQ’ and ‘anti-(I/T) IWPNV’ provided S ₁- to S ₉-amplicon (product), but could not discriminate the S ₂ from the S ₉ of ca. 1.3 kb. The S ₂ and S ₉ were distinguished by digestion with AflII and BstBI, respectively. The digestion with nine S-allele-specific restriction endonucleases, SfcI, AflII, PpuMI, NdeI,AlwNI, HincII, AccII, NruI and BstBI, distinguished S ₁ to S ₉, establishing that this PCR-RFLP system is useful for S-genotype assignments in Japanese pear harboring S ₁- to S ₉-allele. ‘Shinkou’, ‘Shinsei’, ‘Nangetsu’ and ‘Nansui’ assigned as S ₄ S ₉ were determined to be cross incompatible. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of S‐allele genetic diversity in Sicilian almond germplasm comparing different molecular methods

Italian almond germplasm is characterized by a wide diversity in several growing areas among which Sicily is one of the most important. Analysis with consensus and specific primers and DNA sequencing was performed to investigate S‐RNase genetic diversity and to elucidate the homology rate within a genetic pool of 27 Italian accessions. Interestingly, some of the self‐compatible cultivars did not show the presence of S_f allele. Amplicons from consensus and allele‐specific PCR primers revealed a high level of variability. Sequencing of all the S‐RNase amplicons derived from consensus primers allowed the identification of two new S‐RNase alleles (S₅₁ and S₅₂). Surprisingly, despite the AA replacement mutation, S₅₁ did not exhibit any change of its S‐RNase function. Additionally, several mutations, with no effect on amino acid composition, were detected in the intron and/or in the ORF of four known alleles (S_g, S₁₀, S₃₁ and S₃₅). Genetic variation, regarding point mutations and only detected by sequencing, was revealed among 11 of 27 tested cultivars. The new sources of variability might have an interest for product traceability.     

Development of molecular markers linked to the <Emphasis Type="Italic">Fom-1</Emphasis> locus for resistance to Fusarium race 2 in melon

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (F.o.m), is a worldwide soil-borne disease of melon (Cucumis melo L.). The most effective control measure available is the use of resistant varieties. Resistance to races 0 and 2 of this fungal pathogen is conditioned by the dominant gene Fom-1. An F₂ population derived from the ‘Charentais-Fom1’ × ‘TRG-1551’ cross was used in combination with bulked segregant analysis utilizing the random amplified polymorphic DNA (RAPD) markers, in order to develop molecular markers linked to the locus Fom-1. Four hundred decamer primers were screened to identify three RAPD markers (B17₆₄₉, V01₅₇₈, and V06₁₀₉₂) linked to Fom-1 locus. Fragments amplified by primers B17₆₄₉ and V01₅₇₈ were linked in coupling phase to Fom1, at 3.5 and 4 cM respectively, whereas V06₁₀₉₂ marker was linked in repulsion to the same dominant resistant allele at 15.1 cM from the Fom-1 locus. These RAPDs were cloned and sequenced in order to design primers that would amplify only the target fragment. The derived sequence characterized amplified region (SCAR) markers SB17₆₄₅ and SV01₅₇₄ (645 and 574 bp, respectively) were present only in the resistant parent. The SV06₁₀₉₂ marker amplified a band of 1092 bp only in the susceptible parent. These markers are more universal than the CAPS markers developed by Brotman et al. (Theor Appl Genet 10:337–345, 2005). The analysis of 24 melon accessions, representing several melon types, with these markers revealed that different melon types behaved differently with the developed markers supporting the theory of multiple, independent origins of resistance to races 0 and 2 of F.o.m.