A comparative study of melting and non-melting flesh peach cultivars reveals that during fruit ripening endo-polygalacturonase (endo-PG) is mainly involved in pericarp textural changes, not in firmness reduction

Peach softening is usually attributed to the dismantling of the cell wall in which endo-polygalacturonase (endo-PG)-catalysed depolymerization of pectins plays a central role. In this study, the hypothesis that the function of endo-PG is critical for achieving a melting flesh fruit texture but not for reducing fruit firmness was tested by comparing pericarp morphology and endo-PG expression and localization in melting (MF) and non-melting flesh (NMF) fruit at successive stages of ripening. MF Bolero, Springbelle, and Springcrest, and NMF Oro-A and Jonia cultivars were analysed. Both MF and NMF fruit were left to ripen on the tree and reached a firmness of <10 Newtons (N). The image analysis of pericarp tissues revealed that during softening the loss of cell turgidity was a process common to mesocarp cells of all MF and NMF fruit and was clearly visible in peaches with a firmness of less than ～20?N. In contrast, the loss of cell adhesion was a feature exclusively observed in ripe MF fruit pericarp. In this ripe fruit, large numbers of endo-PG isoforms were highly expressed and the enzyme localization corresponded to the middle lamella. As a consequence, wide apoplastic spaces characterized the pericarp of ripe MF peaches. In contrast, no loss of cell adhesion was observed in any NMF fruit or in unripe MF peaches. Accordingly, no endo-PG was detected in unripe NMF fruit, whereas few and poorly expressed enzyme isoforms were revealed in ripe NMF and in unripe MF peaches. In this fruit, the poorly expressed endo-PG localized mainly in vesicles within the cytoplasm and inner primary cell wall. On the whole the results suggested that endo-PG function was needed to achieve melting flesh texture, which was characterized by wide apoplastic spaces and partially deflated mesocarp cells. Conversely, endo-PG activity had no critical influence on the reduction of fruit firmness given the capacity of NMF peaches to soften, reaching values of 5-10?N. As in tomato, the change of symplast/apoplast water status seems to be the main process through which peach fruit regulates its firmness.     

基于引物“随机组合”构建观赏桃SSR指纹图谱

近年来,我国观赏桃新品种日渐繁多、名称混乱、市场难以监管,同时用以区分品种的SSR指纹图谱的构建方法在研究界无统一的科学标准,尤其是构成最终引物组合的核心引物的确定,具体操作流程层出不穷、五花八门。为探索筛选SSR指纹图谱核心引物的科学方法,同时构建观赏桃SSR指纹图谱,该研究选用35对已报道的SSR引物对22份观赏桃种质进行试验。结果表明:通过PCR扩增与分析,多态性较高的8对引物——候选引物总共扩增出31个多态性条带,变幅为3~5个,PIC值变幅为0.458~0.668,MI值变幅为1.374~3.340。采用"随机组合"法对8对引物进行C_8~1、C_8~2、C_8~3…依次分析,得到区分能力最强的3种不同的最少引物组合方式——候选组合,并能区分出18份种质,从中发现区分能力最强的3种引物组合方式并不都是由引物PIC值、alleles数量或MI值等多态性指标最高的引物组成,而是由互补性最强的引物组成。选用组合内各引物多态性条带总数最多的组合方式"4-3"(BPPCT001+BPPCT015a+BPPCT017+BPPCT025)为22份观赏桃种质构建了指纹图谱。基于此,通过常规多态性指标筛选候选引物可以确定出单对引物鉴别能力最强的少量引物;通过"随机组合"筛选候选组合可以进一步确定出引物之间互补性最强的几种组合方式;根据组合内各引物的多态性条带总数确定最终核心引物可以确定出可扩容性最大的引物组合。该研究最终建立了候选引物——候选组合——核心引物组合"三步法"确定SSR指纹图谱核心引物组合的科学方法,不仅为22份供试观赏桃种质构建了SSR指纹图谱,也为其它作物SSR指纹图谱的构建提供了新的思路。     

Genetic Relationships of Ornamental Cultivars of Ginkgo biloba Analyzed by AFLP Techniques

Eight primer combinations that produced clear and a large number of polymorphic bands were screened from 64 EcoR I/Mse I primer combinations (Mse I fluorescent labeled). The genetic relationships of 21 ornamental cultivars of Ginkgo biloba L. from the United States of America, Holland, Japan, France, and China were analyzed. These primer combinations produced a total of 1 119 bands, 229 specific loci (including 54 absent bands, and 175 monomorphic bands). Among them, 983 polymorphic bands (PPB), accounting for 88%, were detected. The percentage of identification per primer combination was as high as 100%. The average PPB of 14 foreign cultivars was 35.86% and the average PPB of seven domestic cultivars was 31.51%. Genetic similarity coefficient (SC) among all cultivars varied from 0.4899 to 0.8499, and all cultivars were divided into the four clusters when SC was set at 0.7300. The cultivars from the same origin did not fall into the same group. The cultivars from France and China were classified into three groups. According to the comprehensive analyses based on specific loci, similarity coefficient, and clustering results, eight cultivars ‘Fastigiata’, ‘Tit’, ‘Tubifolia’, ‘Daeryinxing’, ‘Variegata’, ‘Horizontalis, ‘Pendula’, and ‘Yiyuanyeziyinxing’ were considered to be important germplasms of ornamental cultivars of Ginkgo biloba.     

Genome-wide characterization and selection of expressed sequence tag simple sequence repeat primers for optimized marker distribution and reliability in peach

Simple sequence repeats (SSR) in Prunus expressed sequence tags (EST) were mined, and flanking primers designed and used for genome-wide characterization and selection of primers to optimize marker distribution and reliability in peach. A total of 4,770 and 9,029 SSRs were identified from 12,618 contigs and 34,238 singlets, from which 3,695 and 6,849 primers were designed, respectively. Alignment of the 10,544 forward and reverse primer sequences (21,088 queries) against the peach reference genome at 9e-03 resulted in 23,553 hits (96,621 alignments) with 16,885 queries, and “no hits found” (NHF) for the remaining 4,203 queries. A majority of aligned primers had only one hit/alignment on the peach scaffolds, and the distribution of the 5,500 singly aligned primers (pairs) on each 500-kb genome interval was determined. The average number of ESR-SSR primers per 500-kb interval was 10.8. The primers were categorized into eight subgroups based on the difference between the genome amplicon size and expressed amplicon size of each primer, with 288 primers of optimized distribution and reliability selected for genotype evaluation. Only 2 of the 288 primers failed in all 4 peach cultivars screened, with an overall successful primer/sample rate of 97.2 %. The average number of alleles detected in the four cultivars was 3.84. The polymorphism information content (PIC) values suggested that a majority of the 288 primers had a high rate of allele polymorphism among the four peach cultivars. The advantages of genome-wide analysis of EST-SSR primers and options to improve the polymorphism rate are discussed.     

Peach fruit ripening: A proteomic comparative analysis of the mesocarp of two cultivars with different flesh firmness at two ripening stages

A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the “unripe” to the “ripe” phase.The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; ‘Oro A’) and a melting flesh (MF; ‘Bolero’) cultivar, at “unripe” and “ripe” stages as defined by some parameters typical of ripening, were then analyzed.The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage (“unripe” versus “ripe”) and/or the genetic background of the cultivar (‘Oro A’ versus ‘Bolero’).Thirty out of the 53 differently abundant spots were identified by LC-ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress.Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric (“unripe”) to the climacteric (“ripe”) phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status.The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics.     

Molecular Characterization of Phytoplasmas Associated with Peach Diseases in Iran

Recently, peach trees showing leaf rolling, little leaf, rosetting, yellowing, bronzing of foliage and tattered and shot‐holed leaves symptoms were observed in peach growing areas in the central and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR using phytoplasma universal primer pairs P1/Tint, R16F2/R2, PA2F/R and NPA2F/R were employed to detect phytoplasmas. The nested PCR assays detected phytoplasma infections in 51% of symptomatic peach trees in the major peach production areas in East Azerbaijan, Isfahan, ChaharMahal‐O‐Bakhtiari and Tehran provinces. Restriction fragment length polymorphism (RFLP) analyses of 485 bp fragments amplified using primer pair NPA2F/R in nested PCR revealed that the phytoplasmas associated with infected peaches were genetically different and they were distinct from phytoplasmas that have been associated with peach and almond witches’‐broom diseases in the south of Iran. Sequence analyses of partial 16S rDNA and 16S–23S rDNA intergenic spacer regions demonstrated that ‘Candidatus Phytoplasma aurantifolia’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ are prevalent in peach growing areas in the central and north‐western regions of Iran.     

Development of retrotransposon primers and their utilization for germplasm identification in Diospyros spp. (Ebenaceae)

Retrotransposons play an important role in plant genetic instability and genome evolution. Retrotransposon-based molecular markers are valuable tools to reveal the behavior of retrotransposons in their host genome. In this study, suppression polymerase chain reaction was used, for the first time, to develop retrotransposon long terminal repeat (LTR) and polypurine tract (PPT) primers in Japanese persimmon (Diospyros kaki Thunb.), which were then employed for germplasm identification by means of interretrotransposon-amplified polymorphism (IRAP), sequence-specific amplified polymorphism (SSAP) and retrotransposon-microsatellite-amplified polymorphism (REMAP) molecular markers. The results showed that 16 out of 26 primers produced expected amplifications and abundant polymorphisms by IRAP in 28 genotypes of Diospyros. Moreover, some of these primers were further successfully used in REMAP and SSAP analysis. Each type of molecular markers produced unique fingerprint in 28 genotypes analyzed. Among the primers/primer combinations, two IRAP primers and four SSAP primer combinations could discriminate all of the germplasm solely. Further comparative analysis indicated that IRAP was the most sensitive marker system for detecting variability. High level of retrotransposon insertion polymorphisms between bud sports were detected by IRAP and SSAP, and the primers/primer combinations with powerful discrimination capacity for two pairs of bud sports lines were further obtained. Additionally, possible genetic relationships between several Japanese persimmon were discussed. To our knowledge, this is the first report on the development of retrotransposon LTR and PPT primers in Diospyros, and the retrotransposon primers developed herein might open new avenue for research in the future.     

利用电子鼻评价桃果实香气

为了明确不同桃品种资源果实香气差异,对桃果实香气评价和品质改良提供参考,本研究利用电子鼻系统对桃品种资源果实整果香气进行测定和区分.通过PEN 3.5电子鼻系统采集74份不同品种资源桃果实芳香成分并得到了不同传感器的响应值,采用主成分(PCA)、线性判别法(LDA)和负荷加载(LO)方法分析数据.LO分析结果显示,硫化...     

银杏观赏品种遗传关系的AFLP分析

从64对EcoRⅠ／MseⅠ引物（其中MseⅠ引物为荧光标记物）中筛选出8对扩增产物多态性高、谱带清晰的引物,对来自美国、荷兰、日本、法国和我国的21个银杏观赏品种的遗传关系进行了研究。结果表明：8对引物共产生1117条谱带,229个特异位点（其中缺失带54条、单态带175条）,多态带983条,多态带的比例为88％;每对引物鉴别效率为100％。14个国外银杏观赏品种平均多态带的比例35．86％,7个国内品种平均多态带的比例31．51％。21个观赏品种之间相似系数为0．4899-0．8499。当相似系数为0．7300时,供试观赏品种可分为四类,来源地相同的品种并不单独聚成一类,中国和法国的品种分别属于其中的三类。根据对观赏品种的特异位点、相似系数、聚类结果等进行综合分析表明,‘塔形银杏’、‘垂乳银杏’、‘筒叶银杏’、‘大耳银杏’、‘斑叶银杏’、‘展冠银杏’、‘垂枝银杏’、‘沂源叶籽银杏’这8个品种是银杏观赏品种中的重要特异种质。     

八个贵州地方桃品种果实甜酸风味品质分析

为了评价贵州地方桃品种果实的甜酸风味品质,以主栽桃品种‘燕红’作对照,采用高效液相色谱法测定了贵州8个地方桃品种的果肉糖酸组分及含量。结果表明:(1)8个地方桃品种果肉中糖主要由蔗糖、葡萄糖、果糖和山梨醇组成,其中蔗糖的平均含量最高(55.62 mg/g),约占总糖的71.30%,但变异系数仅为17.92%;8个地方桃品种中,‘米桃’的葡萄糖与果糖含量差异较大,其果糖/葡萄糖的比值为1.21,而其它7个品种的葡萄糖与果糖含量相近。(2)8个地方桃品种果肉中有机酸主要由苹果酸、柠檬酸、奎宁酸和莽草酸组成,其中苹果酸含量最高,约占总酸的60.61%,但在‘白花桃’果实中有机酸含量以奎宁酸为主。(3)对8个地方桃品种果实的糖酸组分进行主成分分析发现,苹果酸含量和山梨醇含量的载荷系数分别为0.910和0.897,说明它们是影响果实甜酸风味的主导因素,且对改善果实的甜酸风味品质具有重要作用。8个贵州地方桃果实的甜酸风味分别为:‘黄腊桃’为甜,‘血桃’、‘青桃’、‘镇远桃’、‘红枫桃’为酸甜,‘白花桃’、‘西桃’和‘米桃’为酸。     

Establishment of AFLP technique and assessment of primer combinations for mei flower

Mei flower is one of the most famous ornamental flowers in eastern Asia for its blossoming in early spring. Amplified fragment length polymorphism (AFLP) is one of the most frequently used techniques for analysis of genetic variation and is used herein for the first time inPrunus mume. This research provides a detailed and modified AFLP protocol for Mei genomic DNA digested withEcoRI/PstI restriction endonuclease combinations. The 10 best primer pairs of high polymorphism were screened from 256 primer combinations that could reliably and repetitively distinguish 14 Mei samples and would be suitable for genetic analysis of more cultivars. Ten primer pairs produced up to a total of 524 AFLP bands and up to 233 polymorphic bands. The ratio of polymorphic bands scoped from 35.71% to 59.67%, and the average ratio was 44.46% in the 10 primers. AFLP is an effective, inexpensive, and timesaving technique for the genetic differentiation of the Mei cultivars, as evidenced in this study.     

Development and characterization of polymorphic microsatellites from Prunus avium‘Napoleon’

Primers were developed for 21 microsatellite loci isolated by enrichment from Prunus avium‘Napoleon’. Twelve loci contained uninterrupted dinucleotide repeats and nine were more complex. Nineteen primer pairs (EMPA001–019) showed single locus polymorphisms in a cultivar survey of 14 sweet cherries, with two to seven alleles per locus. Three primer pairs in combination (EMPA014, 015 and 018) discriminated all cultivars. Two primer pairs for loci monomorphic in P. avium were included: EMPA020 revealed segregation in an interspecific progeny and EMPA021 revealed polymorphism in P. dulcis. Twelve primer pairs reliably amplified products in three peach cultivars of which seven revealed polymorphisms.     

Two forms of exopolygalacturonase increase as peach fruits ripen

Abstract. Freestone peach cultivars are distinguished from clingstone cultivars by a more extensive softening of the mesocarp tissue, and by the separation of mesocarp and endocarp during ripening. Cultivars of both types have been reported to develop exopolygalacturonase activity during ripening, but the enzyme has not been characterized in any detail. During development of freestone peaches ( Prunus persica L. var Coronet), two exopolygalacturonase enzymes were detected 42, 65 and 85 d after full bloom and in ripe fruit. During ripening one enzyme (exoPG 1) increased 36-fold and the other (exoPG 2) 90-fold but exoPG 2 accounted for a 73% of the total activity in ripe fruit. ExoPG 1 was purified 24-fold and exoPG 2 540-fold. ExoPG 2 is a slightly acidic glycoprotein. ExoPG 1 and exoPG 2 differ slightly in their pH optima and in their responses to calcium: each produces monogalacturonic acid as a reaction product. Similar enzymes were found in Flavorerest, a semi-freestone peach.     

Population structure and marker–trait associations for pomological traits in peach and nectarine cultivars

Marker–trait associations based on populations from controlled crosses have been established in peach using markers mapped on the peach consensus map. In this study, we explored the utility of unstructured populations for association mapping to determine useful marker–trait associations in peach/nectarine cultivars. We used 94 peach cultivars representing local Spanish and modern cultivars from international breeding programs that are maintained at the Experimental Station of Aula Dei, Spain. This collection was characterized for pomological traits and was screened with 40 SSR markers that span the peach genome. Population structure analysis using STRUCTURE software identified two subpopulations, the local and modern cultivars, with admixture within both groups. The local Spanish cultivars were somewhat less diverse than modern cultivars. Marker–trait associations were determined in TASSEL with and without modelling coefficient of membership (Q) values as covariates. The results showed significant associations with pomological traits. We chose three markers on LG4 because of their proximity to the endoPG locus (freestone–melting flesh) that strongly affects pomological traits. Two genotypes of BPPCT015 marker showed significant associations with harvest date, flavonoids and sorbitol. Also, two genotypes of CPPCT028 showed associations with harvest date, total phenolics, RAC, and total sugars. Finally, two genotypes of endoPG1 showed associations with flesh firmness and total sugars. The analysis of linkage disequilibrium (LD) revealed a high level of LD up to 20 cM, and decay at farther distances. Therefore, association mapping could be a powerful tool for identifying marker–trait associations and would be useful for marker-assisted selection in peach breeding.     

Simple sequence repeat (SSR) analysis for assessment of genetic variability in apricot germplasm

Thirty SSR primer combinations, developed from peach SSR-enriched genomic libraries and BAC libraries of peach [ Prunus persica (L.) Batsch.], were tested for cross amplification with 74 apricot ( Prunus armeniaca L.) germplasm accessions. Twelve primer pairs amplified 14 polymorphic SSR loci useful for discriminating most apricot cultivars, as well as for investigating patterns of variation in apricot germplasm. Levels of polymorphism were higher than the levels described using other codominant marker systems (i.e., isozymes, RFLP markers). Overall, 107 alleles were identified, and all but 11 accessions were unambiguously discriminated. Genetic differentiation of native germplasm into traditional ecogeographical groups was low, with a high level of genetic identity (> 0.75) between the groups. However, neighbor joining cluster analysis of marker distances between cultivars reflected the complex history of apricot domestication, producing groupings not evidently based on the geographical origin of the cultivars. Distant positioning of Chinese cultivars on UPGMA and neighbor joining dendrograms supports the authors' consideration of Chinese apricots as subspecies, Prunus armeniaca var. ansu Maxim., rather than a separate species.     

部分两用桃品种(系)指纹图谱的建立

以近亲个体'顺10-16'、'青北10-7'和'贺春'为材料建立了两用桃AFLP研究体系,从64对引物组合中筛选出了E-AAT/M-ACT、E-AAT/M-CTG、E-ACA/M-CTG和E-ACA/M-CTT等4个多态性好、分辨率高的引物组合;应用该体系对'锦春'等23个两用桃品种(系)进行AFLP分析,结果共扩增出127条带,其中多态性带62条,多态性百分率48.8%,以其中特异性较高的23个多态性条带构建了这23个两用桃品系的指纹图谱,为两用桃品种鉴定及保护奠定了基础.     

Studies on Paeonia cultivars and hybrids identification based on SRAP analysis

Plants of Paeonia are valuable for their ornamental and medicinal values. Genetic relations and hybrids identification among different sections of Paeonia were studied using sequence related amplified polymorphism (SRAP) markers. A total of 29 cultivars including 2 intersectional hybrids, 13 sect. Moutan and 14 from sect. Paeonia were used. A total of 197 bands were produced using 24 primer combinations, among which 187 bands showed polymorphism. From the bands amplified, we can identify the peony cultivars using unique SRAP markers and specific primer combinations. Fourteen peony cultivars were distinguished among each other by using totally 35 SRAP markers, which were generated by 16 primer pairs. Two specific primer pairs of Me8/Em8 and Me8/Em1 can be used to identify cultivars from different sections. The mean genetic similarity coefficient (GS), the gene diversity (GD), and the Shannon's information index of peony cultivars were 0.45, 0.19 and 0.32, respectively. Both UPGMA (unweighted pair-group method of arithmetic average) dendrogram and PCA (principle component analysis) analysis showed clear genetic relationships among the 29 peony cultivars, and within section and its intersectional hybrids. The above results are valuable for estimating and analyzing genetic background of Paeonia, parent selection in crossing breeding programs, molecular marker assisted selection (MAS) breeding for further germplasm innovation programs.