Random BAC FISH of monocot plants reveals differential distribution of repetitive DNA elements in small and large chromosome species.

BAC FISH (fluorescence in situ hybridization using bacterial artificial chromosome probes) is a useful cytogenetic technique for physical mapping, chromosome marker screening, and comparative genomics. As a large genomic fragment with repetitive sequences is inserted in each BAC clone, random BAC FISH without adding competitive DNA can unveil complex chromosome organization of the repetitive elements in plants. Here we performed the comparative analysis of the random BAC FISH in monocot plants including species having small chromosomes (rice and asparagus) and those having large chromosomes (hexaploid wheat, onion, and spider lily) in order to understand a whole view of the repetitive element organization in Poales and Asparagales monocots. More unique and less dense dispersed signals of BAC FISH were observed in species with smaller chromosomes in both the Poales and Asparagales species. In the case of large-chromosome species, 75-85% of the BAC clones were detected as dispersed repetitive FISH signals along entire chromosomes. The BAC FISH of Lycoris did not even show localized repetitive patterns (e.g., centromeric localization) of signals.

Polymorphic microsatellite loci for the genetic analysis of Lycoris radiata (Amaryllidaceae) and cross-amplification in other congeneric species.

Lycoris radiata is a perennial herb that has been used in traditional Chinese medicine for a long time and has two main medicinal components in its bulb, lycorine and galanthamine. However, the original microsatellite loci have not been developed for any species of Lycoris. Total genomic DNA was extracted from fresh bulbs using a modified CTAB protocol. We isolated 10 microsatellite loci from 21 L. radiata individuals of a natural population from Yellow Mountain in Anhui Province, China. The number of alleles ranged from two to nine. The observed and expected heterozygosities ranged from 0.238 to 0.952 and from 0.455 to 0.784, respectively. One locus significantly deviated from Hardy-Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these microsatellite loci was characterized in additional five species (L. sprengeri, L. anhuiensis, L. albiflora, L. longituba, and L. chinensis) of Lycoris. The results suggest that these microsatellite markers would contribute to the population genetic studies of L. radiata and other related species.

[Genetic variations in trnL-F sequence and phylogenetic clustering of Lycoris species].

OBJECTIVE: To identify some closely related Lycoris species and evaluate interspecific relationships among them. METHOD: The cpDNA trnL-F sequence of 20 taxa representing 15 species of Lycoris and Narcissus tazaetta var. chinensis as one out-group were determined by using direct sequencing of PCR product, and they were analyzed by means of the software of CLUSTRAL and MEGA. RESULT: The length of trnL-F of all taxa was 905 - 1 036 bp. When the gaps were always treated as missing, there were 14 variable sites and 10 parsim-info sites, which could be used to identify some species of Lycoris. Four nucleotides inserteions/deletions were significant different among Lycoris and two species of Narcissus. Phylogeny tree was constructed with the maximum parsimony methods by bootstrap test. Three infrageneric clades of all Lycoris species were resolved. The classification was basically consistent with that of morphology except for L. longituba, L. aurea, and L. straminea. CONCLUSION: The tree suggested that L. anhuiensis can not be taken as an independent species, while it may be of a variety or a hybrid of L. longituba. Regarding the hybrid origin species, the materal parent of L. rosea and L. haywardii was revealed to be L. sprengeri. There were some variations in the trnL-F sequence, which were good molecular markers for identification species of Lycoris.

[The analysis of differential expression and cloning of genes related to raised secondary lateral veins mutant of Lycoris aurea].

Lycoris aurea exhibits parallel venation, the main vein with many lateral veins in a longitudinal parallel arrangement. There are secondary lateral veins (SLV) between each longitudinal veins. In general, SLVs are not remarkable. In this paper, the material was one kind of Lycoris aurea mutant called Raised Secondary Lateral Veins mutant (RSLV), because many Raised Secondary Lateral Veins are in abaxial surface of its leaves. Its growing potential is weaker than that of wild type and its blades are very thin. Moreover, the stamens of RSLV degenerate completely. Two cDNA libraries were constructed from RSLV mutant and wild type (WT) leaves. From the libraries, 3,122 ESTs, which are longer than 100 bp each after vector sequence removed, were acquired by single-pass sequencing from the 5'end. Following a multistep selection, 512 70-mer oligo-DNA probes were designed for attachment on the microarray slide based on the ESTs. The gene expression profile of RSLV mutant and WT leaves was compared through the microarray at transcriptional level. The microarray experiment results were further confirmed by Quantitative Real-Time PCR (QRT-PCR). We identified 5 genes whose expressions changed more than 2-fold between RSLV mutant and WT leaves. They encode phloem protein 2 (PP2), ferritin, pectin methyl esterase (PME), chlorophyll a/b binding protein (CAB protein) and pyruvate decarboxylase (PDC), respectively. Furthermore, the full-length cDNA sequences of the 5 genes were separately obtained from RSLV and WT by RACE. The relationship between differential expressions of the genes and the formation of the RSLV mutant phenotype were discussed.

[The localization of galanthamine in vegetative organ of Lycoris aurea Herb].

Using laser confocal microscopical techniques, we observed the anatomical structure of mature root, bulb, and leaf of Lycoris aurea Herb., and also did some research on the localization of galanthamine in the above-mentioned vegetative organ. The results are as follows: In the mature root, the galanthamine distributes mainly in cell wall, especially in cell wall of exodermis and endodermis and vessel wall. In the mature leaf, galanthamine exist in cell wall of vascular bundle, mesophyll cell between vascular bundles and epidermis cells. The scale leaf is the essential accumulational organ. Plenty of galanthamine distribute in the adaxial parenchyma cell, epidermis cell wall, and also in the clingy cell of abaxial epidermis cell.