Differentially expressed genes under cold acclimation in Physcomitrella patens.

Cold acclimation improves freezing tolerance in plants. In higher plants, many advances have been made toward identifying the signaling and regulatory pathways that direct the low-temperature stress response; however, similar insights have not yet been gained for simple nonvascular plants, such as bryophytes. To elucidate the pathways that regulate cold acclimation in bryophytes, we used two PCR-based differential screening techniques, cDNA amplified fragment length polymorphism (cDNA-AFLP) and suppression subtractive hybridization (SSH), to isolate 510 ESTs that are differentially expressed during cold acclimation in Physcomitrella patens. We used realtime RT-PCR to further analyze expression of 29 of these transcripts during cold acclimation. Our results show that cold acclimation in the bryophyte Physcomitrella patens is not only largely similar to higher plants but also displays distinct differences, suggests significant alteration during the evolution of land plants.

SSR allelic variation in almond (Prunus dulcis Mill.).

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach.

[Cloning and characterization of two MADS box genes from peach (Prunus persica)].

With the aim of finding genes involved in the floral transition of Prunus species (Prunus sp.), the EST (expressed sequence tags) sequences were extracted from the public databases. Eight MADS box genes' cDNAs were obtained. Two of them, PpMADS4 and PpMADS6 (The accession numbers in GenBank are AY705972 and AY705973), were cloned from peach (Prunus persica). The full length cDNA of PpMADS4 is 850 bp long. It contains an open reading frame of 732 bp, coding for a polypeptide of 243 amino acids. The full length cDNA of PpMADS6 is 1,190 bp long. It contains an open reading frame of 768 bp coding for a polypeptide of 256 amino acids. PpMADS4 closely resembles the Arabidopsis AGAMOUS gene. It is an AGAMOUS-like C class MADS box gene, and it expresses in petal, carpel, fruit and nutlet as demonstrated by RT-PCR analysis. PpMADS6 is likely to be the peach orthologue of the Petunia PFG genes and it is an A class MADS box gene. It has been shown with RT-PCR that it expresses in leaf, sepal, petal, carpel and fruit. It may be involved in the transition from the juvenile to the adult stage.

Development of SSR markers for the phylogenetic analysis of almond trees from China and the Mediterranean region.

Expressed sequence tag (EST) derived simple sequence repeats (SSRs, microsatellites) were screened and identified from 3863 almond and 10 185 peach EST sequences, and the spectra of SSRs in the non-redundant EST sequences were investigated after sequence assembly. One hundred seventy-eight (12.07%) almond SSRs and 497 (9.97%) peach SSRs were detected. The EST-SSR occurs every 4.97 kb in almond ESTs and 6.57 kb in peach, and SSRs with di- and trinucleotide repeat motifs are the most abundant in both almond and peach ESTs. Twenty one EST-SSRs were thereafter, developed and used together with 7 genomic SSRs, to study the genetic relationship among 36 almond (P. communis Fritsch.) cultivars from China and the Mediterranean area, as well as 8 accessions of other related species from the genus Prunus. Both EST-derived and genomic SSR markers showed high cross-species transferability in the genus. Out of the 112 polymorphic alleles detected in the 36 cultivated almonds, 28 are specific to Chinese cultivars and 25 to the others. The 44 accessions were clustered into 4 groups in the phylogenetic tree and the 36 almond cultivars formed two distinct subgroups, one containing only Chinese cultivars and one of unknown origin and the other only those originating from the Mediterranean area, indicating that Chinese almond cultivars have a distinct evolutionary history from the Mediterranean almond. Our preliminary results indicated that common almond was more closely related to peach (P. persica (L.) Batsch.) than to the four wild species of almond, (P. mongolica Maxim., P. ledebouriana Schleche, P. tangutica Batal., and P. triloba Lindl.). The implications of these SSR markers for evolutionary analysis and molecular mapping of Prunus species are discussed.

Analysis of genetic variation in Ganoderma lucidum after space flight.

A modified CTAB method was used in the extraction of total cellular DNA of Ganoderma lucidum. Four strains Cx, Ch, C3 and C4, and their counterparts, four space flown strains Sx, Xh, S3 and S4, were analysed by amplified fragment length polymorphism (AFLP) with several primer combinations. Polymorphic bands were detected between Sx and Cx, S3 and C3, respectively. Somatic incompatibility tests further confirmed their heterogeneity. However, no disparity between Sh and Ch, S4 and C4 was detectable. The results suggest that spaceflight may be used to accelerate breeding of Ganoderma lucidum strains for commercial cultivation.