Genome-wide characterization of graft-transmissible mRNA-coding P450 genes of cucumber(Cucumis sativus L.)

P450(cytochrome P450)is a supergene family,which is involved in various metabolic pathways in plants.Based on previous study,we found some of cucumber P450 mRNAs were systemic mobile in cucumber/pumpkin grafts.However,the reason that why P450 mRNAs were endorsed as signaling,and what specific motif(s)did they harbored is not clear yet.Here,we first identified 221 CsaP450 genes in cucumber genomewide level.Combining with graft-transmissiblemRNAs datasets in cucumber,we elucidated 15mobile-CsaP450-coding genes,of which 5 and 10 belonged to A-type and non-A type respectively.Compared with Arabidopsis and pumpkin(Cucubit moschata)graft-induced-transmissible P450 mRNAs,a phylogenetic treewas constructed and divided into eight clans by usingmultiple-sequence alignment.Gene ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)annotations indicated that the expression patterns of the mobile-mRNA-coding CsaP450 genes in different tissues of cucumber was specifically enriched in oxidoreductase activity and secondary metabolic pathways.The structures and motifs of these 15 mobile-mRNA-coding CsaP450 genes and their types of regulatory elements told that the proportion of CU-rich motifs was higher than nonmobile-mRNA-coding CsaP450 genes.The integrated analysis of mobility direction and mRNA abundance of 15 mobilemRNA-coding CsaP450 genes allowed to conclude that there was rarely relationship between them.The study provided a new insight into the relationship between the motifs and functional characterization of mobile-mRNA-coding P450 genes of cucumber in genome-wide levels.

Telomere-to-telomere genome assembly of melon(Cucumis melo L.var.inodorus)provides a high-quality reference for meta-QTL analysis of important traits

Melon is an important horticultural crop with extensive diversity in many horticultural groups.To explore its genomic diversity,it is necessary to assemble more high-quality complete genomes from different melon accessions.Meanwhile,a large number of QTLs have been mapped in several studies.Integration of the published QTLs onto a complete genome can provide more accurate information for candidate gene cloning.To address these problems,a telomere-to-telomere(T2T)genome of the elite melon landrace Kuizilikjiz(Cucumis melo L.var.inodorus)was de novo assembled and all the published QTLs were projected onto it in this study.The results showed that a high-quality Kuizilikjiz genome with the size of 379.2 Mb and N50 of 31.7 Mb was de novo assembled using the combination of short reads,PacBio high-fidelity long reads,Hi-C data,and a high-density genetic map.Each chromosome contained the centromere and telomeres at both ends.A large number of structural variations were observed between Kuizilikjiz and the other published genomes.A total of 1294 QTLs published in 67 studies were collected and projected onto the T2T genome.Several clustered,co-localized,and overlapped QTLs were determined.Furthermore,20 stable meta-QTLs were identified,which significantly reduced the mapping intervals of the initial QTLs and greatly facilitated identification of the candidate genes.Collectively,the T2T genome assembly together with the numerous projected QTLs will not only broaden the high-quality genome resources but also provide valuable and abundant QTL information for cloning the genes controlling important traits in melon.

Effect of Algae on Melon(Cucumis melo subsp.agrestis var.conomon)Growth and Development under Drought-Stress Conditions

Due to climate change,it is necessary to develop plant varieties that are resilient to climate conditions and resistant to abiotic and biotic stresses.The use of microalgae,which are microorganisms that contain carbohydrates,proteins,lipids,and vitamins,against drought tolerance is a new approach.The aim of the current study was to determine the drought-related mechanisms in the conomon melon genotype and develop drought-tolerant melon cultivars.Morphological,physiological,pomological,and molecular analyses were carried out on the algae-treated genotypes.It has been determined that commercial algae application provides the best results in leaf temperature,leaf relative water contents(LRWC),plant height,fruit length,fruit diameter,and yield,while Cag Cag(a special river in the region)stream algae application gives better results in main stem diameter.It was determined that the number of nodes in the control(without algae)plots was higher than in the other two treatments.Yield and LRWC and plant height values of genotype 7 were the best values among other genotypes.The leaf temperature measurement was lower on genotype 9 than on the other genotypes.While the highest fruit length value was measured in genotype 1,genotype 8 was superior in the main stem diameter,fruit diameter,and the number of nodes among the remaining plant materials.In terms of yield,it was determined that the interaction between genotype 2 and the commercial algae resulted in the best outcomes.In addition,the results of the genetic evaluation revealed that the materials used were not genetically distant from each other and more detailed genetic evaluations are needed.The molecular kinship analysis revealed that the genotypes used in the study were divided into three distinct groups,with individuals within each group exhibiting a high degree of relatedness to one another.As a result of the study,it was found that the application of microalgae had significant effects on improving the drought tolerance of Cucumis melo subsp.agrestis var.conomon genotypes.

连作土灭菌对黄瓜(Cucumis sativus)生长和土壤微生物区系的影响

采用盆栽试验的方法,对黄瓜的生长状况、抗病相关酶及土壤微生物区系特征进行了测定。结果表明:连作土灭菌能在较大程度上改善黄瓜的生长,灭菌土上生长的黄瓜的叶绿素含量、株高、叶面积和鲜重均大于连作土,但随氨基酸肥料(AAF)施用量的增加,灭菌土与连作土之间的差异变小。黄瓜叶片的SOD和POD活性在不施用AAF条件下灭菌土显著高于连作土,然而随着AAF施用量的增加,活性则相反。CAT活性及可溶性蛋白含量灭菌土所有处理均极显著高于相对应的连作土,MDA含量则显著低于相对应的连作土。灭菌土的细菌、真菌数量分别比连作土增加1.89～3.03倍和6.96～2.43倍,放线菌的增加幅度相对较小,真菌/细菌比值也显著提高。随AAF施用量的增加,连作土中的黄瓜枯萎病的致病菌(尖孢镰刀菌)减少,而灭菌土的所有处理均未分离到。土壤细菌的PCR-DGGE图谱分析表明,灭菌土DGGE图谱带明显比连作土增加,土壤间带谱的相似性降低,土壤微生物多样性指数增加。灭菌土上的黄瓜未见枯萎病发生,而连作土的黄瓜发病率达31.25%～68.75%。

外源NO对NaCl胁迫下黄瓜(Cucumis sativus L.)幼苗生长和谷胱甘肽抗氧化酶系统的影响

采用营养液水培,研究了外源一氧化氮(NO)对黄瓜(Cucumis sativusL.)幼苗生长和叶片谷胱甘肽抗氧化酶系统的影响。结果表明,正常生长条件下添加NO能促进黄瓜幼苗生长,而添加NO信号传递途径关键酶鸟苷酸环化酶(cGC)抑制剂亚甲基蓝(MB-1)显著抑制了黄瓜幼苗的生长;添加NO显著缓解了盐胁迫对黄瓜幼苗生长的抑制,提高了叶片谷胱甘肽还原酶(GR)活性、脱氢抗坏血酸还原酶(DHAR)活性、抗坏血酸过氧化物酶(APX)及还原型谷胱甘肽(GSH)、抗坏血酸(ASA)含量,降低了氧化型谷胱甘肽(GSSG)含量,提高了GSH/GSSG,对单脱氢抗坏血酸还原酶(MDAR)活性无显著影响;NaCl胁迫下添加NO的同时添加MB-1抑制了GR活性的提高,GSH和ASA含量、GSH/GSSG均降低,GSSG含量提高,但对MDAR、APX和DHAR活性无显著影响,表明NaCl胁迫下NO对GR活性、GSH和ASA含量、GSH/GSSG的调节可能是通过cGC介导的,对MDAR无明显的调节作用,对DHAR、APX的调节还存在其它途径。

Cucumis属植物种间正反杂交差异的研究

在形态和分子两个水平上对栽培黄瓜 (CucumissativusL .,2n =14)与酸黄瓜 (C .hystrixChakr.,2n =2 4)正反杂交而成的双单倍体 ( 2n =19)、双二倍体 ( 2n =38)植株的形态和育性差异进行了比较研究。结果表明 :在形态上植株的分枝数、第 1朵雌花节位偏向于父性遗传 ,主蔓节间长则偏向于母性遗传 ,另外一些性状如主蔓直径呈中间型。通过RAPD标记分析表明 ,在所选的 2 1条随机引物中 ,15条引物 (占 71%)显示出正反交之间的差异 ;其中一些条带表现出父性遗传现象。在育性上 ,正反交植株的表现截然不同。当以黄瓜为母本时 ,杂种植株呈现雌雄高度不育 ;而以酸黄瓜为母本时 ,雌雄育性在染色体加倍后均得到恢复。

两个黄瓜(Cucumis sativus L.)叶色突变体的比较分析研究

通过对黄瓜黄绿叶突变体9110Gt和NCG-042植株表型观测、遗传分析和分子标记验证,证明9110Gt是区别于NCG-042的新叶色突变体。这两个黄绿叶突变体在表型上存在一定的区别:9110Gt在苗期表现叶色黄化,而NCG-042的心叶在整个生育期都表现黄化。遗传分析证明,两个突变体的叶色突变性状分别由两个不同的等位基因控制,且两基因间存在互补作用。分子标记的检测结果也进一步证实了这一结论。

Cucumis属3种不同倍性种间杂交后代的同工酶分析

以3种不同倍性的Cucum is属种间杂交后代[异源四倍体C.hy tivus(2n=4x=38,HHCC)、异源三倍体(2n=3x=26,HCC)和正反交种间杂种F1(2n=2x=19,HC/CH)]及其双亲为试材,比较研究了过氧化物酶(POD)和酯酶(EST)同工酶在不同器官中的酶谱表达特性.结果表明,花蕾中的POD和EST同工酶谱带都比叶片中的丰富,表现出明显的组织特异性.在相同器官的同一酶系统中,3种不同倍性种间杂交后代的酶谱基本一致,主要表现为互补双亲的酶带,同时出现了双亲所没有的酶带(POD4c和EST3b等),表明远缘杂交扩大了黄瓜的遗传基础.此外,在幼叶和花蕾的POD同工酶中,大部分酶带活性随染色体倍性增加而减弱,表明基因剂量与POD同工酶酶谱的表达呈负相关.

Cucumis属双二倍体种小孢子发生和雄配子体发育的细胞学研究

采用染色体制片技术对Cucumis属双二倍体种 CucumishytivusChenetKirkbride 小孢子发生和雄配子体发育进行了细胞学研究.结果显示:在小孢子发生过程中,约31%的花粉母细胞在减数分裂中期 具有正常的19 ,约69%的花粉母细胞染色体构型复杂,平均构型0.41 +14.69 +0.06 +0.93 +0.62 +0.07 ;在四分体时期,形成约8.78%的四分孢子,其余为各种异常的多分孢子;在雄配子体发育过程中,约10%的小孢子可进行有丝分裂,最终发育为正常的两细胞、三孔花粉,其余90%的小孢子最终成为败育花粉.此外,还观察到了减数分裂后期 的染色体组分离和新种花粉形态的变异等特殊现象.

Cucumis hytivus染色体组间交换重组及其对雄配子育性的影响

采用染色体压片技术对双二倍体种Cucumishytivus染色体组间交换重组及其对雄配子育性的影响进行了细胞学研究。找到了该物种染色体组间交换重组的细胞学证据:包括前期的"8"字形、"十"字形结构和中期环状、链状多价体。研究还发现各种可能导致遗传物质不均衡分离的异常结构如染色体桥、染色体滞后、染色体组分离、微核等。染色体组间广泛的交换和重组导致约93%的多分体及部分异常四分体的形成,多分体中大量的畸形小孢子和四分体中遗传物质不平衡的小孢子因不能正常发育而最终形成败育的雄配子,直接影响到Cucumishytivus育性。

Inheritance of Powdery Mildew Resistance in Cucumber (Cucumis sativus L.) and Development of an AFLP Marker for Resistance Detection

Cucumber powdery mildew is one of the most destructive diseases of cucumber throughout the world. In the present study, inheritance of powdery mildew resistance in three crosses, and linkage of resistance with amplified fragment length polymorphism （AFLP） markers are studied to formulate efficient strategies for breeding cultivars resistant to powdery mildew. The joint analysis of multiple generations and AFLP technique has been applied in this study. The best model is the one with two major genes, additive, dominant, and epistatic effects, plus polygenes with additive, dominant, and epistatic effects （E-l-0 model）. The heritabilities of the major genes varied from 64.26% to 97.82%, and susceptibility was incompletely dominant for the two major genes in the three crosses studied. The additive effects of the two major genes and the dominant effect of the second major gene were high, and the epistatic effect of the additive-dominant between the two major genes was the highest in cross I . In cross II, the absolute value of the additive effect, dominant effect, and potential ratio of the first major gene were far higher than those of the second major gene, and the epistatic effect of the additive-additive was the highest. The genetic parameters of the two major genes in cross III were similar to those in cross II. Correlation and regression analyses showed that marker E25/M63-103 was linked to a susceptible gene controlling powdery mildew resistance. The marker could account for 19.98% of the phenotypic variation. When the marker was tested on a diverse set of 29 cucumber lines, the correlation between phenotype and genotype was not significant, which suggested cultivar specialty of gene expression or different methods of resistance to powdery mildew. The target DNA fragment was 103 bp in length, and only a small part was found to be homologous to DNA in the other species evaluated, which indicated that it was unique to the cucumber genome.

Asynchronous meiosis in Cucumis hystrix–cucumber synthetic tetraploids resulting in low male fertility

Interspecific hybridization and allopolyploidization contribute to the improvement of many important crops. Recently, we successfully developed an amphidiploid from an interspecific cross between cucumber(Cucumis sativus, 2n = 2x = 14) and its relative C. hystrix(2n = 2x = 24) followed by chemical induction of chromosome doubling. The resulting allotetraploid plant was self-pollinated for three generations. The fertility and seed set of the amphidiploid plants were very low. In this study, we investigated the meiotic chromosome behavior in pollen mother cells with the aid of fluorescence in situ hybridization, aiming to identify the reasons for the low fertility and seed set in the amphidiploid plants. Homologous chromosome pairing appeared normal, but chromosome laggards were common, owing primarily to asynchronous meiosis of chromosomes from the two donor genomes. We suggest that asynchronous meiotic rhythm between the two parental genomes is the main reason for the low fertility and low seed set of the C. hystrix–cucumber amphidiploid plants.

Current status of genetic transformation technology developed in cucumber(Cucumis sativus L.)

Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major approach for cucumber, and its frequency has been up to 23%. For example, significantly enhancement of the transformation efficiency of this plant species was achieved from the cotyledon explants of the cultivar Poinsett 76 infected by Agrobacterium strains EHA105 with efficient positive selection system in lots of experiments. This review is to summarize some key factors influencing cucumber regeneration and genetic transformation, including target genes, selection systems and the ways of transgene introduction, and then to put forward some strategies for the increasing of cucumber transformation efficiency. In the future, it is high possible for cucumber to be potential bioreactor to produce vaccine and biomaterials for human beings.

Breeding melon(Cucumis melo)with resistance to powdery mildew and downy mildew

Melon(Cucumis melo L.)production is often restricted by a plethora of pests and diseases,including powdery mildew and downy mildew caused respectively by the fungal species Podosphaera xanthii/Golovinomyces orontii and oomycete species Pseudoperonospora cubensis.Many efforts have been directed on identification of resistant sources by screening(wild)melon germplasm.In the current review,we summarized such efforts from various publications of the last 50 plus years.Resistance to powdery mildew has been identified in 239 melon accessions and downy mildew resistance in 452 accessions of both C.melo and the wild relative species C.figarei.Among the resistance sources,C.melo var.cantalupensis accessions PMR 45,PMR 5,PMR 6,and WMR 29 as well as C.melo var.momordica accessions PI 124111,PI 124112,and PI 414723 have been considered as the most valuable germplasm because multiple resistance genes have been identified from these accessions and are widely used in melon resistance breeding.Further genetic mapping in a number of resistant sources has enabled identification of 25 dominant genes,two recessive genes and seven QTLs conferring powdery mildew resistance,as well as eight dominant genes and 11 QTLs for downy mildew resistances.Based on the reported sequences of associated markers,we anchored physically(many of)these genes and QTLs to chromosomes of the melon cv.DHL92 genome.In addition to presenting a comprehensive overview on powdery mildew and downy mildew resistance in(wild)melon germplasm,we suggest strategies aiming at breeding melon with durable and broad-spectrum resistance to pathogens and pests.

Cytogenetic Analysis of the Primary Amphidiploid Derived from Interspecific Hybridization in Cucumis and Its Selfed Progenies

Studies on the reproduction and cytogenetic characterization of a primary amphidiploid Cucumis species C. hytivus Chen and Kirkbride (2n = 4x = 38) indicated that a more comprehensive cytogenetic analysis of this species and its first selfed progeny would increase its potential utility in cucumber improvement. With tendrils used as source materials for mitotic analysis, chromosome numbers in all selfed progenies were 2n = 38, confirming chromosomal stability in this synthetic amphidiploid species. Detailed meiotic processes were described by comparing the primary and the selfed amphidiploids. Meiotic abnormalities, such as chromosome lagging, unequal separation, chromosome multi-polarization and polyads were observed frequently in all amphidiploid plants except for the selfed no.8, in which meiosis was arrested prior to metaphase Ⅰ. Generally, the frequency of multivalents was higher and the configurations were more complex in the selfed progenies, demonstrating a more extensive genetic exchange between cucumber and C. hystrix Chakr. Genome separation between cucumber and C. hystrix was observed through prophase Ⅰ to anaphase Ⅰ in both generations of the amphidiploids. Consequently, in addition to n = 19, a new gamete with n = 7 was produced, which was confirmed by the chromosome counts 2n = 14 in the backcrossing progenies from cucumber × amphidiploid mating. Fertility varied among the selfed amphidiploid plants. The selfed plant no.1 was found to have an improved fertility (e.g., pollen staining ability 40.8% and 25.6 seeds per fruit) and then was used as source germplasm in further introgression and gene exchange experiments.

Effect of Low Concentration of Yttrium on Physiological Characteristics of Cucumber (Cucumis Sativus L.)

There is no doubt that rare earth elements stand an important position among the essential elements of plant growth and it is long time since they are first used as plant growth promoters. Given their effects on microstructure, most reports are focused on the toxicology rather than promotion. Using cucumis sativus L. (Jin Chun No.5) as experiment material, we try to find out the nutritional effects of low Y3+ concentrations on cucumber seedlings′ leaves. The present paper suggests that the rare earth elements act as micronutrients at low concentrations while they give rise to toxicity at high concentration. Benefits defeat toxicity with concentration ranging from 5 to 25 μmol·L-1. Through careful study, at the Y(NO3)3 concentration of 10 μmol·L-1 the content of chlorophyll as well as the activities of SOD, Cu-Zn SOD and the POD are the highest. It indicates 10 μmol·L-1 is the optimum concentration of yttrium for promoting the cucumber growth.

Cytological Analyses on Development of Male and Female Gametophytes in an Interspecific Hybrid F1 from Cucumis hystrix Chakr. x Cucumis sativus L.

An interspecific hybrid F1 of Cucumis hystrix Chakr. x Cucumis sativus L. （NC4406） was used to establish the developmental sequence and to characterize the male and female gametophytes at cytological level for further understanding of the phylogenic relationship and the mechanism of fertility or sterility in the interspecific hybrid F1 The development of male and female gametophytes was studied through meiotic analysis and paraffin section observation technique, respectively. Meanwhile, the fertility level was assessed through hybrid F1 backcrossing to cultivated cucumber 4406. Variable chromosome configurations were observed in the pollen mother cells （PMCs） of hybrid F1 at metaphase Ⅰ , e.g., univalents, bivalents, trivalents, quadravalents, etc. At anaphase Ⅰ and Ⅱ, chromosome lagging and bridges were frequently observed as well, which led to the formation of polyads and only a partial number of microspores could develop into fertile pollen grains （about 23.3%）. Observations of the paraffin sections showed numerous degenerated and abnormal embryo sacs during the development of female gametophytes, and only 40% of the female gametophytes could develop into normal eight-nuclear megaspore. On an average, 22.8 and 6.3 seeds per fruit could be obtained from the reciprocal backcross. The interspecific hybrid F1 of C. hystrix x NC4406 was partially fertile; however, the meiotic behaviors of hybrid F1 showed a high level of intergenomic recombination between C. hystrix and C. sativus chromosomes, which indicated that it plays an important role for introgression of useful traits from C. hystrix into C. sativus.

Growth promotion of interspecific hybrid embryos between Cucumis anguria and Melon(C. melo) by fruit heating

Wild species of the genus Cucumis, such as C. anguria, are resistant to various melon(C. melo) diseases such as Fusarium wilt race 1.2 and are anticipated as genetic resources of melon. However, in the interspecific crosses between C. anguria and melon, the abortion of the embryos was observed in the early growth stage and could not be regenerated even when cultured. Therefore, for embryo rescue, it is necessary to promote embryo growth during abortion occurrence. This study investigated the effects of fruit heating on embryo growth in interspecific hybrids of C. anguria and melon. In the heating device, the minimum night temperature around the fruit was maintained above 21.5 °C. The ovule grain area of C. anguria × MR-1 with fruit heating was significantly enlarged compared to that without fruit-heating. It was also significantly enlarged compared with the ovule grain area of C. anguria self-pollination. Although, in C. anguria × MR-1 without fruit-heating, no embryo was observed until 10 days after pollination(DAP), and pre-embryos were observed at 13 DAP. Conversely, in C. anguria × MR-1 with fruit-heating, pre-embryos were even observed at 10 DAP. These results suggest that the possibility of embryo or ovule culture regeneration has increased due to embryo growth promotion.

Nodulin 26-like intrinsic protein Cs NIP2;2 is a silicon influx transporter in Cucumis sativus L.

Nodulin 26-like intrinsic proteins(NIPs) are a family of channel-forming transmembrane proteins that function in the transport of water and other small molecules.Some NIPs can mediate silicon transport across plasma membranes and lead to silicon accumulation in plants,which is beneficial for the growth and development of plants.Cucumber is one of the most widely consumed vegetables;however,the functions of NIPs in this crop are still largely unknown.Here,we report the functional characteristics of Cs NIP2;2.It was found that Cs NIP2;2 is a tandem repeat of Cs NIP2;1,which had been demonstrated to be a silicon influx transporter gene.Cs NIP2;2 has a selectivity filter composed of cysteine,serine,glycine and arginine(CSGR),which is different from all previously characterized silicon influx transporters in higher plants at the second helix position.Xenopus laevis oocytes injected with Cs NIP2;2 c RNA demonstrated a higher uptake of silicon than the control,and the uptake remained unchanged under low temperature.Cs NIP2;2 was found to be expressed in the root,stem,lamina and petiole,and exogenous silicon treatment decreased its expression in the stem but not in other tissues.Transient expression of Cs NIP2;2-e GFP fusion sequence in onion epidermal cells showed that Cs NIP2;2 was localized to the cell nucleus,plasma membrane and an unknown structure inside the cell.The results suggest that Cs NIP2;2 is a silicon influx transporter in cucumber,and its subcellular localization and the selectivity filter are different from those of the previously characterized silicon influx transporters in other plants.These findings may be helpful for understanding the functions of NIPs in cucumber plants.

Fine Genetic Mapping of Dwarf Trait in Cucumber(Cucumis sativus L.)Using a RIL Population

Plant height in cucumber is not only an important trait for breeding,but also one of the model traits suitable for the study of developmental biology.Amplified fragment length polymorphism(AFLP)and simple sequence repeat(SSR)markers techniques were applied for the construction of genetic linkage maps in cucumber.To understand the dwarf trait genetic basis in cucumber,the quantitative trait loci(QTL)were identified using F_6 recombinant inbred line populations(RILs)comprising 336 lines from the cross between the two cultivars D0462(the dwarf)and DN129(the vine).In total,six SSR markers and 15 AFLP markers were detected on the five linkage groups covering 152.8 cM with a mean marker interval of 7.28 cM.Only one QTL was found to be linked with plant height and the dwarf trait locus remained in the fourth linkage group.The contributory percentage of the single QTL to plant height was 11.39%.