Determination of self-incompatible genotypes in 21 cultivated sweet cherry cultivars in Greece and implications for orchard cultivation

Summary

Sweet cherry is self-incompatible due to having a gametophytic self-incompatibility system. S alleles in the style and pollen determine possible crossing relationships. Knowledge of the S allele constitution of cultivars is important for sweet cherry growers and breeders. Recently, molecular methods have been developed to distinguish between S alleles in sweet cherry.The S allele genotypes of 21 sweet cherry cultivars widely grown in Greece, including 19 not previously genotyped, were determined based on their S-RNase gene sequences using PCR analysis. Eight different S alleles in ten combinations were distinguished and two new S-genotypes (S₁S₁₃ and S₄S₃₀) were documented. Four alleles, S₁, S₃, S₄, and S₉ were widespread and together were responsible for 85% of the S-haplotypes. Therefore many of the cultivar combinations were semi-compatible. In Greece, semi-compatibility was shown to correlate with low yields. However, the cultivar ‘Hybrid Tragana Edessis x Unknown’ (S₃S₁₃) and the cultivar ‘Kapsiotika’ (S₂S₅) carry rare S-haplotypes and are therefore fully cross-compatible with most of the cultivars analysed.     

Determination and confirmation of S-RNase genotypes of apple pollinators and cultivars

Summary

We analysed the S-RNase genotypes of 23 crab apple (Malus spp.) pollinators and 102 cultivars of domestic apple (Malus pumila Mill.) by PCR amplification and digestion. Within the 23 pollinators, four pollinators, ‘Hopa’, ‘Jack’, ‘Pink Perfection’ and ‘Profusion B’, each had two unidentified S-RNase alleles. These cultivars should be useful pollinators for all domestic cultivars. Twenty-one of the domestic cultivars exhibited S-genotypes contrary to those expected from their supposed parentage, suggesting that one or both reported parents were wrong. We confirmed many of the S-RNase genotypes by pollination tests.     

Identification of S-haplotypes of European cultivars of sour cherry

The sour cherry is known to exhibit the phenomenon of gametophytic self-incompatibility which prevents self-fertilization. In sour cherry, besides self-incompatible cultivars, there often occur self-compatible cultivars. This is due to the occurrence of natural mutations of the S-RNase or SFB genes and, consequently, loss of functionality of S-alleles. Here we present the results of the identification of S-haplotypes of 21 cultivars of sour cherry from various regions of Europe. The analyses were performed using methods based on the amplification of intron I and intron II of the S-RNase gene and fragments specific to the individual alleles of the S-RNase or SFB genes. The tested cultivars were found to contain 15 S-haplotypes: S₁, S_1?, S₄, S₆, S_6m, S_6m2, S₉, S₁₂, S₁₃, S_13?, S₂₆, S₃₅, S_36a, S_36b, and S_36b2. The most frequently occurring S-haplotypes were S_13? (61.9%), S_36a (57.1%), and S₂₆ (47.6%). On the basis of the results, 17 of the 21 cultivars were deduced to be self-compatible. The results will be of use in the production of sour cherry fruit by facilitating the selection of suitable pollinating cultivars. The results are also expected to be useful in the breeding of new cultivars of sour cherry when selecting genotypes for crosses.     

Identification of S-genotypes and novel S-RNase alleles in Japanese apricot cultivars native to China

Information on S-genotypes is essential for designing orchards of Japanese apricot (Prunus mume Sieb. et Zucc.) and for hybridization. However, this information is lacking for most cultivars grown in China. Thus, in this work, the S-genotypes of 24 Japanese apricot cultivars native to China were identified by sequencing the PCR products obtained from allele-specific polymerase chain reaction (AS-PCR). Seventeen S-RNase alleles were amplified, ten of them for the first time. The new S-RNase alleles were submitted to GenBank and denoted them as S¹⁷, S¹⁸, S¹⁹, S²⁰, S²¹, S²², S²³, S²⁴, S²⁵ and S²⁶. Furthermore, the S-genotypes of four Japanese apricot cultivars were confirmed by field-testing cross-pollination.     

Cross-compatibility of apple cultivars possessing S-RNase alleles of similar sequence

Summary

We investigated the functional differences in three pairs of S-RNase alleles having similar sequences. It was confirmed using pollination tests that S₁₉ and S₂₈ behaved as different alleles, while S₁₇ and S₁₉ appeared to be the same allele. Our previous new allele designation of S_6b in place of S₁₇ and S₁₉, and not S₁₉ and S₂₈, from S-RNase sequence analyses was therefore confirmed. The deduced amino acid sequences of S₃ and S₁₀, although showing high similarity (94%), were functionally distinct.     

S-RNase genotypes of apple (Malus domestica Borkh.) including new cultivars,lineages, and triploid progenies

Summary

We have determined the S-RNase genotypes of 33 new apple cultivars and lineages produced in Japan, 44 unknown cultivars and two lineages, and 22 triploid progenies. We have speculated on the putative parentage of new cultivars and lineages based on their S-RNase genotypes and also identified mistaken parents. In the case of the triploid progenies, the breeding of new cultivars using a triploid paternal parent may pose problems due to its low pollen viability. Nevertheless, diploid and triploid progenies were obtained using a triploid maternal parent. We have compiled a database of 516 apple S-RNase genotypes, including those previously investigated, which included a survey system for cultivar combinations, showing those that were fully-incompatible, semi-compatible, or fully-compatible, together with information on the PCR-RFLP method used for the identification of S-RNase genotypes and S-RNase allele designation (available at http://www.agr.nagoya-u.ac.jp/~0hort/apple/).     

S-genotyping of 25 sweet cherry (Prunus avium L.) cultivars from the Czech Republic

Sweet cherry (Prunus avium L.) is a self-incompatible species. Determination of the S-genotypes of cherry cultivars is crucial for breeding and to select appropriate cultivars for cross-fertilisation and fruit set. In this study, we characterised the S-genotypes of 25 sweet cherry cultivars, some of which had being bred at the Research and Breeding Institute of Pomology (RBIP), Holovousy, Czech Republic, and others were European cultivars in the RBIP collection. S-genotyping was carried out by polymerase chain reaction using consensus primers for the S-RNase and SFB genes, and capillary electrophoresis. Nine different known S-haplotypes (S₁, S₂, S₃, S₄, S₅, S₆, S₉, S₁₃, and S₁₆) were identified and the cultivars were assigned to 12 incompatibility groups. One local cultivar, ‘Pta?ka z Plzně’, originated from a wild forest seedling and used as a pollinator, was assigned to Group 0 of universal donors. The pedigree of some cultivars was confirmed by their S-genotype. This study represents the ?rst comprehensive S-genotype screening of sweet cherry genetic resources in the Czech Republic and will be useful for the design of crossing programmes and orchard management of these sweet cherry cultivars.     

S_allele identification and genetic diversity analysis of apricot cultivars

In this study, the sexual incompatibility and S-allele diversity of 24 Turkish apricot cultivars, Paviot and Sak?t-1 as parents and 127 F₁ progenies were identified by polymerase chain reaction (PCR) and sequencing techniques. Additionally, genetic diversity and relatedness among the 24 cultivars were determined using 18 simple sequence repeat (SSR) markers from the genus Prunus. PCR for S_alleles identified nine different S-RNase alleles in the 24 apricot cultivars, namely S_c, S₁, S₂, S₈, S₉, S₂₀, S₂₄, S₅₂, and S₅₃. All primers amplified only one S_allele in the cultivars Adilcevaz-1, Adilcevaz-3, Ethembey, Pasamismisi, Canakkale, and Soganci. Most of the Turkish cultivars were self-incompatible. The S_c allele was present in only three cultivars (Canakkale, Ethembey, Imrahor) that are, therefore, self-compatible. The S_alleles of cultivars Paviot and Sak?t-1 displayed homology with the S_c, S₂ and S₂₀, and S₅₂ alleles. In the 127 F₁ genotypes, the two S_alleles of Paviot were inherited by roughly half of the offspring, while about 76% of the offspring inherited the S₅₂ allele from Sak?t-1, and less than 24% inherited S₂₀. The amplification using all SSR 18 primers was successful and produced 128 polymorphic alleles with an average of 7.11 alleles per locus. Among the apricot cultivars studied, expected heterozygosity (He) ranged from 0.33 to 0.72, observed heterozygosity (Ho) ranged from 0.42 to 1.00, PIC values were between 0.28 and 0.89, and similarity rates were between 0.30 and 0.68. The cultivars Levent and Ozal were genetically closest (0.68) while cultivars Sak?t-3 and Soganc? were the most distinct (0.30).     

Development of SCAR markers to differentiate between mume (Prunus mume Sieb. et Zucc.) and apricot (P. armeniaca L.)

Summary

Mume (Prunus mume Sieb. et Zucc.) and apricot (P. armeniaca L.) are similar in fruit and tree morphology, and exhibit high cross- and graft-compatibility with each other. It is therefore difficult to differentiate mume and apricot cultivars on the basis of morphological and phenotypical characteristics. Molecular markers were developed to differentiate nine mume from ten apricot cultivars. Four dominant, random amplified polymorphic DNA (RAPD) markers that can discriminate between mume and apricot cultivars (designated OPA15₆₂₈, OPO10₅₅₀, OPO20₂₅₉, and OPU03₄₁₅) were identified from 21 decamer primers. Two RAPD markers (OPO10₅₅₀ and OPU03₄₁₅) were developed into dominant sequence-characterised amplified region (SCAR) markers (SCO10 and SCU03). These SCAR markers could differentiate between all mume and apricot cultivars.     

The Use of Dinitrocresol-Mineral Oil Sprays for the Control of Prolonged Rest in Apple Orchards

Summary

Pluots are putative hybrids between plums (Prunus salicina Lindl.) and apricots (P. armeniaca L.). The capability to distinguish among plum and pluot cultivars is important in breeding and cultivation. We investigated the genetic diversity among 14 plums, 6 pluots and one plumcot representing commercial cultivars in California, with 28 microsatellite markers. We also tested seven apricot cultivars as a reference to ®nd evidence of apricot in the ancestry of pluots and plumcot. The parental material used in the original cross that produced the pluot and plumcot was not available. Of the 28 SSR markers, 25 were from sweet cherry (Prunus avium L.) and three from peach (Prunus persica L.). Approximately 80% of the cherry primers generated ampli®cation products in plum and pluots, showing transportability between these Prunus species. One to eight putative alleles per locus were displayed by the tested SSRs in plums and pluots. In plum and pluot samples a total of 100 alleles were identi®ed with an average of 4.3 alleles per primer combination. The SSR markers were successfully used for the discrimination of all tested cultivars. In pluots, 76 alleles were found in which 63 (83%) were speci®cally coming from plum, 9 (12%) were common in plum, pluots and apricot while no allele in the pluots was observed that was contributed from apricot. In plumcot, 49 alleles were observed in which 25 (51%) were from plum, 18 (36%) were speci®cally from apricot and 6 (12%) were common in plum, plumcot and apricot. Relationships among the 28 plum, pluot and apricot cultivars were represented by a dendrogram, constructed on the basis of 168 SSR markers. The dendrogram showed the plums and pluots form a cluster distinct from the apricots, with pluot cultivars interspersed among plum cultivars and more closely related to plum than to apricot. Plumcot made a separate branch and was placed between the plum and apricot cluster. These results suggest that the SSR markers are valuable tools for identi®cation of cultivars and diversity analyses in plum.     

Diversity of S-RNase genes and S-haplotypes in Japanese plum (Prunus salicina Lindl.)

Summary

This report demonstrates the diversity of S-haplotypes in Japanese plum by molecular cloning of genomic DNAs and cDNAs that encode S-RNases. Nine different DNA fragments, designated as S^a–Sⁱ, were obtained from 17 Japanese plum cultivars by PCR with an S-RNase gene-specific primer set, Pru-C2 and PCE-R. Eleven different S-haplotypes were found in these cultivars. The banding patterns obtained with another S-RNase gene-specific primer set, Pru-T2 and PCE-R, corresponded to the S-haplotypes predicted from the Pru-C2 and PCE-R primer set. Several cultivars had the same S-haplotypes. Partial genomic DNAs for eight S-RNase genes and cDNAs for two S-RNases were cloned and sequenced. Deduced amino acid sequences contained conserved regions among the rosaceous S-RNases. Comparisons of the sequences from cDNAs and genomic DNAs revealed the presence of two introns in the S-RNase genes of Japanese plum as in other Prunus S-RNase genes. Pollination incompatibility groups and self-compatibility in Japanese plum were discussed with reference to the S-haplotypes.     

Simultaneous down-regulation of DORMANCY-ASSOCIATED MADS-box6 and SOC1 during dormancy release in Japanese apricot (Prunus mume) flower buds

In temperate deciduous fruit crops such as Prunus spp., bud endodormancy is an important physiological phase affecting the timing of blooming and subsequent fruit development. Japanese apricot (Prunus mume) bears unmixed flower buds, separate from vegetative buds, that bloom slightly more than a month before vegetative bud burst. Seasonal expression of Prunus mume DORMANCY ASSOCIATED MADS-box genes (PmDAMs) has previously been analyzed only in vegetative buds, with an association between these genes and flower bud endodormancy release not yet confirmed. In this study, we performed a seasonal expression analysis of PmDAM1–6 genes in flower buds of two Japanese apricot genotypes – namely, high-chill and low-chill cultivars. The analysis revealed that PmDAM3, PmDAM5, and PmDAM6 expressions are closely associated with dormancy release in both flower and vegetative buds. In addition, a yeast two-hybrid screening demonstrated that PmDAM6 can interact in yeast with the homolog of Arabidopsis SOC1 (PmSOC1). Synchronized expression patterns were detected in PmDAM6 and PmSOC1 during dormancy release in flower buds of the two genotypes. Taken together, these results suggest that the dimer of PmDAM6 and PmSOC1 may play a role in the regulation of dormancy transition and blooming time in Japanese apricot flower buds.     

新疆野生樱桃李S-RNase基因分离与鉴定的初步研究

 从4对已报道的李属树种S-RNase基因引物组合中筛选出扩增效果较好的PruC2和PruC4R组合,首次对新疆野生樱桃李（Prunus cerasifera Ehrh.）的45个株系的基因组DNA进行S-RNase基因特异性PCR扩增,并对其PCR产物进行克隆测序。这些核酸序列及其相应的氨基酸序列在GenBank中进行比对, 皆与李属的S-RNase基因有最大同源性,为新疆野生樱桃李的4种S-RNase基因,分别命名为S₁ （511 bp）,S₂ （787 bp）,S₃ （1859 bp）和S₄ （464 bp）,在GenBank的登录号分别为EF638726、EF641276、EF661873和EF661874。45个株系中,43个株系的S基因型分别为S₁S₂、S₁S₃、S₂S₃、S₂S₄和S₃S₄,而10号和15号株系分别只鉴定了一种S-RNase基因,其S基因型组成有待于进一步研究。     

Self- and cross-(in)compatibility between important apricot cultivars in northwest Iran

Summary

Knowledge of the self-(in)compatibility trait in commercial apricot cultivars is of great importance for breeders and growers. Five commercial apricot cultivars, widely grown in Iran, were self- and cross-pollinated to determine their pollen and stylar compatibility. Fruit-set in the orchard and pollen tube growth in pistils, from flowers pollinated in the laboratory, were evaluated. In addition, specific primers previously designed to amplify fragments of the S alleles responsible for the incompatibility trait, were used to amplify DNA extracted from the five cultivars.All results agreed and confirmed that three out of the five cultivars studied were self-incompatible, two of which were cross-incompatible and therefore had the same genotype. The cultivars, ‘Ghorban-e-Marageh’ and ‘Ghermez-e-Shahroodi’ were self-compatible and, interestingly, shared a PCR band with all Spanish self-compatible apricot cultivars examined to date.     

Physiological Breakdown in Stored Monarch Plums

Summary

Several pollination studies carried out on different, self-incompatible almond cultivars and seedlings have shown the presence of variable levels of fruit set following self-pollination that could be attributed to partial self-incompatibility (PSI). PSI is an intermediate reproductive behaviour, described in some angiosperm species, which indicates that self-incompatibility is a quantitative and plastic trait. The present study was performed to substantiate the occurrence of PSI in almond by studying ten cultivars that are traditionally considered to be self-incompatible (four of which had previously shown fruit set after self-pollination). These cultivars were analysed by microscopic observations of the progression of pollen tubes through the pistil following controlled self-pollination, determinations of fruit set after bagging flower buds, and molecular identification of the parentage of the fruits obtained using consensus PCR-primers for Prunus S-RNase alleles. The results showed that, in nearly all cases, the pollen tubes did not enter the ovary and, from a total of 5,349 bagged flower buds, only 17 fruits were obtained. In all cases, PCR analysis of the plants obtained after germination of these seeds showed a band corresponding to an S-RNase allele not present in the maternal progenitor, clearly indicating that they could only have arisen from cross-pollination.Therefore, the low fruit set values observed in some of the cultivars studied were not due to a breakdown in the self-incompatibility response that confers PSI, but to very low rates of contamination with foreign pollen. These results corroborate the self-incompatibility phenotype of the cultivars studied here, and highlight the importance of ascertaining the identity of the parentage in any progeny obtained after bagging.     

Application of simple sequence repeat (SSR) markers in apricot breeding: molecular characterization,protection, and genetic relationships

Seventeen peach simple sequence repeat (SSR) markers have been used in the molecular characterization of 8 apricot (Prunus armeniaca L.) cultivars from Spain, North America, France, and Greece; a new breeding line from the apricot breeding program of INRA (Avignon, France); and 13 breeding lines and 3 new releases from the apricot breeding program of CEBAS-CSIC (Murcia, Spain). DNA fingerprints have been developed establishing the genetic relatedness among cultivars, new releases, and breeding lines. Amplification of SSR loci was obtained for all 17 primer pairs and 14 of them produced polymorphic amplification. The number of presumed alleles revealed by the SSR analysis ranged from one to nine, although most primers revealed three alleles or more. The mean number of alleles per locus was 4.1. Results allowed the molecular identification of all the apricot genotypes assayed. Apricot genotypes clustered into seven principal groups in accordance with their origin and pedigree. The implications of these results for apricot breeding programs in terms of protection of new release and design of new crosses are also discussed.     

S-RNase based S-genotyping of Japanese plum (Prunus salicina Lindl.) and its implication on the assortment of cultivar-couples in the orchard

Determination of the genetic compatibility between self-incompatible cultivars is crucial in agriculture. The Rosaceae family carries the S-RNase-mediated gametophytic self-incompatibility (GSI) system. Each haplotype is conferred by an S-locus. The S-locus contains two highly polymorphic genes, S-RNase and SFB, which are characteristic of each haplotype and therefore these genes are ideal markers for molecular S-genotyping. In this study 43 Japanese plum cultivars grown in Israel were S-genotyped based on their S-RNase gene sequences. Four alleles, S^b, S^c, S^e and S^h are widespread and together are responsible for 87% of the S-haplotypes therefore many of the cultivar combinations are semi-compatible. In Israel semi-compatibility was shown to correlate with low yield. However, two cultivars, ‘Wickson’ S^fS^k and ‘Shiro’ S^fS^g carry rare S-haplotypes and, therefore, are fully compatible with most of the analyzed cultivars.     

Genetic diversity of apricot revealed by a set of SSR markers from linkage group G1

Eight polymorphic simple sequence repeat (SSR) markers located in the G1 linkage group of apricot (Prunus armeniaca L.) were previously developed and evaluated in a small set of cultivars. Those primers were used for studying variability in 77 apricot cultivars belonging to five different geographical groups, such as Chinese, Asian (Irano-Caucasian and Central Asian), North American, Mediterranean and Western European as well as Middle European cultivars. Six of the markers were polymorphic and revealed a total of 71 alleles ranging from 5 (aprigms11) to 20 (aprigms1) alleles per locus with a mean value of 11.83 alleles per locus. In conclusion, the SSR loci located in the G1 linkage group show a level of polymorphism which is similar to loci dispersed throughout the entire genome. The total number of alleles and the number of unique alleles were the highest in Chinese apricots and the lowest in Middle European cultivars. Heterozygosity also showed a decrease from Asia and China to Middle Europe. No association could have been observed between any SSR markers tested and plum pox virus (PPV) resistant phenotype of cultivars. PPV resistant cultivars did not form a separate clade on the dendrogram obtained by UPGMA cluster analysis. Middle European and Chinese cultivars formed separate clusters while other genotypes formed smaller multiple sub-groups or scattered among different clusters. Our results support previous hypotheses on the origin of PPV resistance in North American apricots. The allele data was also presented in a form that allowed the easy observation of allele frequencies in each geographical group at each locus. Using this data field, differences and similarities between cultivar groups can be easily assessed. The analysis demonstrated the links between the North American and Mediterranean apricot germplasm and confirmed that the Chinese and Eastern European cultivars are distantly related.