Androgenetic plants of Anemone coronaria derived through anther culture

The genus Anemone (Ranunculaceae) includes many species cultivated for ornamental purposes. Most cut flower cultivars belong to A. coronaria L. and are multiplied by seed and sold for cultivation as 1‐year‐old tubers. As cultivars represent a population of hybrid individuals derived from crosses between heterozygous parents, the use of a true F₁ hybrid would improve the uniformity and quality of the product. As a first step towards the development of pure‐breeding lines, anther cultures were established from elite cultivars of A. coronaria. Somatic embryos and plantlets were regenerated from five elite cultivars, and up to 16.9 regenerants per 100 cultured anthers were obtained. Cytological analysis identified that 11 of 19 regenerants had either a 2x = 16 karyotype, or were mixoploids. RAPD‐based DNA fingerprinting showed that all the regenerants tested differed genetically from their anther donor, confirming their androgenetic origin. The shortening to 15 months for the time required to produce homozygous lines may convince seed companies to invest in F₁ hybrid breeding.     

Evaluation of variability in Striga aspera, Striga hermonthica and their hybrids using morphological characters and random amplified polymorphic DNA markers

Striga aspera and Striga hermonthica are recognized as separate species, but their close morphological similarity causes difficulty in distinguishing between them in areas where they coexist in Africa. In this study, crosses between the species were made using randomly selected morphologically typical parental plants collected from different locations in Nigeria. Genetic analysis of both species and their reciprocal F₁ hybrids were determined using cluster analysis of DNA profiles derived from genetic polymorphism (RAPD)-polymerase chain reaction (PCR) markers. Principal component and hierarchical cluster analyses were used to separate parental and hybrid populations based on 13 morphological characteristics. Morphological data from wild samples of both species were compared with the hand-pollinated parental, F₁ and F₂ hybrids, and back-crosses. Results showed that S. aspera and S. hermonthica were genetically and morphologically distinct. Morphological and genetic analyses revealed two major clusters: a S. aspera cluster and a S. hermonthica cluster. Genetically, the F₁ hybrids showed closer affinity to their maternal parents, while morphologically, the F₁ hybrids formed distinct clusters intermediate to the parents. Most F₂ plants and back-crosses were morphologically similar to S. hermonthica . Comparative morphological analysis of wild and hand-pollinated populations showed some samples from the wild clustered with the hybrids, suggesting that hybrids may exist in nature.     

Development of Strain-specific Primers for a Strain of Gliocladium catenulatum Used in Biological Control

The randomly amplified polymorphic DNA (RAPD) technique was used to develop strain-specific primers for Gliocladium catenulatum strain J1446, which is promising in biological control. One of the primer pairs developed proved to be strain-specific; strain J1446 was differentiated from 16 G. catenulatum strains and six other strains of two Gliocladium species, as well as from Trichoderma virens, and isolates of Nectria spp. and Fusarium spp. Specific primers were also tested with DNA isolated from cucumber leaves, treated or untreated with a solution made from Gliocladium powder. The expected amplification product was produced only from treated leaves. DNA isolated from Gliocladium-treated potato tubers and fungi grown in peat was also used in amplification reactions. Strain-specific primers detected strain J1446 when the amount of DNA was 5pg or more. Some variation between the Gliocladium strains was found by the random amplified microsatellites method (RAMS) and the universally primed polymerase chain reaction method (UP-PCR), but no clear fragments specific to strain J1446 were produced. Cross-blot hybridisation of UP-PCR products differentiated strain J1446 from T. virens, but not from the Gliocladium isolates. The 28S rDNA sequences and -tubulin sequences were identical or very similar in all Gliocladium strains. Thus, it is possible that the Gliocladium strains of the present study are conspecific, which means that a revision in the taxonomy of Gliocladium species may be necessary.     

Phylogenetic relationships and pathogenicity of Colletotrichum acutatum isolates from grape in subtropical Australia

The identity of Colletotrichum acutatum as the causal pathogen of grape ripe-rot, which causes yield loss and a bitter taint that lowers wine quality in Australian subtropical wine-grape regions, was confirmed using species-specific primers. Cultural, morphological and molecular methods (RAPD-PCR and sequencing of parts of the 5·8S-ITS regions and the β-tubulin-2 gene) were used to determine the phylogenetic relationships of Australian C. acutatum isolates from wine grapes and other horticultural crops. A combination of RAPD-PCR and β-tubulin-2 gene data showed that all wine-grape ripe-rot isolates from northern regions of New South Wales (NSW) and Queensland belong to a proposed new C. acutatum group (A9), together with isolates from Australian strawberry, mango, blueberry and olive. The 5·8S-ITS sequences for these grape pathogens were identical to published sequences for an isolate from Cyclamen (the Netherlands) and differed by 1 bp from isolates from Capsicum (Taiwan) and orange (Costa Rica). The grape ripe-rot isolates from the Shoalhaven Valley (southern NSW) were clustered within two other C. acutatum groups: A2 and A5. In vitro infection studies showed that Australian C. acutatum isolates from almond, blueberry, chilli, grape, mango, olive, strawberry and tomato were able to infect grape and could also infect blueberry and strawberry, indicating a lack of host specificity. This lack of host specificity, the genetic similarity with non-grape isolates, and the fact that many of the non-grape hosts were isolated from wine-growing regions, suggest the potential for cross-infection between grape and other horticultural crops.     

Phenotypic and Genotypic Characterisation of Northern Ireland Isolates Of Phytophthora Infestans

Genetic and phenotypic diversity of the population of the late blight pathogen Phytophthora infestans in Northern Ireland was assessed from 223 single-lesion isolates collected from 29 sites in 1995 and 1996. The proportion of metalaxyl-resistant isolates was approximately 14%. The A2 mating type was not detected. Allozyme analyses revealed that the Northern Ireland isolates were monomorphic and homozygous at the loci coding for allozymes of glucose-6-phosphate isomerase and peptidase (Gpi 100/100, Pep 100/100). The majority of isolates tested (156 out of 162) were mitochondrial DNA haplotype IIa. Among the remaining isolates, four were haplotype Ia, two were the rare IIb haplotype and none was of the old Ib type. RAPD-PCR analysis of selected isolates revealed relatively little diversity among the Northern Ireland isolates, which clustered separately from isolates from GB and the European mainland.     

Analysis of genetic diversity and phylogeny in Saccharum and related genera using RAPD markers

Molecular diversity in Saccharum complex was studied using 195 RAPD markers generated by 12 random primers. Among the Saccharum species, S. officinarum showed a low level of genetic diversity while S. sinense was found to be more diverse. Six taxonomical groups were clearly resolved in the cluster analysis. S. officinarum, S. robustum, S. spontaneum and Erianthus spp. formed discrete groups. S. barberi and S. sinense formed a single cluster, so also Narenga and Sclerostachya. S. officinarum was found to be closer to S. robustum and distant from S. spontaneum. Among the related genera, Sclerostachya was closer to Saccharum while Erianthus was found to be highly divergent from all the Saccharum species. Six of the primers used generated RAPD fragments unique to Erianthus. It is suggested that the Erianthus spp. can contribute substantially towards sugarcane varietal improvement in view of its greater divergence with Saccharum.     

不同寄主植物上烟蚜DNA多态性的RAPD-PCR分析

用ＲＡＰＤ－ＰＣＲ技术研究了桃树,油菜和烟草上烟蚜的ＤＮＡ多态性。结果表明,用相似性指数和Ｎｅｉ的遗传距离及聚类分析对本研究筛选的三个引物ＯＰＸ－０４,ＰＯＸ－０６和ＯＰＸ－１９的扩增结果进行比较,除ＯＰＸ－０６的Ｄ值聚类可将各寄主植物上的烟蚜分开外,其余均可将样本分为两类,即来自桃树的烟蚜和来自油菜,烟草上的烟虾。     

Genetic Variation Among and Within Monilinia Species Causing Brown Rot of Stone and Pome Fruits

Nucleotide sequence analysis of the internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA (rDNA) divided the three brown rot pathogens Monilinia laxa, M. fructicola and M. fructigena into four distinct groups. Isolates of M. fructigena received from Japan, which varied by 5 base substitutions in the ITS region from the European M. fructigena isolates, formed the fourth group. Four of five Japanese isolates of M. fructicola tested varied from the New World isolates in that they did not possess a group-I intron in the small subunit (SSU) rDNA. RAPD-PCR data indicated that isolates of M. laxa varied but were randomly distributed worldwide; ITS data indicated no apparent distinction between those from Malus spp. and those from Prunus spp. M. fructigena similarly did not cluster according to geographic origin. In contrast, M. fructicola isolates tended to be clustered according to their origin; Japanese isolates of M. fructicola clustered together and showed similarity to some of the New Zealand isolates. Isolates from USA and Australia were more variable.     

亚洲百合DNA的提取及RAPD-PCR反应体系的优化

为了促进亚洲百合分子水平的研究,以亚洲百合幼嫩叶片为材料,采用改良CTAB法提取亚洲百合基因组DNA,得到的DNA质量较高,适合于RAPD -PCR分析。通过单因素优化得到在25μl反应体系中,亚洲百合RAPD 分析的最佳反应体系：160μmol/ L dNTPs,0.3μmol/L随机引物,Taq DNA聚合酶1.5U,Mg2＋浓度2.0mmol/L。     

苦瓜枯萎病菌的鉴定及其遗传多样性分析

 对分离获得的32株苦瓜枯萎病菌菌株进行形态学特征和寄主专化型测定, 结果表明, 测试的苦瓜枯萎病菌株均为尖孢镰刀菌苦瓜专化型 (Fusarium oxysporum f. sp. momordicae), 这些菌株可以侵染苦瓜和瓠瓜幼苗, 但不侵染其他葫芦科瓜类作物。对苦瓜枯萎病菌菌株的rDNA-ITS区 (ITS1、5.8S和ITS2)序列进行扩增测序, 结果显示其序列长度均为456 bp;聚类分析表明测序菌株与镰刀菌属中尖孢镰刀菌不同专化型的菌株聚为一群。利用RAPD标记技术分析苦瓜枯萎病菌的遗传多样性, 结果显示苦瓜枯萎病菌株与其他葫芦科瓜类作物枯萎病菌株间的遗传相似系数范围为0.59~0.99, 当遗传相似系数为0.85时, 供试的48个菌株分成10个类群 (G_1~10)。在RAPD聚类树中所有苦瓜枯萎病菌株聚在一个分支上 (G₁群), 菌株间的遗传相似系数范围为0.92~1.00, 具有较高的遗传相似性, 且菌株的聚群与地理来源存在一定的相关性。