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.     

槜李等15个李品种S基因型鉴定及其多态性分析

利用李属S-RNase基因特异性引物,对15个供试李品种进行PCR扩增,共获得30个目的条带。对这些目的条带进行测序鉴定出15个李品种的S基因型。通过与NCBI中利用BLASTn与GenBank+EMBL+DDBJ+PDB等数据库中的序列比对,结果表明,其中9个为新S-RNases基因,对9个新S-RNases核苷酸序列进行分析发现,位于高变区内的内含子大小为141～1758bp,其同源性为33.9%(S-18～S-19)～81.6%(S-20～S-21),表现出丰富的长度和序列多态性;编码区的核苷酸序列比对结果,其同源性为73.3%(S-16～S-19)～91.7%(S-17～S-22);其推导氨基酸序列相似性为67.3%(S-16～S-19)～89.1%(S-17～S-22);包含李属S-RNase一级结构所共有的C2、C3保守区和高变区(RHV)。系统进化分析表明,9个新S-RNases与李属其它树种S-RNases聚类在一起,归属为李亚科(Prunoideae)。     

Identification of a new self-incompatibility allele (S31) in a Hungarian almond cultivar and its reliable detection

Self-incompatibility of almond [Prunus dulcis (Mill.) D.A. Webb] is controlled by the S-locus with 30 described allelic variants. In this study, PCR amplification, cloning and DNA sequence analysis revealed a new S-RNase allele in a Hungarian cultivar, ‘Tétényi bőtermő’. This new allele was labelled as S₃₁. Since S₃₁ is characterized by almost identical intron sizes as S₉, consensus PCR was not successful in discrimination of the alleles, even if fluorescently labelled fragments were sized on an automated sequencer. Therefore, an allele-specific forward primer (PdS31-F) was designed to anneal selectively within the second intron of the S₃₁-RNase gene and used in combination with the EM-PC3consRD consensus primer. This allowed for the successful discrimination of S₃₁ from S₉. The PdS31-F primer and allele-specific PCR in general might be useful in the identification of different alleles with matching intron sizes that might occur during screening for S-alleles in a more diverse population, e.g. local cultivars from Central Europe to Asia.     

新疆主栽杏品种自交不亲和SFB基因的检测

 为系统鉴定新疆主栽杏品种自交不亲和SFB基因型,以新疆25个主栽杏品种为试材,利用蔷薇科保守序列设计引物对叶片基因组DNA进行SFB基因特异PCR扩增,筛选出有效的扩增引物;对成功扩增的杏品种的特异条带克隆测序,在GenBank上进行BLASTN比对,确定各品种的SFB基因型。结果显示：引物组合Ⅱ-1、Ⅳ-2,1-Ⅰ、1-Ⅱ对新疆杏品种的扩增效果最好,成功地在18个品种上扩增出了5种大小不同的条带,14种不同的基因型,其中两     

中国樱桃S-RNase基因PCR扩增技术体系的建立

通过对TaqDNA聚合酶、Mg2+、dNTP、通用引物、模板DNA浓度等反应参数的系统研究,建立了中国樱桃S-RNase基因特异PCR扩增体系。该体系反应的总体积25μL,其中Taq酶1.25U,MgCl22.5mmol/L,dNTP0.15mmol/L,通用引物0.25μmol/L,模板DNA 50 ng。反应程序为:94℃预变性3 min;33个循环的94℃变性1 min,56℃退火45 s,72℃延伸1.5 min;最后72℃延伸10 min。利用优化的扩增体系在中国樱桃的不同品种中获得有效扩增,进一步证明了该体系具有良好的稳定性和重复性。     

Investigation of the fertilization biology of some sweet cherry cultivars grown in the Central Northern Anatolian Region of Turkey

The experiments were carried out between 2003 and 2005 in the Province of Amasya. The first objective of the study was to determine the best pollinizer for the cultivar ‘0900 Ziraat’ which is the most popular sweet cherry cultivar (Prunus avium L.) in Turkey. The ‘0900 Ziraat’ was used as a female parent and ‘Türko?lu’, ‘Köro?lu’, ‘Kargayüre?i’, ‘Hac? Ali’, ‘Starks Gold’ and ‘Geçkiraz’ were used as pollinizer cultivars. In 2003, for the hand cross pollination combinations, fruit set was between 31.25% and 42.80%. In 2004, fruit set was reduced by spring frost and was between 9.69% and 15.24%. Additionally, the blooming periods of the experimental cultivars, fruit set of open pollination and hand self pollination were examined. The fruit set ratios of all tested cultivars under open pollination conditions varied between 26.73% and 53.49% in 2003 and 4.38% and 38.35% in 2004. ‘Geçkiraz’ had the highest fruit set owing to its late flowering date. For open pollination, fruit set values of ‘0900 Ziraat’ were lower than hand cross pollination with other cultivars in 2003 when the results of both treatments are compared. On the contrary, fruit set was higher than hand cross pollination in 2004. In the present study, pollen germination values varied greatly among year, cultivar and sucrose concentration. Viability percentages were generally high, not less than 66%. According to phenological observations, partial or complete overlapping of the flowering period of ‘0900 Ziraat’ and its possible pollinizer cultivars were observed in 2003 and 2005. But in 2004, the flowering period overlapped for only ‘0900 Ziraat’ and ‘Geçkiraz’ cultivars. When blooming times are taken into consideration, ‘Geçkiraz’ was found to be the best pollinizer cultivar.     

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).     

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。     

中国樱桃泰山干樱S-RNase基因的克隆及序列分析

以中国樱桃品种泰山干樱为试材,利用李属植物C2、C5保守区引物,扩增花柱S-RNase基因,获得4个S等位基因,测序结果表明序列大小分别为:1608、950、796、504bp。根据同源比较发现大小为796bp的等位基因与基因库中登录的S1-RNase为同一基因,其它3个S-RNase基因为首次报道,依序列大小分别命名为S2(1608bp,登陆号EF541168)、S4(950bp,登陆号EF541173)和S6(504bp,登陆号EF541172)。序列分析表明S2-RNase在C3区存在终止密码子,导致翻译提早终止;S4-RNase的C5区前有插入片断;S6-RNase在高变区比其它S等位基因少一个氨基酸残基。氨基酸序列同源性比较分析表明,中国樱桃S-RNase与樱花、扁桃的相似性高。在系统进化树中中国樱桃的4个S-RNase基因的氨基酸序列和樱花、扁桃、甜樱桃、酸樱桃等一起归于李亚科。     

‘奥嗄二十世纪’梨自交亲和性分子机制及遗传特性研究

 ‘奥嗄二十世纪’是自交不亲和性梨品种'二十世纪'的自交亲和性花柱突变体,花粉自交不亲和性功能正常,其自交亲和性突变的分子机制及遗传特性目前仍有争议。本研究通过田间自花及相互授粉以及基因组、mRNA转录和蛋白质水平比较,分析‘奥嗄二十世纪’、‘二十世纪’及其后代S-RNase基因的存在与否、表达特性及其在后代中的遗传。结果显示,‘奥嗄二十世纪’花柱S₂-RNase基因的核苷酸序列和表达特性与其原始品种‘二十世纪’的完全一样;而S4-RNase基因信号比其原始品种‘二十世纪’的弱,而且也在花柱中正常表达(包括转录和翻译水平),但表达量低;然而在其自交亲和后代基因组中检测不到S₄-RNase基因。本研究表明,‘奥嗄二十世纪’基因组中存在花柱S₄-RNase基因,但不能遗传给后代。     

中国杏自交不亲和花粉特异SFB基因的鉴定与序列分析

利用花粉SFB基因的同源扩增结合内切酶酶切的方法,在9个中国杏品种中首次克隆了6个花粉SFB基因,对应花柱S-RNase基因序号将其分别命名为Par-SFB8、Par-SFB9、Par-SFB11、Par-SFB17、Par-SFB23和Par-SFB26,在GenBank上的登录号分别为EU652883、EU935588、EU652884、EU652885、EU652886和EU652887。序列分析表明杏的花粉SFB基因具有蔷薇科李属植物SFB基因的典型结构特征;其内含子位于5′端非翻译区,长度90 ~ 108 bp,核苷酸序列的同源性为63.9% ~ 93.3%。氨基酸序列的同源性比较表明,杏花粉SFB基因的种内同源性为73.7% ~ 85.3%;与李属其他植物花粉SFB基因的种间同源性为75.2% ~ 97.7%。利用特异PCR扩增进一步确定了杏的S9、S11、S17和S26单元型花柱S-RNase与花粉SFB基因间距离,表明花柱S-RNase与花粉SFB基因紧密连锁;同时组织特异性表达分析确定SFB基因在花粉组织中特异表达。以上结果说明获得的SFB基因为杏的花粉候选S基因。     

野生欧洲李嫁接育苗技术研究

为探索野生欧洲李嫁接繁殖技术,提高其嫁接成活率,以野生欧洲李为研究对象,采用4种不同嫁接方法,研究不同嫁接砧木、嫁接时间、嫁接方法、砧木粗度和嫁接高度对接穗成活率及新梢生长量的影响。结果表明:适合野生欧洲李嫁接的4种不同砧木的优先顺序依次为:栽培欧洲李品种‘女神’>野生樱桃李>野杏>山桃。春季4月中旬以野生樱桃李为砧木嫁接时,适合采用劈接法和嵌芽接法,最高成活率达90.37%;夏季6月中旬、7月中旬嫁接则适合采用T形芽接法,成活率最高为90.93%,且新梢生长快。根据接穗嫁接成活率和成活后生长量筛选出适合野生欧洲李嫁接的砧木粗度为大于14.00 mm。野生欧洲李在7月中旬以‘女神’作砧木,采用T形芽接法嫁接后成活率最高达93.52%。     

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.     

Carbohydrate profiles in the graft union of young sweet cherry trees grown on dwarfing and vigorous rootstocks

In composite (scion–rootstock) dwarfing fruit trees, an overgrowth at the graft union is often observed, the severity of which is correlated with degree of dwarfing. The graft union of dwarfing sweet cherry (Prunus avium L.) rootstocks may limit soluble sugar transport or starch mobilization, leading to localized accumulation. Soluble sugars and starch were measured in the tissues surrounding the graft union of young ‘Rainier’ (2002) and ‘Lapins’ (2003) sweet cherry trees on ‘Gisela 5’ (‘Gi 5’; dwarfing) and ‘Colt’ (vigorous) rootstocks. Separate rootstock shank, rootstock, graft union, and scion tissues were analyzed for both starch and soluble sugar content throughout the growing season in both years. Starch concentrations did not vary among locations within the graft union for ‘Rainier’ on either the dwarfing or vigorous rootstock, or for ‘Lapins’ on the dwarfing stock. However, for ‘Lapins’ on ‘Colt’, starch was highest in the rootstock shank and declined vertically (rootstock shank ? rootstock > union > scion). Soluble sugar concentrations were generally similar to or higher in scions on ‘Gi 5’ than on ‘Colt’, and were similar to or lower in the rootstock and rootstock shank on ‘Gi 5’ than on ‘Colt’. Results suggest that rootstock has a significant effect of localized accumulations of carbohydrates above and within the graft union of ‘Gi 5’ and below the graft union of ‘Colt’.     

Phenotypic variability and genetic structure in plum (Prunus domestica L.), cherry plum (P. cerasifera Ehrh.) and sloe (P. spinosa L.)

In the present study, phenotypic variability of 80 plum (Prunus domestica L.) varieties maintained in the French National Plum Collection was evaluated with 19 quantitative traits. In addition, genetic diversity and genetic structure was studied in three plum species (P. domestica L., Prunus cerasifera Ehrh. and Prunus spinosa L.) using chloroplast DNA (cpDNA) markers and five single sequence repeat (SSR) loci. Based on phenotypic traits, some varieties, such as mirabelle plums, grouped together. Bayesian structure analysis was used to identify different genetic groups, whereby damson plums were clearly distinguished from greengage plums. When examining the three species together, a higher level of cpDNA allelic richness was found in P. cerasifera and in P. spinosa than in P. domestica where only five cpDNA haplotypes were detected in the national plum collection, with one main haplotype that accounted for 80% of the varieties studied. P. domestica cpDNA haplotypes tended to group together with P. cerasifera haplotypes whereas most of P. spinosa haplotypes formed a separate cluster. SSR markers were somewhat able to distinguish the three species. These results provide some clues as to the origin of plum and the various plum varieties. Our results also provide useful information for the management of plum genetic resources.     

甜樱桃花芽分化后期特征观察

通过品种比较、地区比较和人工环境模拟3个关联的试验设计,观察甜樱桃从落叶后至谢花后的花芽分化发育的特征,探求其南引试栽、结实困难的原因。结果表明,郑州的早红宝石、红灯和拉宾斯,烟台、郑州和金华的红灯,以及花芽分化期置于露地和日光温室中的早红宝石在多个物候期阶段的最终分化发育的基本特征一致。因此,造成甜樱桃南引试栽、结实困难的主要原因可能与生长季的花芽分化无关,不能仅限于与花前高温引发的胚珠、胚囊发育不良有关,低温累积量不足也是不可被忽视的因素。     

Genetic relatedness of sweet cherry (Prunus avium L.) cultivars from Ukraine determined by microsatellite markers

Microsatellite (SSR) markers were used to characterise 23 sweet cherry cultivars of Ukrainian, and four cultivars of non-Ukrainian, origin. To assess their genetic diversity and relatedness, 11 pairs of primers were applied to microsatellite loci, resulting in amplification of 66 SSR alleles. The mean value of the number of different alleles, and the polymorphic index content, amount to 7.333 and 0.700, respectively, demonstrating a significant genetic diversity of the investigated sweet cherry cultivars. Four highly polymorphic SSR loci (EMPAS02, EMPAS06, PceGA34, UDP98-412), which belong to the list recommended by the European Cooperative Program for Plant Genetic Resources, can be used as a minimum genetic marker set for identification of the majority of the studied cultivars; however, for successful discrimination of the most similar cultivars, more markers, located on all chromosomes of sweet cherry, appear to be necessary. Application of unweighted variable-group method using averages clustering allowed elucidation of the relatedness among the sweet cherry varieties, and showed that the Ukrainian cultivars combine genetic material of local, western European, and probably Caucasian origin; however, the origin of several cultivars still remains unclear, and should be studied additionally.     

梨S基因芯片的试制及分子杂交条件的优化

 为研究寡核苷酸芯片在确定梨品种S基因型中的应用价值,根据梨自交不亲和基因的结构特点设计了部分S基因的检测探针,并制成寡核苷酸芯片;采用引物Cy3荧光标记法标记检测样品的PCR产物并进行杂交,以检测不同样品的S基因型。结果表明：通过对不同杂交温度、杂交时间等的探索获得了较好的反应条件并用制备的芯片检测了已知S基因型的梨样品,检测结果与各品种的已知基因型相符。证明寡核苷酸芯片检测梨品种的S基因型是一种切实可行的检测方法,若对芯片进一步完善,则今后在梨的自交不亲和性状的机理研究及梨自交不亲和性状的利用等方面将有着广阔的应用前景。     

Uniconazole is an effective blossom thinner for ‘Bing’ cherry (Prunus avium)

Small fruit size is one of the limiting factors in marketing cherries (Prunus avium L.). Several techniques have been used to improve fruit size; among them, blossom and fruit thinning in order to reduce crop load have proved effective. Unfortunately, hand thinning is very costly, and chemical thinning has proved ineffective. In the present study, carried out in two consecutive years (2006–2007), application of uniconazole (E-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl-1-penten-3-ol)) at 100–125 mg l⁻¹, in the form of 0.2–0.25% Magic™, at 30% full bloom, reduced the fruit set of ‘Bing’ cherry fruit. As a result time spent in hand thinning was reduced by about 50% and the crop load by about 40%, and there was a considerable shift to larger fruit size (>26 mm), provided the crop load was not too low. No negative effects were observed on the return bloom and yield in the following year.