Rick Kesseli

PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp.

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp. Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L. sativa and 13 accessions of the wild species L. serriola, L. saligna, L. virosa, L. perennis, and L. indica. The same genotypes were analyzed as in a previous study that had utilized RFLP markers. The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data. The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis. There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data. AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels.

Population structure delineated with microsatellite markers in fragmented populations of a tropical tree, Carapa guianensis (Meliaceae)

Deforestation and selective logging in the tropics may have serious consequences on genetic processes in tropical tree populations, affecting long‐term survival of a given species as well as tropical forest communities. Because understanding the effects of human‐induced changes on genetic processes is of utmost importance in formulating sound conservation and management plans for tropical forest communities, we developed microsatellite or simple sequence repeat (SSR) markers for the tropical tree Carapa guianensis (Meliaceae) and assessed the polymorphism of SSRs in adult and sapling populations in a large contiguous forest and in selectively logged and fragmented forests. The number of alleles in polymorphic loci ranged between 4 and 28. No inbreeding was detected in saplings or adult cohorts, but the allelic richness was lower in the sapling cohort of the isolated fragment. Genetic distances, Nei’s D and (δµ)2, and RST values among saplings were greater than among adult cohorts, suggesting restriction of gene flow due to deforestation and habitat fragmentation. These SSR loci may be used to address many related questions regarding the population and conservation genetics of tropical trees.

Conservation of microsatellites among tropical trees (Leguminosae)

Although microsatellites or simple sequence repeats (SSRs) have become a popular tool in genetic mapping and gene flow studies, their utility is limited due to paucity of information about DNA sequences in plants. We tested the utility of microsatellite markers characterized for the tropical tree Pithecellobium elegans as a genetic tool for related species. The results indicate that SSR loci are conserved among closely related species, and SSR primers developed for P. elegans could be successfully used as a genetic tool in several species of the tribe Ingeae. This study indicates that there is high potential for the transfer of SSR markers among closely related taxa, circumventing laborious cloning and screening procedures involved in characterizing SSR loci for many species.

Sexual reproduction in the invasive species Fallopia japonica (Polygonaceae)

Fallopia japonica (Polygonaceae) is an invasive perennial plant, well known in North America for its ability to spread aggressively via vegetative reproduction. The contribution of sexual reproduction to the distribution of this species is not well documented, and as a result, F. japonica is treated solely as a clonal species. To investigate the role of sexual reproduction in this species, germination experiments were conducted using seed collected from 29 parents from field sites in Massachusetts and from four greenhouse-grown cultivars. Results showed that wild F. japonica produce large quantities of seed that typically have high germinability. This seed is viable whether sown immediately after collection or subjected to various conditions during the winter season and germinated the following spring. Cultivars of F. japonica also produce viable seed and can thus contribute to the invasiveness of this species. In addition, wild F. japonica seedlings were observed at several field sites, with several of these seedlings surviving the winter and resprouting the following spring. That sexual reproduction and seedling survival occur in the wild has strong implications for the development of management strategies for this species.

Genetic diversity and genomic distribution of homologs encoding NBS-LRR disease resistance proteins in sunflower

Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encode nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from sunflower (Helianthus annuus L.), and most of the previously identified homologs are members of two large NBS-LRR clusters harboring downy mildew R-genes. We mined the sunflower EST database and used comparative genomics approaches to develop a deeper understanding of the diversity and distribution of NBS-LRR homologs in the sunflower genome. Collectively, 630 NBS-LRR homologs were identified, 88 by mining a database of 284,241 sunflower ESTs and 542 by sequencing 1,248 genomic DNA amplicons isolated from common and wild sunflower species. DNA markers were developed from 196 unique NBS-LRR sequences and facilitated genetic mapping of 167 NBS-LRR loci. The latter were distributed throughout the sunflower genome in 44 clusters or singletons. Wild species ESTs were a particularly rich source of novel NBS-LRR homologs, many of which were tightly linked to previously mapped downy mildew, rust, and broomrape R-genes. The DNA sequence and mapping resources described here should facilitate the discovery and isolation of recognition-dependent R-genes guarding sunflower from a broad spectrum of economically important diseases.

Sources and genetic structure of a cluster of genes for resistance to three pathogens in lettuce

The second largest cluster of resistance genes in lettuce contains at least two downy mildew resistance specificities, Dm5/8 and Dm10, as well as Tu, providing resistance against turnip mosaic virus, and plr, a recessive gene conferring resistance against Plasmopara lactucae-radicis, a root infecting downy mildew. In the present paper four additional genetic markers have been added to this cluster, three RAPD markers and one RFLP marker, CL1795. CL1795 is a member of a multigene family related to triose phosphate isomerase; other members of this family map to the other two major clusters of resistance genes in lettuce. Seven RAPD markers in the region were converted into sequence characterized amplified regions (SCARs) and used in the further analysis of the region and the mapping of Dm10. Three different segregating populations were used to map the four resistance genes relative to molecular markers. There were no significant differences in gene order or rate of recombination between the three crosses. This cluster of resistance genes spans 6.4 cM, with Dm10 1.2 cM from Dm8. Marker analysis of 20 cultivars confirmed multiple origins for Dm5/8 specificity. Two different Lactuca serriola origins for the Du5/8 specificity had previously been described and originally designated as either Dm5 or Dm8. Some ancient cultivars also had the same specificity. Previously, due to lack of recombination in genetic analyses and the same resistance specificities, it was assumed that Dm5 and Dm8 were determined by the same gene. However, molecular marker analysis clearly identified genotypes characteristic of each source. Therefore, Dm5/8 specificity is either ancient and widespread in L. serriola and some L. sativa, or else has arisen on multiple occasions as alleles at the same locus or at linked loci.

Universal markers for comparative mapping and phylogenetic analysis in the Asteraceae (Compositae)

The development of universal markers that can be assayed across taxa, but which are polymorphic within taxa, can facilitate both comparative map-based studies and phylogenetic analyses. Here we describe the development of such markers for use in the Asteraceae, which includes the crops lettuce, sunflower, and safflower as well as dozens of locally important crop and weed species. Using alignments of a conserved orthologous set (COS) of ESTs from lettuce and sunflower and genomic sequences of Arabidopsis, we designed a suite of primer pairs that are conserved across species, but which are predicted to flank introns. We then tested 192 such primer pairs in 8 species from across the family. Of these, 163 produced an amplicon in at least 1 taxon, and 125 amplified in at least half of the taxa surveyed. Thirty-nine amplified in all 8 species. Comparisons amongst sequences within the lettuce and sunflower EST databases indicate that the vast majority of these loci will be polymorphic. As a direct test of the utility of these markers outside the lettuce and sunflower subfamilies, we sequenced a subset of ten loci from a panel of cultivated safflower individuals. All 10 loci proved to be single-locus, and nine of the 10 loci were polymorphic with an average of 12.8 SNPs per kb. Taken together, these loci will provide an initial backbone for comparative genetic analyses within the Asteraceae. Moreover, our results indicate that these loci are phylogenetically informative, and hence can be used to resolve evolutionary relationships between taxa within the family as well as within species.

Genomics of Compositae weeds: EST libraries, microarrays, and evidence of introgression.

PREMISE OF STUDY Weeds cause considerable environmental and economic damage. However, genomic characterization of weeds has lagged behind that of model plants and crop species. Here we describe the development of genomic tools and resources for 11 weeds from the Compositae family that will serve as a basis for subsequent population and comparative genomic analyses. Because hybridization has been suggested as a stimulus for the evolution of invasiveness, we also analyze these genomic data for evidence of hybridization. METHODS We generated 22 expressed sequence tag (EST) libraries for the 11 targeted weeds using Sanger, 454, and Illumina sequencing, compared the coverage and quality of sequence assemblies, and developed NimbleGen microarrays for expression analyses in five taxa. When possible, we also compared the distributions of Ks values between orthologs of congeneric taxa to detect and quantify hybridization and introgression. RESULTS Gene discovery was enhanced by sequencing from multiple tissues, normalization of cDNA libraries, and especially greater sequencing depth. However, assemblies from short sequence reads sometimes failed to resolve close paralogs. Substantial introgression was detected in Centaurea and Helianthus, but not in Ambrosia and Lactuca. CONCLUSIONS Transcriptome sequencing using next-generation platforms has greatly reduced the cost of genomic studies of nonmodel organisms, and the ESTs and microarrays reported here will accelerate evolutionary and molecular investigations of Compositae weeds. Our study also shows how ortholog comparisons can be used to approximately estimate the genome-wide extent of introgression and to identify genes that have been exchanged between hybridizing taxa.

Rapid growth and out-crossing promote female development in zebrafish (Danio rerio)

Sex determination in fishes is often enigmatic, a situation that is often made even more complex by the fact that the process of sexual differentiation in many species may be influenced by environmental conditions. This situation is typified in zebrafish, a popular model organism. Despite the vast array of information available for the species, the genetic controls of sex are unknown. Further, environmental parameters, such as rearing densities, seem to exert an influence on the sex ratios of captive stocks. In an effort to dissect the genetic and environmental controls underlying the expression of sex in this species, we manipulated growth of pure-bred and out-crossed zebrafish by varying their food supply during development. Faster-growing zebrafish were more likely to be female than siblings that were fed less, and out-crossed broods had higher proportions of females than broods from pure-bred crosses. The dependence of sex ratio on feeding rate is readily understood in terms of adaptive sex allocation: zebrafish life history seems to confer the greater pay-off for large size on females. A similar male/female difference in the pay-off for hybrid vigor could similarly account for the female bias of out-crossed broods—and it could be a manifestation of Haldane’s rule.

Genetic diversity and clonal vs. sexual reproduction in Fallopia spp. (Polygonaceae)

Although fundamental to the study of invasion mechanisms, the relationship between mode of reproduction and plant invasion is not well understood. Fallopia japonica (Japanese knotweed), a highly aggressive invasive plant in both Europe and North America, serves as a model species for examining this relationship. In Britain, F. japonica var. japonica is a single female clone reproducing solely through vegetative growth or obligate hybridization with other Fallopia spp. In the U.S., however, there is more evidence for sexual reproduction. Here, simple sequence repeat (SSR) markers were developed, and three Massachusetts populations were sampled at regular intervals. The amount of sexual and clonal reproduction in each population was determined based on within-population genetic diversity. Clonal growth was apparent, but the populations together contained 26 genotypes and had evidence of sexual reproduction. One genotype that was present in all populations matched the single aggressive British clone of F. japonica var. japonica. Also, a potentially diagnostic marker for the F. sachalinensis genome provided evidence of inter- and intraspecific sexual reproduction and introgression. These differences observed in U.S. populations compared to European populations have significant implications for management of Fallopia spp. in the U.S. and underscore the importance of regional studies of invasive species.