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.     

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.     

SSR markers reveal the uniqueness of olive cultivars from the Italian region of Liguria

Twenty-three important Ligurian olive accessions corresponding to 16 cultivars were studied using 12 SSR markers and 40 Mediterranean cultivars were included in the study in order to investigate the relationships between Ligurian and Mediterranean germplasm. All SSRs produced polymorphic amplifications. One hundred and forty-nine alleles were found in the 63 accessions analysed. Twenty-two alleles were specific to germplasm from Liguria and of these 12 were unique to single cultivars. Heterozygosity and discriminating power calculated in this regional germplasm were high on average (0.70 and 0.74) and not so much lower than the values in the total sample that includes cultivars from different Mediterranean countries (0.77 and 0.88 respectively). No cases of genetic identities were found between Ligurian and Mediterranean accessions. Several cases of homonyms and synonyms within the Ligurian germplasm were explained. Cluster analysis generally revealed a clear discrimination of the profiles from Liguria and Italy with respect to the cultivars from other Mediterranean countries. Only one Ligurian cultivar, “Negrea”, appeared to have a different origin, grouping with the Mediterranean cultivars.     

Genetic diversity of Iranian and some European grapes as revealed by nuclear and chloroplast microsatellite and SNP molecular markers

Genetic and chloroplast haplotype variations of 35 Iranian genotypes and 10 European grape cultivars were investigated using 9 nuclear simple sequence repeats (nSSRs), 4 chloroplast simple sequence repeats (cpSSRs) and 46 single nucleotide polymorphism (SNP) markers. In total, 83 alleles were detected at nine nSSRs, giving a mean of 15.66 alleles per locus and polymorphism information content (PIC) values ≥0.75 ranged from 0.75 to 0.90. For SNP markers, PIC values varied from 0.30 to 0.39 with an average of 0.34. Analysis of molecular variance revealed 97 and 93% of partitioned genetic diversity within populations using nSSRs and SNPs markers, respectively. Un-weighted neighbour-joining (NJ) cluster analysis grouped grapes into 10 and 9 major clusters using SSR and SNP markers, respectively. Synonyms and homonyms were identified among the Iranian genotypes. Close genetic relationship among Farkhi and Bidane-Sefid genotypes may probably propose a common ancestor and mutational evolution. Most European cultivars were differentiated from Iranian genotypes, however, clustering of some Iranian genotypes with European cultivars in the same clusters suggests that clonally propagated materials have probably been exchanged from the Middle East to West or vice versa. C and D chloroplast haplotypes were the most frequent within the Iranian genotypes, while A chloroplast haplotype was exclusively observed among European cultivars.     

SSR-based identification key of cultivars of Olea europaea L. diffused in Southern-Italy

Olive (Olea europaea L.) is an important fruit species in Italy and Mediterranean basin constituted by a wide germplasm with a large number of cultivars present in all the Mediterranean area. SSRs are becoming the markers of choice for variability studies in olives as they are simple to perform, transferable, hypervariable, highly polymorphic and show a high information content.Olive genetic diversity was studied using 16 SSR markers on 30 cultivars diffused in Southern-Italy. Resolving Power (RP) and Power of Discrimination (PD) were calculated to evaluate the efficiency of the SSR markers investigated in studies of cultivars fingerprinting. Based on their high efficiency, two SSR markers, UDO43 and DCA16 were chosen to set up an identification key to distinguish the entire set of cultivars, confirming the high biodiversity of the Southern-Italian olive germplasm and the suitability of SSR markers in studies of cultivar diagnosis.     

Analysis of genetic structure in a large sample of pomegranate (Punica granatum L.) using fluorescent SSR markers

Molecular markers are helpful to efficiently protect the plant breeders’ rights. Fluorescent simple sequence repeats (SSR) based on DNA sequencing is a powerful and automated platform for the detection of genetic polymorphism. Here, we first reported 13 SSRs using a fluorescent SSR panel according to the reference genome information, and detected the genetic diversity and population structure of 136 pomegranate varieties. The average number of alleles per locus was 6.31, gene diversity ranged from 0.16 to 0.37 with an average of 0.28, and polymorphic information content (PIC) varied from 0.14 to 0.29 with a mean value of 0.22. The mean genetic distance (GD) between varieties was 0.32. Population structure analysis divided the varieties into three different subpopulations (Q1, Q2, and Q3). The cultivars in Q1 all came from China, and almost all cultivars came from foreign countries combined with Chinese were grouped into Q2. The only accession originated from Myanmar was grouped to Q3. The results indicated that 13 SSR markers were polymorphic. The varieties originating from the same geographical region were more closely related, and extensive gene flow had taken place between the varieties from different regions. The results may be useful for the efficient selection breeding and variety protection.     

Microsatellite markers based assessment of genetic diversity in Iranian olive (Olea europaea L.) collections

To study the genetic variation in Iranian olive collections and some foreign olive cultivars, 47 accessions of 18 local cultivars from 6 olive collections of Iran (Roudbar, Zanjan, Ahvaz, Dezful, Kazeroon and Shiraz), were analyzed along with 30 imported cultivars using 16 microsatellite primer pairs. All the used microsatellite loci revealed polymorphism in the studied genotypes, except GAPU14 and GAPU113 markers. Fourteen microsatellite primers amplified 126 polymorphic alleles in the 87 selected olive accessions. The average number of alleles per locus was 9, ranging from 3 to 14. Polymorphic information content (PIC) was 0.85. The genetic similarity based on Jaccard coefficient ranged from 0.15 to 1. The genetic relationships among accessions were investigated using cluster analysis and principal component analysis (PCA). Most of the accessions with the same name were grouped together; some exceptions were also observed. As expected, close relationship was observed among accessions within same cultivar. Most of the Iranian olive accessions were clustered to a main distinct group. Two-dimensional scatter plot of principal component analysis revealed a clear separation of most of the Iranian olives from Syrian and other introduced cultivars. These suggest that Iranian cultivars have different origin related to West Mediterranean basin cultivars and have evolved independently from the others. Between and within Iranian and foreign cultivars (cultivars including three or more accessions) genetic diversity was analyzed using analysis of molecular variance (AMOVA). AMOVA revealed higher within cultivar genetic variation (62.76%) as compare to that between cultivar variations (37.24%). The intra- and inter-cultivar variance tested by permutation test showed significant genetic variation at both levels. The high level within cultivar genetic variance could be due to mislabeling and presence of homonyms in cultivars produced by vegetative propagation from original plants.     

Molecular characterization and genetic relationship among almond cultivars assessed by RAPD and SSR markers

RAPDs and SSRs were used to study the genetic diversity of Iranian almond cultivars and their relationship to important foreign cultivars and three related species. Eight unidentified almond Shahrodi cultivars and three wild almonds (Prunus communis, Prunus orientalis and Prunus scoparia) were also included. Of the primers tested, 42 (out of 80) RAPD and 18 (out of 26) SSR primers were selected for their reproducibility and high polymorphism. A total of 664 polymorphic RAPD bands were detected out of 729 bands. The number of presumed alleles revealed by the SSR analysis ranged from 3 to 10 alleles per locus with a mean value of 6.64 alleles per locus. Both techniques discriminated the genotypes very effectively, but only RAPDs were able to discriminate the cultivars Monagha and Sefied. Results demonstrated an extensive genetic variability within the tested cultivars as well as the value of SSR markers developed in peach for characterization of almond and related species of Prunus. Dice similarity coefficient was calculated for all pair wise comparisons and was used to construct a UPGMA dendrogram. For both markers a high similarity in dendrogram topologies was obtained although some differences were observed. All dendrograms, including that obtained by the combined use of both the marker data, depicted the phenetic relationships among the cultivars and species, depending upon their geographic region and/or pedigree information. Almond cultivars clustered with accession of P. communis showing their close relationship. P. orientalis and P. scoparia were clustered out of the rest of P. dulcis.     

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.     

Development and characterization of SSR markers in Chinese jujube (Ziziphus jujuba Mill.) and its related species

In order to study the extensively genetic diversities of more than 700 cultivars of Chinese jujube, it is necessary to utilize various informative DNA markers. SSR markers are highly polymorphic, co-dominant, locus-specific markers widely used in genetic studies, but less used in Chinese jujube because of no specific primers available. In this study, we used the approach of selectively amplified microsatellite (SAM) to develop SSR markers for Chinese jujube and its related species. Three cultivars (Dongzao, Dalilongzao and Jinsixiaozao) were selected to perform the approach of SAM with CT repeats. There were totally 25 primers obtained, of which we selected 16 primers available to detect the polymorphism in populations of 24 Chinese jujube cultivars, two wild jujube varieties and two Indian jujube cultivars. Based on these primers, genetic relationships of the 28 samples were constructed in a dendrogram according to the UPGMA cluster analysis. The samples were clustered into three main groups, including Chinese jujube, wild jujube and Indian jujube as expected. The 16 sequence-specific SSR primers could efficiently distinguish all the 24 cultivars of Chinese jujube, except for two cultivars, Jinsixiaozao and its ‘stoneless’ mutant, Wuhexiaozao. As a result, SAM was a very efficient method in targeted developing sequence-specific SSR primers in Chinese jujube. Furthermore, SAMs could also be used as high polymorphic molecular markers independently. The further study would focus on developing other oligonucleotide repeat types and applying more SSRs available in the genetic research of Chinese jujube.     

Microsatellite marker-based identification of mother plants for the reliable propagation of olive (Olea europaea L.) cultivars in Australia

Summary

Olive production in Australia has continued to increase in recent years, however there remains a high degree of confusion on the genetic identities of the cultivars being grown. In the present study, seven microsatellite (simple sequence repeat; SSR) loci were used to identify a set of 53 olive tree samples from different sources. The microsatellite DNA profiles of all 53 tree samples, including seven unknown trees, were compared with the SSR profiles of 14 reference olive cultivars. A total of 60 fragments (alleles), averaging 8.57 alleles per microsatellite locus, were amplified. High average values were found for the observed heterozygosity, the expected heterozygosity, and the polymorphic information content (0.73, 0.74, and 0.72, respectively). While all seven microsatellite markers proved useful for characterisation and identification purposes, a combination of three SSR primer pairs (DCA9, DCA18, and EM030) was sufficient to distinguish all 53 olive samples. The microsatellite allelic profiles allowed the 53 tree samples to be grouped into 23 genotypes. The allelic profiles of 14 of these genotypes matched with their reference cultivars, while the genetic identities of the remaining nine genotypes could not be confirmed. Some of these unknown genotypes may have been derived from feral olive trees, or were due to mislabelling and/or planting errors among Australian olive cultivars. Our results confirm the usefulness of microsatellite markers as a tool for cultivar differentiation and identification, and indicate the need for reliable identification of mother plants for commercial propagation.     

Genetic assessment of apple germplasm in Bosnia and Herzegovina using microsatellite and morphologic markers

Traditional apple cultivars from Bosnia and Herzegovina (B&H), potentially diverse due to specific geographic location and history of the country, represent a possible source of valuable traits for future breeding efforts and sustainable fruit growing. A total of 39 accessions, 24 traditional B&H cultivars and 15 modern international cultivars, maintained at the ex situ apple collection “Srebrenik” in Northeast Bosnia were, investigated using 10 SSR (simple sequence repeats) markers and 23 morphologic characteristics. All the used primer pairs manage to amplify clearly distinguishable and highly polymorphic SSR alleles, in average 10.4 alleles per locus. More than two different alleles per locus were detected for seven accessions (five traditional B&H cultivars and two international cultivars). Forty one unique alleles were exclusively present within the B&H cultivars, while seven unique alleles were only detected within international cultivars. The differentiation between traditional B&H and international cultivars (Fst = 0.060; P < 0.0001) was significant, also confirmed by analyses of molecular variance (AMOVA) (f_CT = 0.092; P < 0.001). Cluster analyses of 39 apple accessions, based on 10 SSR loci, revealed that only two traditional B&H cultivars grouped tightly with international cultivars (Ljepocvjetka and Bobovec Jon), while the rest formed separate clusters. Multivariate analyses of variance (MANOVA), nonparametric multivariate analyses of variance (NPMANOVA) and analyses of similarity (ANOSIM) showed statistically significant difference in morphologic characteristics between traditional B&H cultivars and the international cultivars. Cluster analyses of 39 apple accessions, based on the morphologic data, displayed less differentiation between traditional and international accessions, in comparison to the cluster analyses based on molecular data. No correlation between the molecular and morphologic data set was detected using the Mantel test. Many of the morphologic characteristics which have been analyzed in this study have significant commercial importance, we can assume that unlike the microsatellites these traits have been under agronomic selection pressure.     

香蕉A基因组品种间遗传关系的SSR检测

应用SSR技术,对32个香蕉A基因组类型品种(系)的遗传关系进行了检测。40对SSR引物在32个品种(系)中分别扩增带数在3～15个,平均每个SSR座位可检测2.99个多态性带;引物的多态信息量(PIC)在0.00～0.88,平均0.62。依据SSR数据计算的品种间遗传距离在0.00%～34.27%,平均12.45%,大多数品种间的遗传变异非常有限,但也存在着遗传差异突出的品种:FHIA25、Yangambi KM5、Pisang Jari Buaya、Rose和皇帝蕉。依据26%的遗传距离,除了FHIA25和Pisang Jari Buaya单独化成1组外,其它30个品种可以分为2组:品种间遗传差异相对较高的组I和品种间遗传差异相对较低的组Ⅱ。Williams与引进的洪都拉斯3号、M931之间,洪都拉斯1号和洪都拉斯2号之间,高脚青芽蕉和高脚顿地雷分别没有区分开来,这可能是同物异名,也可能是同一品种未能分辨的突变体。     

36 份枣品种SSR 指纹图谱的构建

 利用12 对SSR 引物对36 个枣品种进行分析,采用荧光M13 毛细管电泳技术进行多态检测,共检测到99 个多态位点,每对引物的多态位点数达到8.25,PIC 值变幅为0.62 ~ 0.85,平均为0.75。依据12 对SSR 引物在36 个品种中扩增的特异带型组合,采用引物-带型组合法构建了36 个枣品种的指纹图谱,并对这36 份枣品种做聚类分析,遗传相似系数在0.6667 ~ 0.9444 之间。研究结果为枣树分类、种质鉴定和分子育种提供了重要的工具。     

普通杏群体遗传结构的荧光AFLP分析

以准噶尔—伊犁生态群（新疆野杏）、中亚生态群（新疆栽培杏和李光杏）、欧洲生态群和华北生态群的45个普通杏品种或材料为试材,以梅杏、辽梅杏和辽杏为外组,利用荧光AFLP标记对普通杏4个生态群的群体遗传结构进行了研究,结果表明：7对EcoRI/MseI引物（其中MseI引物为FAM荧光标记物）平均扩增多态带数为130.86,平均多态带为60.58%。4个生态群的多态带百分比比较表明准噶尔—伊犁新疆野杏生态群（P = 43.59%）＞中亚南疆栽培杏生态群（P = 41.27%）＞华北生态群（P = 39.42%）＞欧洲生态群（P = 39.42%）＞中亚李光杏生态群（P = 37.57%）;普通杏在种级水平Nei's基因多样度（H = 0.143）和Shannon信息指数（I = 0.226）显著或极显著高于群体水平。在群体水平上,准噶尔—伊犁新疆野杏生态群的Nei's基因多样度和Shannon信息指数（H = 0.131;I = 0.202）高于中亚生态群（南疆栽培杏）（H = 0.127;I = 0.195）、欧洲生态群（H = 0.124;I = 0.189）和华北生态群（H = 0.116;I = 0.180）,但无显著性差异,显著高于中亚李光杏生态群（H = 0.113;I = 0.173）;普通杏4个生态群的遗传分化系数（G_ST = 0.147）显示,普通杏的遗传变异主要存在于群体内,占总变异的85.3％;通过遗传分化系数计算得G_ST基因流N_m = 2.901,说明普通杏4个生态群存在适度的基因交流,人为引种可能是产生基因交流的主要原因,而地理隔离可能是阻碍基因交流的主要因素。对普通杏4个生态群的群体遗传多样性和群体遗传结构的分析初步认为,普通杏起源于准噶尔—伊犁新疆野杏生态群,通过人为驯化,在新疆南部形成栽培杏中心,并形成中亚南疆栽培杏生态群,之后通过人为引种向东传播形成华北生态群,向西传播形成欧洲生态群。     

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.     

基于SSR标记的板栗地方品种遗传多样性与关联分析

采用SSR标记对山东等10个省份95个板栗地方品种的遗传多样性、群体结构进行分析,并进行板栗18个农艺性状与SSR标记关联分析。结果表明：（1）17对SSR引物在95个板栗品种中检测出44个等位位点,平均为2.6个,Shannon’s指数（I）和多态性信息含量（PIC）平均值分别为0.67和0.352,遗传多样性较为丰富;（2）山东群体和江苏群体的多态性位点比率（P）最高,均达到94.12%,观察等位基因数（Na）分别为2.53和2.18,亦高于其他群体;（3）根据Evanno等统计模型,92个板栗品种被划分为3个亚群,分别包含25、40和27个品种,且均有着复杂的地理起源,另有3个品种没有明确的类群归属;（4）利用GLM和MLM模型进行关联分析,并经过假阳性检验,发现叶柄长度和淀粉含量分别与标记CsCAT 5和CsCAT 22显著关联,关联系数分别为0.4027和0.1869。     

SSR fingerprinting Chinese peach cultivars and landraces (Prunus persica) and analysis of their genetic relationships

Thirty-two Chinese peach landraces/cultivars, a major subset of the core Chinese peach collection, were fingerprinted using seven pairs of SSR primers to assess their genetic diversity and relatedness. The seven primer pairs detected eight loci and revealed an allele richness of 3.125 (average alleles per locus), an expected heterozygosity (He) of 0.450, and a Shannon index of 0.728 among the landraces/cultivars. This level of genetic diversity is lower compared to other fruit trees and Prunus congenus species (cherry and apricot), but it is comparable to previous reports in peaches. A greater level of genetic diversity was observed in landraces than in cultivars, indicating that peach landraces are valuable for germplasm collection. All cultivars and landraces, except two, were unambiguously identified based on multi-locus genotypes. Eight unique alleles were detected among this group of Chinese peaches. UPGMA clustering analysis separated the 32 cultivars/landraces into two distinct groups, which is generally in accordance with the known pedigree information. The results provide accurate genetic information for defined acquisition policy in the repositories, improving the integrity and efficiency of germplasm management and giving evidences for protection of breeder's intellectual rights.     

Characterization of variability and genetic similarity of European pear using microsatellite loci developed in apple

Seven microsatellite loci (SSR) developed in apple were used for the identification of 63 European pear cultivars. A total of 46 fragments were amplified, with an average of 6.6 alleles per SSR. Only one microsatellite amplified more than one locus. The mean expected and observed heterozygosities over the six single-locus SSRs averaged 0.68 and 0.44, respectively, and the number of effective alleles per loci was 3.43. The amplified fragments produced 61 different fingerprinting patterns that allowed to unequivocally distinguish all the varieties analyzed. Only two varieties, which derive from mutations, could not be distinguished from the original variety. Cluster analysis of the estimated genetic similarity, grouped the varieties according to their pedigree and their geographic origin. The variability detected with the SSRs in European pear varieties was low when compared with the variability detected in other fruit crops in the Rosaceae.