采用SSR和RAPD标记研究黄瓜属（葫芦科）的系统发育关系

野黄瓜Cucumis hystrix（2n=24）是在亚洲发现的第一个染色体基数为12的黄瓜属物种。这一发现对现行的以染色体基数和地理分布为基础的黄瓜属分类系统提出了质疑。采用SSR和RAPD两种分子标记对黄瓜属22份不同类型材料的亲缘关系进行了研究。结果表明,野黄瓜C.hystrix与黄瓜C.sativus var.sativus（2n=14）间的遗传距离（SSR:0.59,RAPD:0.57）小于其与甜瓜C.melo var.melo（2n=24）间的距离（SSR:0.87,RAPD:0.70）。SS     

甜瓜属种间杂交线粒体DNA的遗传分析

为了研究甜瓜属种间杂交线粒体DNA的遗传规律,对甜瓜属人工杂交合成的异源四倍体新种(Cucumis hytivus Chen and Kirkbride.)的S5后代及其杂交母本甜瓜属野生种（Cucumis hystrix Chakr.）和父本栽培种黄瓜‘北京截头’(Cucumis sativus L.)线粒体中apocytochrome b (cob), NADH dehydrogenase subunit 1 (nad 1), NADH dehydrogenase subunit 7(nad 7) 基因序列片断进行了测序与分析。结果显示3个种长度为909 bp cob, 943 bp nad 1和880 bp nad 7基因片断序列中分别存在着相应的1、7和17个多态性核苷酸位点,其中新种与父本‘北京截头’相同而与母本野生种不同的多态性碱基位点分别有1、6和14个,nad 1和nad 7中分别只有1和3个多态性位点与双亲都不相同。该结果表明甜瓜属hystrix譻ativus种间杂交后代线粒体DNA多态性碱基位点主要来源于父本而不是母本,线粒体DNA主要表现为父系遗传。     

Linkage map of Cucumis melo including phenotypic traits and sequence-characterized genes.

A new linkage map of Cucumis melo, derived from the F2 progeny of a cross between PI 414723 and C. melo 'TopMark' is presented. The map spans a total of 1421 cM and includes 179 points consisting of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSRs), simple sequence repeats (SSRs), and restriction fragment length polymorphism (RFLP) markers. The map also includes an aphid resistance trait (Vat) and the sex type gene, andromonoecious (a), the two of which are important in resistance breeding and the control of hybrid seed production, as well as a seed-color gene, Wt-2. Most RFLPs represent sequence-characterized cDNA probes from C. melo and Cucumis sativus. These include resistance gene homologues and genes involved in various aspects of plant development and metabolism. A sub-set of our SSR and RFLP markers were also mapped, as part of this study, on additional mapping populations that were published for this species. This provides important reference points ("anchors"), enabling us to identify several linkage groups with respect to other melon maps.     

Reproduction and cytogenetic characterization of interspecific hybrids derived from Cucumis hystrix Chakr. x Cucumis sativus L

Interspecific hybrids between Cucumis hystrix Chakr. (2n = 2 x = 24) and Cucumis sativus L. (2n = 2 x = 14) were produced by means of F(1) (2n = 19) embryo rescue and subsequent chromosome doubling. The hybridity was confirmed by genomic in situ hybridization (GISH) and chromosome analysis. The amphidiploid (2n = 38) was self-pollinated and backcrossed to cucumber resulting in lines with improved crossability to C. sativus. Examination of shape, stainability, and germination rate of pollen grains and yield as a function of mature fruit set per ten pollinated flowers indicated a tendency for increased fertility in BC(1)S(1) progeny when compared to F(1) and amphidiploid offspring. Cytogenetic characterization of F(1) and amphidiploid progeny was performed. Generally normal meioses produced viable pollen grains, and fertilization resulted in partial fertility restoration in amphidiploid progeny. Chromosome anomalies such as "frying-pan trivalent", chromosome lagging and spindle mis-orientation were also observed. In most of the PMCs of the F(1) diploid hybrid progeny, 19 univalents were observed at diakinesis and MI. In the amphidiploid, more than 90% of the configurations at MI consisted of the predicted 19 bivalents and less than 5% contained multivalents [trivalents (2.3%) + quadrivalents (0.3%)], suggesting the presence of preferential pairing, and a distinctive parental genome as well. The chiasmata observed between homoeologous chromosomes further demonstrated the introgression of the C. hystrix genome into that of C. sativus.     

Distinction between Nuclear Satellite DNAs and Chloroplast DNA in Higher Plants

Triton X-100 solubilized chloroplast DNA but not nuclear DNA from a mixture of chloroplasts and nuclei. The buoyant density of chloroplast DNA was different from that of the satellite DNA in all of the species examined (Phaseolus coccineus, Cucumis sativus, Cucumis melo, Antirrhinum majus, Vicia faba, Oenothera fruiticosa youngii). Chloroplast DNA constituted between 4.3% and 0.25% of the total leaf DNA in these species, and was present as 5 to 20 copies in each chloroplast.     

甜瓜属植物种间杂交研究进展

在甜瓜属野生种Cucumis hystrix Chakr. 和栽培黄瓜之间的种间杂交取得成功的基础上,简要回顾了甜瓜属植物种质资源和系统学的研究情况,然后从甜瓜属植物种间杂交的概况、种间杂交的障碍及其解决途径等三个方面,回顾和讨论了甜瓜属植物的种间杂交的研究进展,并对进一步通过种间杂交研究利用甜瓜属野生植物资源的前景进行了展望。     

野黄瓜与栽培黄瓜正反交异源四倍体序列消除的初步研究

不同分类群的异源多倍体在二倍化过程中, 正反交序列消除往往表现出不同特征, 暗示了在不同物种中, 核质互作在多倍体进化过程的作用不同。利用13对EcoRI-NN/MseI-NNN选择性引物, 对野黄瓜Cucumis hystrix (2n=24)与栽培黄瓜C. sativus (2n=14)的正反交F1、异源四倍体及二倍体亲本DNA进行AFLP分析。结果表明: 杂交后代基因组的杂合性诱发了F1与异源四倍体广泛的序列消除; 细胞质可能会影响部分亲本序列消除的频率, 但是正反交在序列消除频率上差异不显著, 并且在序列消除时间(均始于F1代)及消除类型上也表现出一致性, 表明核质互作并不是影响序列消除的主要因素; 实验还发现, 正反交不能影响序列的倾向性丢失, 染色体数少的黄瓜条带易发生丢失。     

Comparison of the distribution of the repetitive DNA sequences in three variants of Cucumis sativus reveals their phylogenetic relationships

Repetitive DNA sequences with variability in copy number or/and sequence polymorphism can be employed as useful molecular markers to study phylogenetics and identify species/chromosomes when combined with fluorescence in situ hybridization (FISH). Cucumis sativus has three variants, Cucumis sativus L. var. sativus, Cucumis sativus L. var. hardwickii and Cucumis sativus L. var. xishuangbannesis. The phylogenetics among these three variants has not been well explored using cytological landmarks. Here, we concentrate on the organization and distribution of highly repetitive DNA sequences in cucumbers, with emphasis on the differences between cultivar and wild cucumber. The diversity of chromosomal karyotypes in cucumber and its relatives was detected in our study. Thereby, sequential FISH with three sets of multi-probe cocktails (combined repetitive DNA with chromosome-specific fosmid clones as probes) were conducted on the same metaphase cell, which helped us to simultaneously identify each of the 7 metaphase chromosomes of wild cucumber C. sativus var. hardwickii. A standardized karyotype of somatic metaphase chromosomes was constructed. Our data also indicated that the relationship between cultivar cucumber and C. s.var. xishuangbannesis was closer than that of C. s. var. xishuangbannesis and C. s. var. hardwickii.     

黄瓜单性花决定基因M的表达分析

植物成花性型决定过程涉及多种机制, 而单一基因位点控制非完全花(单性花)的发育为葫芦科植物所特有, 其中以黄瓜(Cucumis sativus L.)和甜瓜(C. melo L.)研究最深入。文章利用两对黄瓜近等基因系材料, 结合定量PCR技术, 研究黄瓜单性花决定基因M(CsACS2, GenBank登陆号: FJ529216)的表达情况。利用药剂(AgNO3和艾维激素)处理不同基因型黄瓜材料, 研究在内源乙烯作用被干扰的情况下CsACS2基因的表达变化。结果表明: 内源乙烯可以诱导CsACS2基因的表达。在正常生长条件下, 这种内源乙烯可能来自CsACS1G基因(F基因)或CsACS2基因(M基因)本身, 而后一种情况可能涉及一种正回馈激活机制。     

栽培黄瓜与野黄瓜正反杂交的几种同工酶分析

运用天冬氨酸转氨酶(AAT)、苹果酸脱氢酶(MDH)以及酯酶(EST)3种同工酶对栽培黄瓜"长春密刺"Cucumis sativus cv. Changchunmici (2n=14)与野黄瓜C. hystrix (2n=24)的正反交种间杂种F1 (正交: 野黄瓜×栽培黄瓜"长春密刺", 反交: 栽培黄瓜"长春密刺"×野黄瓜)及其双亲进行鉴定和比较研究。结果表明: 正反交种间杂种F1主要表现为双亲酶带的互补, 同时还形成4个杂合带(Aat-1-94、Aat-2-104、Mdh-3-102和Est-5-102)。上述3种酶均能准确地鉴定种间杂种的真实性。研究还发现正反交杂种F1的AAT和MDH的酶谱分别在酶带数目和强弱上表现出一定的差异, 进一步证实了野黄瓜与栽培黄瓜杂交存在正反交差异。     

Comparison of the distribution of the repetitive DNA sequences in three variants of Cucumis sativus reveals their phylogenetic relationships

Repetitive DNA sequences with variability in copy number or/and sequence polymorphism can be employed as useful molecular markers to study phylogenetics and identify species/chromosomes when combined with fluorescence in situ hybridization(FISH).Cucumis sativus has three variants,Cucumis sativus L.var.sativus,Cucumis sativus L.var.hardwickii and Cucumis sativus L.var.xishuangbannesis.The phylogenetics among these three variants has not been well explored using cytological landmarks.Here,we concentrate on...     

Variability and phylogenetic relationships of the Cucumis sativus L. species inferred from NBS profiling and RAPD analysis

Genetic variability of the Cucumis sativus species and its phylogenetic relationsips with other species of the genus were studied on the basis of RAPD marking and analysis of intra- and interspecific polymorphism of the nucleotide sequences of the NBS-LRR gene family in species of the genus Cucumis with the use of the NBS profiling method. According to RAPD analysis, cucumber cultivars from different geographic regions are highly similar, except for accessions k-3835 and k-3833 from Afghanistan. NBS profiling analysis revealed phylogenetically most distinct accessions expected to be characterized by specificity of resistance: k-3845 from Uzbekistan, k-3851 from Kyrgyzstan, line 701, k-3835 and k-3833 from Afghanistan, k-2757 and k-3079 from Netherlands, vr.k. 908 from Canada, k-2926 from Bulgaria, Russian cultivars Monastyrskii, Izyashchnyi, and Lel'. Three essentially different groups of species were distinguished, and the C. sativus species (subgenus Cucumis) was found to be distant from the species belonging to the subgenus Melo.     

Isolation and identification of Triticeae chromosome 1 receptor-like kinase genes (Lrk10) from diploid, tetraploid, and hexaploid species of the genus Avena.

The DNA sequence of an extracellular (EXC) domain of an oat (Avena sativa L.) receptor-like kinase (ALrk10) gene was amplified from 23 accessions of 15 Avena species (6 diploid, 6 tetraploid, and 3 hexaploid). Primers were designed from one partial oat ALrk10 clone that had been used to map the gene in hexaploid oat to linkage groups syntenic to Triticeae chromosome 1 and 3. Cluster (phylogenetic) analyses showed that all of the oat DNA sequences amplified with these primers are orthologous to the wheat and barley sequences that are located on chromosome 1 of the Triticeae species. Triticeae chromosome 3 Lrk10 sequences were not amplified using these primers. Cluster analyses provided evidence for multiple copies at a locus. The analysis divided the ALrk EXC sequences into two groups, one of which included AA and AABB genome species and the other CC, AACC, and CCCC genome species. Both groups of sequences were found in hexaploid AACCDD genome species, but not in all accessions. The C genome group was divided into 3 subgroups: (i) the CC diploids and the perennial autotetraploid, Avena macrostachya (this supports other evidence for the presence of the C in this autotetraploid species); (ii) a sequence from Avena maroccana and Avena murphyi and several sequences from different accessions of A. sativa; and (iii) A. murphyi and sequences from A. sativa and Avena sterilis. This suggests a possible polyphyletic origin for A. sativa from the AACC progenitor tetraploids or an origin from a progenitor of the AACC tetraploids. The sequences of the A genome group were not as clearly divided into subgroups. Although a group of sequences from the accession 'SunII' and a sequence from line Pg3, are clearly different from the others, the A genome diploid sequences were interspersed with tetraploid and hexaploid sequences.     

甜瓜STK类R基因同源序列的克隆与分析

以植物丝氨酸/苏氨酸蛋白激酶类( serine-threonine kinase,STK)抗病基因产物催化结构域I和Ⅸ的保守氨基酸序列( FGK/V/L/SVYK/RG,DY/IYSF/YGV/I/M)设计简并引物,对甜瓜(Cucumis melo L.)基因组DNA进行PCR扩增,得到大约500 bp的目的条带,通过重组质粒克隆并经PCR检测后得到12条不同的DNA序列,命名为tg1～tg12,其中tg2、tg5、tg9和tg12(Genbank登录号为JN646853 ～JN646856)可以编码完整的氨基酸序列.Blast分析结果显示:4条序列均具有ATP结合部位、底物结合部位和激酶结构域的活化环(A-loop)等,属于典型的蛋白激酶基因家族,可能是STK类R基因的同源序列片段;4条序列与蓖麻(Ricinus communisL.)的STK同源性均较高.氨基酸序列比对结果显示tg2、tg5、tg9和tg12均具有R基因的9个保守结构域,为STK类候选抗病基因类序列.分子系统树显示tg2、tg5、tg9和tg12与已知的R基因(Pto、Lr10和Lectin)在氨基酸水平上的相似性仅为33.5％ ～53.4％,且4个甜瓜同源序列的氨基酸相似性也较低,表明甜瓜RGAs标记可能具有较高的特异性.     

Analysis of cytoplasmic variation in a cucumber germplasm collection using chloroplast microsatellite markers

Nine PCR-based markers were developed from the microsatellites in non-coding regions of chloroplast genome of Cucumis sativus and used to detect chloroplast DNA variation. These markers successfully detected intraspecific polymorphism among 37 cucumber accessions containing Chinese native germplasms (CNGs) and non-Chinese germplasms (NCGs). Each marker detected between two and four alleles and the diversity value of the makers ranged from 0.105 to 0.528. Based on the data from allele size variation, a total of 17 distinct haplotypes were identified from the 35 accessions (excluding the two accessions possessing null genes). Three haplotypes were prevalent among CNGs but most NCGs had unique haplotype. No identical haplotype was found between CNGs and NCGs, reflecting lack of exchange of CNGs with others in the 60–80s of last century. A wild species (C. hystrix Chakr.) tested herein shared a haplotype with some CNGs, suggesting that it could be the ancestry of C. sativus or at least had a common ancestral lineage. The genetic relationship among the 37 cucumber accessions was further analyzed through construction of dendrogram based on Jaccard coefficient of similarity obtained from the allele sizes. All the CNGs were clustered into a group (containing the wild accession) that distinctly differed from the other four groups containing NCGs. This result agreed with the findings above obtained from haplotype analysis. Our research documented here will offer useful information for cucumber breeding.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

The potential of Lagenaria rootstock to confer resistance to the carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychidae) in Cucurbitaceae

Antibiosis and resistance of six Cucurbita and two Lagenaria accessions to the carmine spider mite, Tetranychus cinnabarinus Boisduval, were evaluated in the laboratory. Significant differences among accessions were observed three days after the inoculation of detached leaf discs. The Lagenaria accessions, Slawi and Sus, proved to be the most resistant to mites, with average populations of mite eggs, 87 and 95%, respectively less than that of the susceptible C. pepo accession, Orangetti. The Cucurbita accessions, Tace, Brava, Tetsukabuto, Phoenix and TZ-148 had mite egg totals 4, 9, 13, 26 and 40%, respectively, less than those of accession Orangetti. The Sus accession of Lagenaria was resistant to T. cinnabarinus from the four-leaf stage until fruit set in laboratory and field tests. Grafting the susceptible Brava onto Sus rootstock increased the resistance of the scion to the same level as that of non-grafted Sus. Grafting the susceptible Cucumis melo Noy Yizre'el on resistant or susceptible rootstocks of Cucurbita and Lagenaria accessions did not affect its susceptibility to T. cinnabarinus. The results indicate that resistance to T. cinnabarinus can be transferred by grafting from Lagenaria stocks to Cucurbita scions but not in the opposite direction.     

RAPD and organelle specific PCR re-affirms taxonomic relationships within the genus Coffea

The phenetic relationships between 18 Coffea accessions representing 11 of the most important Coffea species employed in current breeding programmes were examined using RAPD markers and chloroplast and mitochondrial genome specific sequence tagged sites (STS). Estimates of variability based on the number of shared RAPD amplification products placed the species into three distinct groups which were consistent with derived chloroplast DNA phenotypes, the geographical origins of the species and previous studies based on morphological characteristics and RFLPs. C. eugenioides (2n = 2x = 22) exhibited the greatest similarity to the cultivated C. arabica (2n = 4x = 44) and may represent its maternal progenitor. The results are discussed in the context of strategies for Coffea improvement.