Molecular diversity and relationships among Cymbidium goeringii cultivars based on inter-simple sequence repeat (ISSR) markers.

Spring orchid (Cymbidium goeringii) is a popular flowering plant species. There have been few molecular studies of the genetic diversity and conservation genetics on this species. An assessment of the level of genetic diversity in cultivated spring orchid would facilitate development of the future germplasm conservation for cultivar improvement. In the present study, DNA markers of intersimple sequence repeats (ISSR) were identified and the ISSR fingerprinting technique was used to evaluate genetic diversity in C. goeringii cultivars. Twenty-five ISSR primers were selected to produce a total of 224 ISSR loci for evaluation of the genetic diversity. A wide genetic variation was found in the 50 tested cultivars with Nei's gene diversity (H = 0.2241) and 93.75% of polymorphic loci. Fifty cultivars were unequivocally distinguished based on ISSR fingerprinting. Cultivar-specific ISSR markers were identified in seven of 50 tested cultivars. Unweighted pair-group mean analysis (UPGMA) and principal coordinates analysis (PCA) grouped them into two clusters: one composed the cultivars mainly from Japan, and the other contained three major subclusters mainly from China. Two Chinese subclusters were generally consistent with horticultural classification, and the third Chinese subcluster contained cultivars from various horticultural groups. Our results suggest that the ISSR technique provides a powerful tool for cultivar identification and establishment of genetic relationships of cultivars in C. goeringii.

[Study on the relationships of Cymbidium by RMAPD technique].

Cymbidium is one of the Orchidaceae genera. There are 70 Cymbidium species in the world, mainly distributed over the tropical and the subtropical area of Asia and northern Australia. In China, approximately 49 species and some variations are distributed over the provinces to the south of Qinling. The orchid market of China is quite wide, not only because of their view and admiration value, but also their officinal value as well. Some species of Cymbidium are morphologically similar, so the identification of Cymbidium species and the understanding of the relationships between them are very important for new varieties breeding of Cymbidium. This study attempts to apply RMAPD to detect the relationships between the Cymbidium species. 456 pairs of RMAPD combinations were screened, but only 12 pairs of them were used to analyze the genetic polymorphism of 16 Cymbidium. 362 bands were produced by 12 pairs of RMAPD primers in total, including 90 polymorphic bands (24.8%). The polymorphic bands of each primer combination ranged from 3 to 11 with the average of 8. Among all species, genetic similarity coefficient ranged from 0.63 to 0.87. The result showed that the genetic distance was closest between C. floribundum and C. aestivum, C. ensifolium and C. sinense, C. maguanense and C. hookerianum, which is almost the same as the traditional classification. It was the first time that species of Cymbidium were detected by RMAPD technique. This study shows RMAPD is an appropriate and effective molecular marker technique to identify the relationships of Cymbidium.

[The genetic diversity of Cymbidium by ISSR].

ISSR was applied to detect the relationship between 16 Cymbidium species, and 836 bands were amplified with 15 primers, including 227 polymorphic bands. The polymorphic percentage is 27.2%. UPGMA results showed that the genetic distance were closest between C.goeringii (Rchb.f.) Rchb.f. and C. goeringii var. longibracteatum, and C.lancifolium Hook. was far away from the other 15 species. This result is quite similar to the traditional classification, indicating that the technique could supplement some information to traditional taxonomy in the molecular level.

[Studies of genetic diversity among 10 species of Ilex based on RAPD and AFLPs].

The techniques of random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLPs) were used to amplify the genomic DNA of 10 species of Ilex, so as to reveal the polymorphism in different species of Ilex. In RAPD analysis, a total of 301 lines were amplified with 11 kinds of primers selected from 100 kinds of 10 bp random primers, and the polymorphism was 98.63%. In AFLPs analysis,a huge diversity was showed by 3 primers combinations carried out on these species. Both RAPD and AFLPs analysis were clustered by UPGMA and the results showed that the genetic distances were the closest between I. purpurea and I. vomitoria Ait, I. Litseaefolia and I. zhejiangensis, I. hylonoma var. glabra and I. triflora var. kanehirai.

Application of Arabidopsis AGAMOUS second intron for the engineered ablation of flower development in transgenic tobacco.

To explore a new approach to generating reproductive sterility in transgenic plants, the barnase gene from Bacillus amyloliquefaciens was placed under the control of an 1853-bp nucleotide sequence from the 3'end of the second intron of Arabidopsis AGAMOUS and CaMV 35S (-60) minimal promoter [AG-I-35S (-60)::Barnase], and was introduced into tobacco through transformation mediated by Agrobacterium tumefaciens. All AG-I-35S (-60)::Barnase transgenic plants showed normal vegetative growth and 28% of the transgenic lines displayed complete ablation of flowering. Two transgenic lines, Bar-5 and Bar-15, were 98.1 and 98.4% sterile, respectively, as determined by seed production and germination. When controlled by AG-I-35S (-60) chimeric promoter, barnase mRNA was detected in the reproductive tissues of transgenic tobacco plants, but not in vegetative parts. This study presents the first application of an AG intron sequence in the engineered ablation of sexual reproduction in plants. The AG-I-35S (-60)::Barnase construct can be useful in diminishing pollen and seed formation in plants, providing a novel bisexual sterility strategy for interception of transgene escape and has other potentially commercial use for transgenic engineering.

[Analysis of genetic diversity among 13 Chinese species of Dendrobium based on AFLP].

Amplified fragment length polymorphism (AFLP) was assessed in 13 Chinese species of Dendrobium. Three primer combinations E+ACT/M+CAC, E+AAC/M+CAC and E+ACA/M+CAC were used for selective amplification. Between 100 bp and 300 bp, a totle of 346 fragments were detected, in which 342 are polymorphic. The rate of polymorphism was 98.8%. The AFLP data were analyzed using Nei & Li similarity coefficient and UPGMA Cluster method. The result indicated that the 13 species were clustered into three groups on coefficient level 0.54. Group I included D. falconeri, D. officinale, D. wilsonii, D. hercoglossum, D. crystallinum. D. hancockii, D. guangxiense, D. primulinum, D. crepidatum, D. thyrsiflorum. Group II included D. chrysotoxum. Group III included D. loddigesii. This outcome was corresponding to the classification result by using the traditional method. It is concluded that AFLP markers can be used on the studies of genetic relationships and classification of Dendrobium.

[Molecular identification of cymbidium mosaic Potexvirus and Odontoglossum ringspot Tobamovirus complex infected Phalaenopsis and its pathological ultrastructural alteration].

Filamentous and rod-shaped virions, and aggregated crystals were observed in infected leaves of by negative staining and ultramicrotomy. Histologic study synchronously showed typical crystal forms of the two virions: the crystals from filamentous particles congregated in strips, arrayed in multilayer and piled in certain angles or helix between layers; while the crystals from rod-shaped particles arrayed in parallel, angle-layer or helix. The two kinds of crystals both presented in parenchyma cells, plasmodesma, and vascular bundles; as an evidence that indicates the existence of short distance transport of viruses between cells, plasmodesmata were produced through piercing the membrane around the reproducing viral crystals; the chloroplasts in the infected cells were hypoplastic, and the filamentous virion were observed within the chloroplasts; the mitochondrions were over-developed, swelled or even cavitated; the nucleus were also swelled and cavitated. Further multiplex RT-PCR and sequencing that the coat protein genes simultaneously expanded to Cymbidium mosaic potexvirus (CymMV) and Odontoglossum ringspot tobamovirus (ORSV) showed homology with available abstracts from GenBank, and the respective percentages of identity are 98% and 99%-100%. The instant and direct identification evidences of CymMV and ORSV complex infection on phalaenopsis are revealed at both cellular and molecular levels, and the character of its pathological ultrastructural alteration as the gist in cellular pathology for pathogenesis are also presented.