Isolation and characterization of microsatellite markers for Dendranthema morifolium (Asteraceae) using next-generation sequencing.

Dendranthema morifolium (Asteraceae) is a perennial herbaceous plant native to China. A long history of artificial crossings may have resulted in complex genetic background and decreased genetic diversity. To protect the genetic diversity of D. morifolium and enabling breeding of new D. morifolium cultivars, we developed a set of molecular markers. We used pyrosequencing of an enriched microsatellite library by Roche 454 FLX+ platform, to isolate D. morifolium simple sequence repeats (SSRs). A total of 32,863 raw reads containing 2251 SSRs were obtained. To test the effectiveness of these SSR markers, we designed primers by randomly selecting 100 novel SSRs, and amplified them across 60 cultivars representing five different petal shape groups. Sixteen SSRs were polymorphic with the number of alleles ranging from 6 to 19, and their expected and observed heterozygosities ranging from 0.477 to 0.848, and 0.250 to 0.804, respectively. The polymorphism information content ranged from 0.459 to 0.854 and the inbreeding coefficient ranged from -0.119 to 0.759. An unweighted pair-group method arithmetic average analysis was performed to survey the phylogenetic relationships of these 60 cultivars and five clusters were identified. These markers can be used for investigating genetic relationships and identifying elite alleles through linkage and association analyses.

Isolation and characterization of microsatellite markers for Osmanthus fragrans (Oleaceae) using 454 sequencing technology.

Osmanthus fragrans (Oleaceae) is an evergreen shrub or small tree that grows in south China. In this study, Roche 454 FLX+ sequencing combined with the magnetic bead enrichment method was used to isolate microsatellite markers from the genome of O. fragrans. A total of 1471 microsatellites that contained enough flanking sequences for primer pair design were identified from 89,633 raw sequencing reads. One hundred primer pairs were randomly chosen to test primer amplification efficiency. Among these tested primer pairs, 20 yielded polymorphic amplification products across 16 individuals from the Albus, Luteus, and Aurantiacus groups. The number of alleles ranged from 2 to 6, with an average of 3.7. The observed heterozygosity ranged from 0 to 0.813, with an average of 0.460. Shannon's information index ranged from 0.463 to 1.707, with an average of 0.975. Six loci (Of 05, Of 06, Of 08, Of 12, Of 15, and Of 19) deviated significantly from Hardy-Weinberg equilibrium (P < 0.05), which was due to an excess of homozygotes or heterozygotes. Nine pairs of loci (Of 01 and Of 05; Of 04 and Of 05; Of 01 and Of 06; Of 04 and Of 12; Of 02 and Of 13; Of 04 and Of 13; Of 12 and Of 13; Of 04 and Of 19; Of 05 and Of 19) showed significant linkage disequilibrium, which indicated significant allelic association between the loci. This set of microsatellite markers will be valuable for molecular marker-assisted breeding in O. fragrans.

Analysis of expressed sequence tags from Chaetomium cupreum grown under conditions associated with mycoparasitism.

AIMS: To elucidate the molecular mechanisms associated with mycoparasitism from Chaetomium cupreum, an effective biocontrol agent with ability against plant pathogenic fungi. METHODS AND RESULTS: One cDNA library was constructed from conditions predicted to resemble mycoparasitic process. A total of 1876 ESTs were generated and assembled into 1035 unigenes. BlastX search revealed that 585 unigenes had similarities with sequences available from public databases. Based on the ESTs abundance, MFS monosaccharide transporter was found as the gene expressed at the highest level. A KEGG analysis allowed mapping of 60 metabolic pathways well represented by the glycolysis/gluconeogenesis, D-arginine and ornithine metabolism, and tryptophan metabolism. The genes related to mycoparasitism were detected. CONCLUSIONS: The results revealed that the cell walls of the fungal pathogen can simulate some aspects of the mycoparasitic interaction between C. cupreum and its targets. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to study genes expression under conditions associated with the mycoparasitic process. The findings contribute to elucidate the molecular mechanisms involved in mycoparasitism and will help to advance our efforts in developing novel strategies for biocontrol of plant fungal diseases.