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.     

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.     

S-allele genotypes of apple pollenizers,cultivars and lineages including those resistant to scab

Summary

We examined S-allele genotypes of ten apple cultivars and species to determine their possible usefulness as pollenizers for all apple cultivars. ‘Dolgo’ did not contain any known S-RNases encoded at the S-locus, suggesting its possible usefulness as a pollenizer for almost all apple cultivars. We also identified and confirmed the S-allele genotypes of 18 apple cultivars by polymerase chain reaction (PCR)-digestion analysis. The S-genotype of ‘Kiou’ (S₁S₇), ‘Korei’ (S₃S₂₈), ‘Korin’ (S₁S₉), ‘Kotoku’ (S₁S₂₈), ‘Kyokkou (S₇S₂₅), ‘Lobo’ (S₁S₇), ’Mahe 7’ (S₂S₇), ‘Mellow’ (S₂S₃), ‘Takahara’ (S₃S₉) and ‘Warabi’ (S₉S₂₈) were confirmed by pollination results. These cultivars seemed not to have originated from the expected seed or pollen parents or, in the case of ‘Lobo’, might have been mislabelled. Finally, we identified the S-allele genotypes of ‘Prima’ (S₂S₁₀), ‘Querina’ (S₃S₉) and ‘Yoko’ × ‘Prima’ (S₃S₁₀), which are resistant to scab.     

Determining the S-genotypes of several sweet cherry cultivars based on PCR-RFLP analysis

Summary

Based on the cDNA sequences encoding sweet cherry self-incompatibility associated ribonucleases (S-RNases), a PCR-based S-allele typing system for sweet cherry cultivars has been recently developed. Using this technique, we determined S-genotypes of the three newly released Japanese cvs Kouka-Nishiki, Beni-Sayaka and Beni-Shuho and one British cv Merton Glory that was classified as a Universal Donor, which is able to be used as a pollen donor for all cultivars in pollen incompatibility groups I to XIII. Furthermore, we also determined the partial sequences of the S-RNase genes of ‘Rainier’ (S¹S⁴)‘ and ‘Sato-Nishiki (S³S⁶)’,which leads to the development of a more reliable S-allele identification method of PCR-RFLP for sweet cherry cultivars. Total DNA isolated from leaves of the four cultivars along with those from ten cultivars with known S-genotypes were PCR amplified with two sets of primers that were designed from DNA sequences encoding the signal peptide (Pru-T2) and two conserved domains (Pru-C2 and Pru-C4R) of sweet cherry S-RNases. By comparing the size of PCR products on agarose gel, the 5-genotypes of ‘Kouka-Nishiki’, ‘Beni-Sayaka’, ‘Beni-Shuho’ and ‘Merton Glory’ were suggested to be S¹S³, S¹S⁶, S⁴S⁶, and S⁴S⁶, respectively. Two of these three S-genotypes (S¹S⁶ and S⁴S⁶) were found for the first time. DNA sequencing of PCR products from S-alleles of ‘Rainier’ and ‘Sato-Nishiki’ revealed that Ban II, Nru I, Apa LI and Ava I sites, respectively, were unique in the S¹-, S³-, S⁴- and S⁶- sequences flanked by Pru-T2 and Pru-C4R primers. RFLP analysis of the PCR products using these enzymes confirmed that S¹-, S³-, S⁴- and S⁶-alleles of the four cultivars contained the respective restriction enzyme recognition sites.     

Determination of S-RNase genotypes and isolation of four novel S-RNase genes in Japanese apricot (Prunus mume Sieb. et Zucc.) native to China

Summary

Japanese apricot (Prunus mume Sieb. et Zucc.) originated in China with more than 500 cultivars. Most cultivars are self-incompatible. It is essential to identify S-genotypes for production and breeding. The S-genotypes of 17 Japanese apricot cultivars native to China were determined using the Prunus S-RNase consensus primer pair, Pru-C2 and PCE-R, which were designed from the second and third conserved regions of Prunus S-RNases, respectively. Eleven S-RNase alleles (S₁ S₂, S₇, S₁₂, S₁₄, S₁₅, S₁₈, S₂₀, S₂₂, S₂₃ and S₂₆) and four new S-RNase alleles (S₃₀, S₃₁, S₃₂ and S₃₃) with GenBank Accession Numbers JN232975, JN232976, JN232977, JN232978 were identified. Furthermore, the S-genotypes of four Japanese apricot cultivars were confirmed by field-testing for cross-pollination. The results of this study enrich the information available on S-genotypes in Japanese apricot and provide a reasonable basis for the appropriate arrangement of pollination trees in orchard practice.     

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.     

Multiple introduction of honeybee colonies increases cross-pollination,fruit-set and yield of ‘Black Diamond’ Japanese plum (Prunus salicina Lindl.)

Summary

Japanese plum (Prunus salicina Lindl.) belongs to the Rosaceae family, which carries the S-RNase-mediated gametophytic self-incompatibility system, which prevents self-fertilisation, and thus promotes out-crossing. The plum cultivar ‘Black Diamond’^® has become, one of the most important cultivars in Israel in the last decade, yet its yield is low in comparison with its known potential. Honeybees (Apis mellifera) are the most important pollinators for plums and several studies have demonstrated an apparent relationship between the number of honeybees and the size of the fruit crop. Therefore, in this study, we focussed on improving bee management in a ‘Black Diamond’ orchard. In four consecutive years of experiments, we examined the effects of increasing the density of bee colonies, and of making multiple introductions of colonies, on honeybee activity and on their effectiveness as pollinators of ‘Black Diamond’. We showed that four separate introductions, each of 0.625 colonies ha^–1, every 2 – 3 d from 10% full bloom to 3 d after full bloom, with a total density of only 2.5 colonies ha^–1, resulted in the greatest level of fruit set.     

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.     

中国原产果梅自交亲和变异品种花粉决定基因SFB的插入突变

 果梅是配子体自交不亲和树种,但在长期进化过程中也出现了自交亲和品种。以果梅典型的自交不亲和品种‘南高’和自交亲和品种‘甲州小梅’为对照,采用田间自交授粉试验对原产于中国的‘四川白梅’和‘长农17’两个果梅品种进行了自交亲和性鉴定,结果表明：其自花授粉坐果率分别为30.73%和16.27%,属于自交亲和品种。进一步通过AS-PCR分析发现花粉SFB基因存在一段插入序列,造成了分子水平的基因突变。推测该基因的突变使‘四川白梅’和‘长农17’SFB功能发生改变,从而自交亲和。     

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-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/).     

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.     

Identification of seven haplotype-specific SFBs in European pear (Pyrus communis) and their use as molecular markers

The European pear (Pyrus communis) carries the S-RNase-mediated gametophytic self-incompatibility (GSI) system. The S-haplotype is conferred by an S-locus, which contains the style-specific expressed S-RNase, and the pollen-specific expressed F-box genes (SFB). Both the S-RNase and the SFB genes are multi-allelic and each is characteristic of one of the S-haplotypes. Therefore, they are ideal markers for molecular S-genotyping. In this work, for the first time, seven haplotype-specific SFBs were isolated from European pears. Particular primers for each of these SFBs were generated, thus providing an additional tool for S-genotyping of European pear cultivars.     

Assessment of genetic variation among thornless blackberries (Rubus spp.) using random amplified polymorphic DNA

Summary

To assess genetic relatedness in thornless blackberry (Rubus spp.), 11 different blackberry cultivars were screened using random amplified polymorphic DNA (RAPD) markers. The blackberries selected represented four different thornless backgrounds. Genetic similarity was estimated using 140 random primers, and the cluster analysis conducted using the RAPD data grouped the cultivars into three distinct clades. Ninety-eight primers produced 113 cultivar-specific RAPD fragments capable of identifying each cultivar. In addition, reproducible polymorphism using two primers was observed within the ‘Evergreen’ (R. laciniatus) clade that consisted of the pure thornless blackberry ‘Everthornless’, the chimeral ‘Thornless Evergreen’, and their thorny progenitor ‘Evergreen’. All three plants are believed to be identical, except for a single mutational event that caused the phenotypic change from thorny to thornless. The R. laciniatus RAPD marker data provide information that may eventually be useful to identify the gene(s) responsible for thornlessness in that species.     

Phenolic Compounds,Antioxidant Activity,Fatty Acids and Volatile Profiles of 18 Different Walnut (Juglans regia L.) Cultivars and Genotypes

The study was carried out to determine phenolic compounds, antioxidant activity, total lipids, saturated fatty acids, unsaturated fatty acids, and volatile organic compounds (VOCs) of 18 different walnut (Juglans regia L.) cultivars and genotypes grown in Usak province, Turkey. The biochemical compounds were identified for each cultivar and genotype and a comparative evaluation was carried out. The results showed that most of the biochemical compounds are significantly varying among each other (p?<?0.05). The existence and abundance of the biochemical compounds in the cultivars and genotypes have not only found to be a cause of differentiation, but they have been important parameters for the similarities among the cultivars and genotypes. Total phenolic content and antioxidant activity ranged from 750.67 to 1245.64?mg GAE ml^?1 and from 42.46 to 56.50% in kernels, respectively. The highest contents of the phenolic compounds were noted from gallic acid (11991?mg kg^?1), ellagic acid (1057.16?mg kg^?1), catechin (425.4?mg kg^?1), and rutin (216.6?mg kg^?1), as an average of all cultivars and genotypes. The highest lipid contents were noted from ‘Franquette’ and ‘Fernette’ as 64.28% and 63.26%, respectively, while the lowest content was noted from ‘Oguzlar 77’ (52.52%). A total of 42 VOCs were described from the 18 different walnut cultivars and genotypes. Overall, results showed that the phenolic profiles, fatty acids composition, and VOCs play an important role in similarities and diversities among the cultivars. According to the results, 7 different clusters were developed from the 18 cultivars and genotypes. Herein, ‘Fernor’, ‘Maras 18’, ‘Arslan Local Type’ and ‘Cisco’ cultivars were found to have the lowest biochemical compositions. The superior cultivars or genotypes, in terms of the abundance of the biochemical composition, were found to be ‘Gulizar Hanim Type’, ‘Lara’, ‘Pedro’, ‘Tulane’, ‘Fernette’, ‘Balkan’ and ‘Franquetta’, where they were found to develop 3 clusters by 1:3:3, respectively.

     

Cloning of the S25 cDNA from ‘McIntosh’ apple and an S25-allele identification method

Summary

An S₂₅-RNase gene in the pistil responsible for self-incompatibility within the apple (Malus × domestica Borkh.) was cloned from a McIntosh cultivar. We developed an S₂₅-allele-specific polymerase chain reaction and digestion (PCR-digestion) method using specific primers and the restriction enzyme BamHI. We investigated the S-allele genotypes of 18 ‘McIntosh’ progeny.     

S-genotype characterisation of 10 Kernel-apricots native to China

The reproductive mechanism of Kernel-apricot involves Gametophytic self-incompatibility (GSI) which is under S-allele control. It encodes a polymorphic pistil ribonuclease (S-RNase), which is multiallelic and ideal markers for S-genotypes characterisation. In this study, six S-RNase primers were used to identify theS-genotypes of 10 Kernel-apricot, including three self-design primers and three universal primers. Ten S-alleles were identified in 10 Kernel-apricot through sequencing and comparing in NCBI. Moreover, some Kernel-apricot S-genotypes were confirmed by experiment of fruit-setting ratio and pollen tube growth. In conclusion, S-genotypes information of Kernel-apricot will be helpful for breeders and growers to plan compatible hybridised combination for ensuring effective pollination and high fruit-setting ratio.     

The relationship between fruit size and cell division and enlargement in cultivated and wild persimmons

Summary

To determine the anatomical basis for differences in fruit size in Japanese persimmon (Diospyros kaki Thunb.), the number and sizes of parenchymal cells were measured in four astringent cultivars and in wild D. lotus over a single season in Japan. Fruit weight at harvest correlated with the final number of parenchymal cells (r = 0.95*) and their size (r = 0.92*). There was also a correlation between fruit diameter and the number of cell layers at full bloom (r = 0.93*), which increased 1.7 – 1.9-fold during fruit development.The duration of cell division and the sizes of the cells at full bloom were similar among the cultivars, whereas mean parenchymal cell lengths increased by 7.7-, 6.7-, 5.4-, 5.0- and 3.8-fold from full bloom to harvest in ‘Otanenashi’ (average fruit diameter = 96 mm), ‘Hiratanenashi’ (77 mm), ‘Saijo’ (54 mm), ‘Tsurunoko’ (44 mm), and D. lotus (20 mm), respectively. The final size of tannin cells was larger in ‘Otanenashi’ and ‘Hiratanenashi’ than in the other cultivars, including the wild species.These results indicate that the size of fruit at harvest was determined by the number of cells at full bloom, and their expansion during fruit development.     

Effects of grafting height of MM106 rootstock on growth,lateral shoot formation and yield in apple trees

Summary

Between 2004 and 2008, the effects of different grafting heights on sylleptic shoots were tested in the apple (Malus domestica Borkh.) cultivars ‘Granny Smith’ and ‘Gloster’, and cumulative fruit yields were evaluated. MM106 apple rootstocks were grafted 10, 20, 40, or 60 cm above soil level in August 2004. The results showed that an increased grafting height significantly decreased tree height in both cultivars. The tallest and shortest trees were observed at grafting heights of 10 cm (153.0 and 170.0 cm) and 60 cm (141.3 and 143.5 cm) in ‘Granny Smith’ and ‘Gloster’, respectively.Among the various grafting heights tested, 60 cm in ‘Granny Smith’ and 20 cm in ‘Gloster’ gave the largest stem diameters (17.6 mm and 16.8 mm, respectively). The number of lateral shoots increased significantly with increased grafting height in both cultivars. The largest numbers of lateral shoots in ‘Granny Smith’ (10.75) and ‘Gloster’ (2.00) were obtained from a grafting height of 60 cm, while 2.55 and zero lateral shoots occurred at 10 cm grafting height in ‘Granny Smith’ and ‘Gloster’, respectively. Shoot lengths decreased significantly by increasing the grafting height. Grafting heights of 10 cm and 60 cm resulted in the tallest and shortest shoots in both cultivars. Cumulative fruit yields were significantly affected by grafting height in both cultivars. The highest yield was found for a 60 cm grafting height in both ‘Granny Smith’ (11.295 kg tree^–1) and ‘Gloster’ (4.818 kg tree^–1).The results of this study suggest that grafting heights of 40 cm and 60 cm have the potential to promote branching and early bearing for apple fruit production in sustainable and organic agricultural systems.     

Agronomic,cytogenetic and molecular studies on hermaphroditism and self-compatibility in the Greek kiwifruit (Actinidia deliciosa) cultivar ‘Tsechelidis’

The development of hermaphrodite cultivars of kiwifruit (Actinidia deliciosa) to overcome dioecism is a priority for all breeding programmes, worldwide. If successful, the number of pollinators in the orchard could be reduced, allowing more space for reproductive vines. Hermaphroditism and the expression of self-compatibility were studied in A. deliciosa, using the new kiwifruit cultivar, ‘Tsechelidis’, as a model. This plant material was chosen for investigation as its female flowers produced viable pollen. The aim of the present study was to investigate the expression and nature of self-compatibility in ‘Tsechelidis’ and to evaluate its potential for breeding purposes. The expression of self-compatibility in ‘Tsechelidis’, between years, was investigated by controlled self-pollination of female flowers in the field and by microscopic studies on pollen viability in the laboratory. The commercial cultivar ‘Hayward’ and the pollinator ‘Matua’ were used as controls. Controlled self-pollination in the field confirmed that ‘Tsechelidis’ exhibited a degree of hermaphroditism and self-compatibility. Its flowers produced viable pollen and could set fruit of an acceptable size and quality after self-pollination, compared to open-pollinated fruit. The average fruit set percentage for ‘Tsechelidis’ was 34%, and ranked from 20% to 60% per vine, while the corresponding rate for ‘Hayward’ was only 4%. Microscopic studies on ‘Tsechelidis’ pollen revealed variations in pollen viability between different vines, which were even greater between different flowers on the same vine, ranging, in some cases, from 0% (absolutely sterile) to 70% (significantly fertile) per flower. Female control ‘Hayward’ vines were found to be practically sterile (0%), while male control ‘Matua’ vines were almost 100% fertile. The nature of self-compatibility in ‘Tsechelidis’ was investigated further by molecular analysis using the sex-linked sequence characterised amplified region (SCAR) markers, SmX, SmY, and SmY1, and by single nucleotide polymorphism (SNP) analysis. Hermaphroditism in ‘Tsechelidis’ was related to sex expression, as observed by SNP analysis, but complete genome mapping would be required to fully understand the mechanisms involved and to exploit this new genetic material for breeding purposes.