Yingnan Chen

The willow genome and divergent evolution from poplar after the common genome duplication

Willows (Salix) and poplars (Populus) are known worldwide as woody species with diverse uses. Although these two genera diverged from each other around the early Eocene, they share numerous traits, including the same chromosome number of 2n = 38 and the common ‘Salicoid’ genome duplication with a high macrosynteny. However, most willow species flower early in their lives with short, small and sometimes indistinct stems, and thus differ from poplars in their life histories and habits. In addition, multiple inter- and intrachromosomal rearrangements have been detected involving chromosomal regions present in both lineages, suggestive of the likely genomic divergence after the common genome duplication.

Transcriptome analysis of differentially expressed genes relevant to variegation in peach flowers.

Background Variegation in flower color is commonly observed in many plant species and also occurs on ornamental peaches (Prunus persica f. versicolor [Sieb.] Voss). Variegated plants are highly valuable in the floricultural market. To gain a global perspective on genes differentially expressed in variegated peach flowers, we performed large-scale transcriptome sequencing of white and red petals separately collected from a variegated peach tree. Results A total of 1,556,597 high-quality reads were obtained, with an average read length of 445 bp. The ESTs were assembled into 16,530 contigs and 42,050 singletons. The resulting unigenes covered about 60% of total predicted genes in the peach genome. These unigenes were further subjected to functional annotation and biochemical pathway analysis. Digital expression analysis identified a total of 514 genes differentially expressed between red and white flower petals. Since peach flower coloration is determined by the expression and regulation of structural genes relevant to flavonoid biosynthesis, a detailed examination detected four key structural genes, including C4H, CHS, CHI and F3H, expressed at a significantly higher level in red than in white petal. Except for the structural genes, we also detected 11 differentially expressed regulatory genes relating to flavonoid biosynthesis. Using the differentially expressed structural genes as the test objects, we validated the digital expression results by using quantitative real-time PCR, and the differential expression of C4H, CHS and F3H were confirmed. Conclusion In this study, we generated a large EST collection from flower petals of a variegated peach. By digital expression analysis, we identified an informative list of candidate genes associated with variegation in peach flowers, which offered a unique opportunity to uncover the genetic mechanisms underlying flower color variegation.

Transcriptome Analysis of the Differentially Expressed Genes in the Male and Female Shrub Willows (Salix suchowensis)

Background The dioecious system is relatively rare in plants. Shrub willow is an annual flowering dioecious woody plant, and possesses many characteristics that lend it as a great model for tracking the missing pieces of sex determination evolution. To gain a global view of the genes differentially expressed in the male and female shrub willows and to develop a database for further studies, we performed a large-scale transcriptome sequencing of flower buds which were separately collected from two types of sexes. Results Totally, 1,201,931 high quality reads were obtained, with an average length of 389 bp and a total length of 467.96 Mb. The ESTs were assembled into 29,048 contigs, and 132,709 singletons. These unigenes were further functionally annotated by comparing their sequences to different proteins and functional domain databases and assigned with Gene Ontology (GO) terms. A biochemical pathway database containing 291 predicted pathways was also created based on the annotations of the unigenes. Digital expression analysis identified 806 differentially expressed genes between the male and female flower buds. And 33 of them located on the incipient sex chromosome of Salicaceae, among which, 12 genes might involve in plant sex determination empirically. These genes were worthy of special notification in future studies. Conclusions In this study, a large number of EST sequences were generated from the flower buds of a male and a female shrub willow. We also reported the differentially expressed genes between the two sex-type flowers. This work provides valuable information and sequence resources for uncovering the sex determining genes and for future functional genomics analysis of Salicaceae spp.

Confirmation of Single-Locus Sex Determination and Female Heterogamety in Willow Based on Linkage Analysis.

In this study, we constructed high-density genetic maps of Salix suchowensis and mapped the gender locus with an F1 pedigree. Genetic maps were separately constructed for the maternal and paternal parents by using amplified fragment length polymorphism (AFLP) markers and the pseudo-testcross strategy. The maternal map consisted of 20 linkage groups that spanned a genetic distance of 2333.3 cM; whereas the paternal map contained 21 linkage groups that covered 2260 cM. Based on the established genetic maps, it was found that the gender of willow was determined by a single locus on linkage group LG_03, and the female was the heterogametic gender. Aligned with mapped SSR markers, linkage group LG_03 was found to be associated with chromosome XV in willow. It is noteworthy that marker density in the vicinity of the gender locus was significantly higher than that expected by chance alone, which indicates severe recombination suppression around the gender locus. In conclusion, this study confirmed the findings on the single-locus sex determination and female heterogamety in willow. It also provided additional evidence that validated the previous studies, which found that different autosomes evolved into sex chromosomes between the sister genera of Salix (willow) and Populus (poplar).

Melampsora larici-populina, the main rust pathogen, causes loss in biomass production of black cottonwood plantations in the south of China

Epidemic outbreaks of rust disease have been observed in most of the black cottonwood (Populus trichocarpa) plantations in the south of China in recent years. However, the exact pathogens that cause rust disease in this area remain largely unknown. In this study we collected rust fungi from black cottonwood plantations at four different places in the south of China. Examination of these fungi by scanning electron microscope (SEM) and light microscopy (LM) revealed that the morphological characteristics of urediniospores for all the collected fungi were very close to that of Melampsora larici-populina. Using species-specific primers, these pathogens were confirmed to be M. larici-populina. In a survey on the resistance to rust disease for 88 genotypes of black cottonwood, nine potential candidate genotypes were detected that may be free from infection of rust pathogen. The results of this study provide essential information for breeding new rust-resistant black cottonwood cultivars.

Marker-Aided Selection of Polyploid Poplars

Polyploid breeding is an important means for creating elite varieties for the development of poplar plantations. However, polyploid poplars are rare in natural stands. In this study, we established an analytical toolkit to perform marker-aided selection of polyploid poplars. This toolkit contains 12 SSR primer pairs with sites located in the exonic DNA regions and resulting amplified microsatellites in the intronic/intergenic regions. Highly conserved primer pairs were selected by testing in eight species from four poplar sections. The amplified loci’s variability was examined using trees from a germplasm collection of Populus deltoides. Subsequently, copy numbers amplified by the highly variable primers were experimentally determined using progeny of a full-sib diploid pedigree. Based on the above tests, a subset of primers were finally selected and used for marker-aided selection of polyploid poplars from a set of natural Populus tomentosa stands. The reliability of the established analytical toolkit was further verified using a flow cytometer. We established a fast and reliable technique to screen polyploid poplars from natural stands.

Sequencing and Analysis of the Pseudomonas fluorescens GcM5-1A Genome: A Pathogen Living in the Surface Coat of Bursaphelenchus xylophilus.

It is known that several bacteria are adherent to the surface coat of pine wood nematode (Bursaphelenchus xylophilus), but their function and role in the pathogenesis of pine wilt disease remains debatable. The Pseudomonas fluorescens GcM5-1A is a bacterium isolated from the surface coat of pine wood nematodes. In previous studies, GcM5-1A was evident in connection with the pathogenicity of pine wilt disease. In this study, we report the de novo sequencing of the GcM5-1A genome. A 600-Mb collection of high-quality reads was obtained and assembled into sequence contigs spanning a 6.01-Mb length. Sequence annotation predicted 5,413 open reading frames, of which 2,988 were homologous to genes in the other four sequenced P. fluorescens isolates (SBW25, WH6, Pf0-1 and Pf-5) and 1,137 were unique to GcM5-1A. Phylogenetic studies and genome comparison revealed that GcM5-1A is more closely related to SBW25 and WH6 isolates than to Pf0-1 and Pf-5 isolates. Towards study of pathogenesis, we identified 79 candidate virulence factors in the genome of GcM5-1A, including the Alg, Fl, Waa gene families, and genes coding the major pathogenic protein fliC. In addition, genes for a complete T3SS system were identified in the genome of GcM5-1A. Such systems have proved to play a critical role in subverting and colonizing the host organisms of many gram-negative pathogenic bacteria. Although the functions of the candidate virulence factors need yet to be deciphered experimentally, the availability of this genome provides a basic platform to obtain informative clues to be addressed in future studies by the pine wilt disease research community.

Natural infectious behavior of the urediniospores of Melampsora larici-populina on poplar leaves

The uredinial stage in the life cycle of Melampsora larici-populina on poplar leaves is the most important pathogenic phase. We captured partial phases of uredinial infection in the wild, aiming to reconstruct the process of uredinial ontogeny by using scanning and transmission electron microscope. At the initial infection stage, germ tubes germinated from the echinulate urediniospores. Germ tubes were frequently seen to merge with the leaf surface and cuticle breakage was observed, indicating direct hyphal penetration. Stomata penetration occurred commonly, sometimes with more than one germ tube penetrating the same stoma. Melampsora larici-populina did not form appressoria in the infection process, implying that infectious behavior of this pathogen may differ from the other rust pathogens. In general, germ tubes branched randomly, and no distinct evidence indicated that stoma could induce or orient germ tube branches. However, oriented germ tube growth has been occasionally observed in other studies. The urediniospores collapsed and finally wizened when they became nutrient stressed. At the last stage of infection, the uredinia erupted from the leaf epidermis and appeared as orange pustules on the leaf surface.

A Highly Dense Genetic Map for Ginkgo biloba Constructed Using Sequence-Based Markers

Ginkgo biloba L. is a well-known living gymnosperm fossil that has medicinal and ornamental value. In this study, a high density genetic map was constructed with megagametophytes of 94 seeds from a single Ginkgo tree by employing the specific-locus amplified fragment (SLAF) sequencing technique. The average sequencing depth was 11.20×, which yielded 538,031 high-quality SLAFs. Among these SLAFs, 204,361 were heterozygous in the maternal tree and segregated in the progeny. The established map contained 12,263 SLAFs that were assigned to 12 linkage groups (LGs). The number of LGs on this map equaled the number of chromosomes in Ginkgo. The total map length was 1,671.77 cM, with an average distance of 0.89 cM between adjacent marker bins. Map evaluation based on the haplotype map and the heat map validated the high quality of the established map. Because Ginkgo is an economically and biologically important tree, strenuous efforts have been exerted to sequence its genome. This new map, built using sequence-based markers, will serve in the future as a fundamental platform for anchoring sequence assemblies along Ginkgo chromosomes. This map also provides a desirable platform for various genetic studies of Ginkgo, including gene/quantitative trait locus mapping and marker-aided selection.

Analyzing the Nucleotide Variations within the Expressed Sequence Tags of Loblolly Pine (Pinus taeda)

Single nucleotide polymorphisms (SNPs) represent the most frequent variations in eukaryotic genomes. For example, the frequency of SNPs is one per kilobase in human [1], one every 78 base pair (bp) in grapevine [2], and one every 43 bp in maize [3]. SNPs have proven to be useful genetic tools in many genetic studies, including molecular breeding, population genomics, genetic diversity, and cultivar identification [4].