Untangling the transcriptome from fungus-infected plant tissues.

The development of sequencing technology allows low-cost generation of sequence data. The huge amount of raw sequence data now available has introduced many challenges associated with analysis of these large-scale data banks. For example, it is very important to distinguish materials of plant and fungal origin in fungus-infected plant tissue. The origin of transcripts that were sequenced from Library 895-M6 (poplar tissue infected by Marssonina brunnea) on Illumina/Solexa GA IIx was determined by combining three methods: (1) based on the taxonomic information of homologous sequences; (2) based on the reference genome sequence; (3) based on the transcriptome sequence of the host and its pathogen obtained from Library 895 (poplar) and Library M6 (M. brunnea) as well as Library 895-M6 (mixture of poplar and M. brunnea). We idenified accurately the origin of 80,978 (99.5%) contigs in the mixed poplar and M. brunnea sample (Library 895-M6) by integrating the results from the three methods. The results of this study demonstrate that a combination of these three approaches described here is an effective strategy for determining the origin of sequences in a mixed pool, and provides a basis for further transcriptome analysis of the mixed sample.

Sequencing the genome of Marssonina brunnea reveals fungus-poplar co-evolution.

BACKGROUND: The fungus Marssonina brunnea is a causal pathogen of Marssonina leaf spot that devastates poplar plantations by defoliating susceptible trees before normal fall leaf drop. RESULTS: We sequence the genome of M. brunnea with a size of 52 Mb assembled into 89 scaffolds, representing the first sequenced Dermateaceae genome. By inoculating this fungus onto a poplar hybrid clone, we investigate how M. brunnea interacts and co-evolves with its host to colonize poplar leaves. While a handful of virulence genes in M. brunnea, mostly from the LysM family, are detected to up-regulate during infection, the poplar down-regulates its resistance genes, such as nucleotide binding site domains and leucine rich repeats, in response to infection. From 10,027 predicted proteins of M. brunnea in a comparison with those from poplar, we identify four poplar transferases that stimulate the host to resist M. brunnea. These transferas-encoding genes may have driven the co-evolution of M. brunnea and Populus during the process of infection and anti-infection. CONCLUSIONS: Our results from the draft sequence of the M. brunnea genome provide evidence for genome-genome interactions that play an important role in poplar-pathogen co-evolution. This knowledge could help to design effective strategies for controlling Marssonina leaf spot in poplar.

Analysis of floral transcription factors from Lycoris longituba.

Transcription factors (TFs) are proteins that bind to specific promoter regions of their target genes and regulate gene transcription. Many of these factors have been found to influence flowering. Lycoris longituba exhibits a great deal of diversity in flower color and flower form, making it a suitable model for the study of floral development. We have identified 338 putative TFs from more than thirty thousand ESTs sequenced from the floral tissue of L. longituba, and validated them using real-time RT-PCR. Fifty-one of the TFs were recognized as being potentially flower-specific, and the expression patterns of some of them during six flowering phases have been elucidated. Homolog annotation and phylogenetic analysis revealed that some TFs that belong to several TF families, such as MADS, MYB-related, NAC, and ABI3-VP1, were suggested to play important roles in the flowering process. Our dataset may be used to identify priority target TF genes for further study.

[Progress in research on forest tree comparative genomics].

Comparative genomics is one of the important research fields in genomics. By comparative genomic studies it was possible to establish a giant genetic system beyond one species which might be significant to tree. Comparative genomics in Salicaceae, Pinaceae, Rosaceae, Fagaceae and Hamamelidaceae had shown that the organization of genomes remained highly conserved over long evolutionary periods and extensive synteny or collinarity or microcollinarity existed in tree genomes. A total of 13,019 pairs of orthologs were identified between genes in Populus and Arabidopsis using the best bidirectional Basic Local Alignment Search Tool (BLAST) hits, with an average mutual coverage of these alignments equal to 93%; 11,654 pairs of orthologs had greater than 90% alignment of gene lengths. The progress of comparative genomics in tree species was reviewed and the prospect of this field was discussed, which might be useful to tree genomics in China.

[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.

[Phylogeny of the genus Arundinaria based on nucleotide sequences of nrDNA ITS region].

The sequences of nrDNA regions of 17 species and Phyllostachys edulis (outgroup) sampled, which are of represent and type species for different taxa in the genus Arundinaria,were analyzed by PCR amplification and direct DNA sequencing. The phylogenetic trees generated from maximum parsimony analysis showed that the sampled bamboos were naturally monophletic, appearing that these species of the bamboos belong to the genus Arundinaria. The internal transcribed spacers (ITS) data indicated that the species were divided into two branches, one including A. oleosa, A. hsienchuensis, A. chino, A. amara, A. yixingensis, A. amabilis, A. fortunei, and A. pygmaea, the other including A. graminea, A. fargesii, A. faberi, A. hupehensis, Pseudosasa japonica cv. Tsutsumiana, P. japonica, Brachystachyum densiflorum, A. oedogonata, and A. sulcata. The result also showed that there was close relationship between A. graminea and A. fargesii, Pseudosasa japonica cv. Tsutsumiana and P. japonica, A. sulcata, Brachystachyum densiflorum and A. oedogonata, (99%, 100% and 82% boot-strap support respectively). Moreover, there was very close relationship between A. amabilis and A. hsienchuensis, indicating that A. amabilis belongs to the genus Arundinaria. It was shown in the phylogenetic tree that A. pygmaea and A. fortunei had close relationship, and were a sister branch to the bamboos of Pleioblastus.

[Analysis of genetic structure in population of Larix kaempferi by chloroplast SSR markers].

Genetic structure of seven populations in Larix kaempferi in Japan was studied by use of cpSSR markers. Ten different length fragments in and ten different kinds of haplotypes were reduced in 197 samples based on 3 pairs of polymorphic primers screened from 11 pairs of primers. There were significant variant haplotypes among the populations. The genetic variation in the populations of Larix kaempferi was detected by using cpSSR with the number of average loci A=3.33, the number of average efficient loci NE=1.20, gene diversity HE=0.17 and 5.37% variation from different populations. The genetic variation was mainly from individuals in population.

[Sequence of the ITS region of nuclear ribosomal DNA(nrDNA) in Xinjiang wild Dianthus and its phylogenetic relationship].

Xinjiang is a center of distribution and differentiation of genus Dianthus in China, and has a great deal of species resources. The sequences of ITS region (including ITS-1, 5.8S rDNA and ITS-2) of nuclear ribosomal DNA from 8 species of genus Dianthus wildly distributed in Xinjiang were determined by direct sequencing of PCR products. The result showed that the size of the ITS of Dianthus is from 617 to 621 bp, and the length variation is only 4 bp. There are very high homogeneous (97.6%-99.8%) sequences between species, and about 80% homogeneous sequences between genus Dianthus and outgroup. The sequences of ITS in genus Dianthus are relatively conservative. In general, there are more conversion than transition in the variation sites among genus Dianthus. The conversion rates are relatively high, and the ratios of conversion/transition are 1.0-3.0. On the basis of phylogenetic analysis of nucleotide sequences the species of Dianthus in China would be divided into three sections. There is a distant relationship between sect. Barbulatum Williams and sect. Dianthus and between sect. Barbulatum Williams and sect. Fimbriatum Williams, and there is a close relationship between sect. Dianthus and sect. Fimbriatum Williams. From the phylogenetic tree of ITS it was found that the origin of sect. Dianthusis is earlier than that of sect. Fimbriatum Williams and sect. Barbulatum Williams.

[Identification of markers linked to resistance locus of Marssonina leaf spot in poplars by bulked segregant analysis(BSA)].

DNA markers linked to resistance locus of Marssonina leaf spot in poplars were found by bulked segregant analysis(BSA). The bulks consisted of individual with a extreme phenotype taken from a population of 91 F1 clones,which is a progeny of Populus deltoides Bartr.cv."Lux"(I-69/55)(Resistance) and P.euramericana cv.I-45(Susceptible). Out of 114 RAPD primers, four markers showed polymorphisms between the resistance-bulk and the susceptible-bulk.By using selective genotype linkage analysis,OPAI17-1550 and OPAI13-900 were found linked to the resistance locus. The genetic distances between the two markers and the resistance locus were 29.9cM and 37.4cM,respectively.

[Analysis of the inter-species relationships on lycoris Amaryllidaceae) by use of RAPD].

The fingerprints of 13 species in genus Lycoris were generated by use of RAPD method. Forty-one primers were screened from 520 random primers, and a total of 350 DNA fragments were amplified ranging from 0.3-3.0 kb, 253 (72.3%) of which were polymorphic. The average number of DNA band produced by each primer was 6.2. Nei's similarity coefficients and genetic distances were calculated by use of the software of TFPGA version 1.3 and dendrogram of Lycoris was constructed using UPGMA. It is indicated that the 13 species of the genus Lycoris were divided into two groups, and five species of the genus including L. rosea, L. haywardii, L. straminea, L. sprengeri and L. radiata with monotype karyotypes (I-shaped) were clustered together respectively. The basic chromosome number was x = 11. The others which have two-types karyotypes (I-shaped and V-shaped) were clustered together respectively. They were L. houdyshelii, L. albiflora, L. chinensis, L. longituba, L. anhuiensis, L. squmigera, L. caldwellii and L. aurea. The closest relationship was between L. rosea and L. haywardii. L. radiata is highly divergent from L. aurea. The results were in consistence with that of the analysis of chromosome karyotype. The present paper discussed the problems whether L. rosea, L. haywardii and L. stramina originated as natural hybrids and taxonomy position of L. albiflora, L. straminea and L. houdyshelii based on the RAPD analysis.