Xinwang Wang

Functional Genomics Analysis of Horseweed (Conyza canadensis) with Special Reference to the Evolution of Non-Target-Site Glyphosate Resistance

Abstract The evolution of glyphosate resistance in weedy species places an environmentally benign herbicide in peril. The first report of a dicot plant with evolved glyphosate resistance was horseweed, which occurred in 2001. Since then, several species have evolved glyphosate resistance and genomic information about nontarget resistance mechanisms in any of them ranges from none to little. Here, we report a study combining iGentifier transcriptome analysis, cDNA sequencing, and a heterologous microarray analysis to explore potential molecular and transcriptomic mechanisms of nontarget glyphosate resistance of horseweed. The results indicate that similar molecular mechanisms might exist for nontarget herbicide resistance across multiple resistant plants from different locations, even though resistance among these resistant plants likely evolved independently and available evidence suggests resistance has evolved at least four separate times. In addition, both the microarray and sequence analyses identified non–target-site resistance candidate genes for follow-on functional genomics analysis. Nomenclature: Glyphosate, horseweed, Conyza canadensis (L.) Cronq. ERICA

Variability of United States Isolates of Macrophomina phaseolina Based on Simple Sequence Repeats and Cross Genus Transferability to Related Genera Within Botryosphaeriaceae

Twelve simple sequence repeat (SSRs) loci were used to evaluate genetic diversity of 109 isolates of Macrophomina phaseolina collected from different geographical regions and host species throughout the United States (US). Genetic diversity was assessed using Nei’s minimum genetic distance, and the usefulness of each locus was determined by calculating the polymorphism information content (PIC). A total of 98 alleles were detected and of these 31 were unique to individual genotypes. Eight of twelve loci were highly informative with PIC values greater than 0.50. The majority of pairwise comparisons of genetic distance were greater than 0.60 indicating moderate to high genetic diversity. Dendrograms based on the genetic dissimilarities were created for the 109 isolates of which 79 were from soybean. Some clustering by host and geography was noted, but, the dendrograms generally grouped isolates independent of host or geography. Additionally, sequencing of the internal transcribed spacer region (ITS) for 10 isolates revealed that all of these isolates were 99% similar. Three SSR loci from M. phaseolina were cross amplified in other genera in the Botryosphaeriaceae. This was the first study of genotyping and assessing genetic diversity of M. phaseolina isolates collected from a widespread host and geographic range across the US with SSRs. With an additional 34 loci publically available for M. phaseolina, the results indicate that previously developed SSRs from one species can be used in future population, ecological, and genetic studies of M. phaseolina and other genera within the Botryosphaeriaceae.

Development and characterization of simple sequence repeats for flowering dogwood (Cornus florida L.)

Abundant, codominant simple sequence repeats (SSRs) markers can be used for constructing genetic linkage maps and in marker-assisted breeding programs. Enrichment methods for SSR motifs were optimized with the ultimate aim of developing numerous loci in flowering dogwood (C. florida L.) genome. Small insert libraries using four motifs (GT, CT, TGG, and AAC) were constructed with C. florida ‘Cherokee Brave’ deoxyribonucleic acid (DNA). Colony polymerase chain reaction (PCR) of 2,208 selected clones with three primers we reported previously indicated that 47% or 1,034 of the clones harbored one of the four targeted SSR motifs. Sequencing the putative positive clones confirmed that nearly 99% (1,021 of 1,034) of them contained the desired motifs. Of the 871 unique SSR loci, 617 were dinucleotide repeats (70.8%), and 254 were trinucleotide or longer repeats (29.2%). In total, 379 SSR loci had perfect structure, 237 had interrupted, and 255 had compound structure. Primer pairs were designed from 351 unique sequences. The ability of the 351 SSR primer pairs to amplify specific loci was evaluated with genomic DNA of ‘Appalachian Spring’ and ‘Cherokee Brave’. Of these primers, 311 successfully amplified product(s) with ‘Cherokee Brave’ DNA, 21 produced weak or faint products, and 19 did not amplify any products. Additionally, 218 of the 311 primers pairs revealed polymorphisms between the two cultivars, and 20 out of 218 primers detected an average of 13.7 alleles from 38 selected Cornus species and hybrids. These SSR loci constitute a valuable resource of ideal markers for both genetic linkage mapping and gene tagging of flowering dogwood.

Genetic diversity of flowering dogwood in the Great Smoky Mountains National Park

In the past three decades, flowering dogwood (Cornus florida) populations have experienced severe declines caused by dogwood anthracnose. Mortality has ranged from 48% to 98%, raising the concern that the genetic diversity of this native tree has been reduced significantly. In this study, we investigated levels of genetic diversity and population structure of flowering dogwood populations in the Great Smoky Mountains National Park (GSMNP). Understanding the factors influencing geographic distribution of genetic variation is one of the major concerns for preserving biodiversity and conservation of native populations. Eighteen microsatellite loci were used to evaluate the level and distribution of genetic variation of native flowering dogwood trees throughout the GSMNP. Significant genetic structure exists at both landscape and local levels. Two genetic clusters exist within the park and are separated by the main dividing ridges of the Great Smoky Mountains. The differentiation of the clusters is subtle, but statistically significant. Gene flow, evident through low-elevation corridors, indicates that nonrandom mating occurs between related individuals despite wide dispersal of seeds. Although high mortality rate and reduced fecundity caused by dogwood anthracnose severely affected native flowering dogwood populations throughout the entire GSMNP, this study confirmed that considerable genetic diversity still exists at the population level. It seems unlikely that recent demographic dynamics have resulted in a depletion of genetic variation.

Analysis of genetic diversity in flowering dogwood natural stands using microsatellites: the effects of dogwood anthracnose.

Flowering dogwood (Cornus florida L.) populations recently have experienced severe declines caused by dogwood anthracnose. Mortality has ranged from 48 to 98%, raising the concern that genetic diversity has been reduced significantly. Microsatellite data were used to evaluate the level and distribution of genetic variation throughout much of the native range of the tree. Genetic variation in areas affected by anthracnose was as high as or higher than areas without die-offs. We found evidence of four widespread, spatially contiguous genetic clusters. However, there was little relationship between geographic distance and genetic difference. These observations suggest that high dispersal rates and large effective population sizes have so far prevented rapid loss of genetic diversity. The effects of anthracnose on demography and community structure are likely to be far more consequential than short-term genetic effects.

Analyzing Microsatellites Using the QIAxcel System

Microsatellites are ubiquitous throughout eukaryotic genomes and are useful in analyzing populations and genetic diversity. The QIAxcel system, an automated capillary electrophoresis device, allows the user to determine the size of microsatellite fragments, to discern allelic polymorphisms among individuals, and to differentiate homozygous and heterozygous individuals. This system provides comparable base pair resolution to more expensive systems at a relatively affordable cost.

Biomass feedstock: diversity as a solution

1Department of Plant Pathology and Microbiology, Texas A & M University, College Station, TX 77843, USA 2Institute for Plant Genomics and Biotechnology, Texas A & M University, College Station, TX 77843, USA 3Advanced Research Institute for Sustainable Energy, Texas A & M University, College Station, TX 77843, USA 4Department of Horticulture, Texas A & M University, College Station, TX 77843, USA 5Texas Agrilife Dallas Center, Texas Agrilife Research, Dallas, TX, USA 6Department of Plant Sciences, University of Tennessee, Knoxville, TN , USA 7BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, USA †Author for correspondence: E-mail: syuan@tamu.edu Biomass-based biofuel production has emerged as a major approach to facilitate energy independence, reduce GHG emissions, revitalize rural communities and enhance sustainable economic development [1–3]. Despite the importance of biomass conversion improvement, feedstock development is also crucial in developing an economically viable and sustainable biofuel production system. Bioenergy feedstock development includes germplasm selection, molecular breeding and genetic modification of proper feedstock for biofuel production [1,4]. The development and implementation of lignocellulosic biofuel is hindered by several major challenges in feedstock development. The first challenge is feedstock availability, which translates into the need for increased use of marginal land and the development of a diverse range of feedstocks suitable for different niches. The second challenge is sustainability. We need biofuels that can ultimately reduce GHG emissions, that have a positive net energy balance Joshua S Yuan†1,2,3, Xinwang Wang4,5 & C Neal Stewart Jr6,7

Microsatellites from the charcoal rot fungus (Macrophomina phaseolina)

Microsatellite loci were identified from the charcoal rot fungus (Macrophomina phaseolina). Primer pairs for 46 loci were developed, and of these, 13 were optimized and screened using genomic DNA from 55 fungal isolates collected predominantly from two soybean fields in Mississippi. Twelve of the optimized loci were polymorphic and the number of alleles per locus ranged from 6 to 22. These microsatellites will be useful in population and pathogenicity studies to correspond with development of potential disease‐resistant soybean and other susceptible crops.

Microsatellites from kousa dogwood (Cornus kousa)

Microsatellite loci were identified from Cornus kousa‘National’. Primer pairs for 86 loci were developed and of these, eight were optimized and screened using genomic DNA from 22 kousa cultivars. All optimized loci were polymorphic and the number of alleles per locus ranged from three to 17. Observed heterozygosity ranged from 0 to 0.3 and expected heterozygosity ranged from 0.38 to 0.91. These microsatellites will be useful in population studies, and a breeding programme for cultivar development of Cornus species.

Transcriptomes That Confer to Plant Defense against Powdery Mildew Disease in Lagerstroemia indica.

Transcriptome analysis was conducted in two popular Lagerstroemia cultivars: “Natchez” (NAT), a white flower and powdery mildew resistant interspecific hybrid and “Carolina Beauty” (CAB), a red flower and powdery mildew susceptible L. indica cultivar. RNA-seq reads were generated from Erysiphe australiana infected leaves and de novo assembled. A total of 37,035 unigenes from 224,443 assembled contigs in both genotypes were identified. Approximately 85% of these unigenes have known function. Of them, 475 KEGG genes were found significantly different between the two genotypes. Five of the top ten differentially expressed genes (DEGs) involved in the biosynthesis of secondary metabolites (plant defense) and four in flavonoid biosynthesis pathway (antioxidant activities or flower coloration). Furthermore, 5 of the 12 assembled unigenes in benzoxazinoid biosynthesis and 7 of 11 in flavonoid biosynthesis showed higher transcript abundance in NAT. The relative abundance of transcripts for 16 candidate DEGs (9 from CAB and 7 from NAT) detected by qRT-PCR showed general agreement with the abundances of the assembled transcripts in NAT. This study provided the first transcriptome analyses in L. indica. The differential transcript abundance between two genotypes indicates that it is possible to identify candidate genes that are associated with the plant defenses or flower coloration.