MULTI-LOCUS SIMPLE SEQUENCE REPEAT (SSR) MARKERS FOR GENOTYPING AND ASSESSING GENETIC DIVERSITY OF XYLELLA FASTIDIOSA IN CALIFORNIA Project Leader:

We have designed and developed Xylella fastidiosa (Xf) Simple Sequence Repeat (SSR) primers. Thirty-four of them have been validated and are available to public (Lin et al. 2005). These primers are Xf-specific and powerful for detecting polymorphism among and within crop-associated Xf strains and can be used for Xf genotyping, population structure and genetic diversity studies. Recently, we used fluorescent-labeled primers for PCR and an ABI 3100 genetic analyzer in combination with our rapid sample preparation protocol to create a high-throughput Xf pathogen diagnostic and genetic analysis platform. We used this marker system to study the geographic population structures of grape Xf strains in California. We also used this marker system as a tool to study interactions between Vitis and Xf in Pierce’s disease (PD) resistant and susceptible grapes. INTRODUCTION Understanding pathogen genetic diversity is critical in developing an effective disease control strategy. Host plant resistance is one of the most important components in integrated crop management. However, the durability of disease resistance depends upon the variability and adaptability of pathogen populations and their interactions with host plant resistance genes. Variation in pathogen population structure can lead to resistance breakdown and disease outbreak under suitable environmental conditions. The molecular basis of plant host-Xf interactions needs to be investigated to better understand the epidemiology of Xf-induced diseases and how Xf strains interact to cause PD in different grape cultivars. Given the fact that diseases caused by Xf are complex patho-systems with a wide range of symptomatic as well as asymptomatic hosts, a number of insect vectors with wide host ranges, and variable environmental factors, the genetic diversity and biological relationships of Xf strains with different grape cultivars needs to be better understood. The goal of this project is to develop a reliable marker system that unambiguously identifies Xf strains from various geographic locations and host plants. Coletta-Filho et al. (2001 and 2003) developed simple sequence repeat markers from CVC Xf sequences and used them for CVC Xf population genetic studies. Here we designed SSR markers from four available Xf genomic sequences that work with all Xf strains. We further developed this marker system into a fluorescent-based multiplex genotyping format. Using a rapid DNA isolation method, we directly analyze Xf from infected plant tissues therefore avoiding the time-consuming bacterial isolation step and reducing the chance of sample loss due to contamination and culture difficulties. This system has proven to be powerful and reliable for distinguishing genetic relatedness. The sensitivity, specificity, and power in detecting polymorphism, as well as its adaptability to a high through-put diagnostic platform makes this system an ideal tool for large scale studies of Xf population genetics and epidemiological risk assessment analyses. OBJECTIVES 1. Develop a high through-put multi-locus Xf genetic analysis system for genotyping and analyzing population structures of Xf in California. 2. Analyze genetic diversity and structure of Xf populations. Construct a large Xf allele frequency database for use as an Xf strain identification system. 3. Use the SSR Marker analysis system to study the interactions between hosts and Xf including adaptation, host selection and pathogenicity of Xf strains. RESULTS Objective 1 To develop an accurate and high through-put system for Xf genetic analysis, we combined fluorescentlabeled primers for PCR and analyzed them with a 16-capillary DNA sequence analyzer (ABI 3100). Each dye set consists of four primers labeled with FAM, NED, HEX and VIC respectively (Figure 1A). Therefore, data output is four times (96 x 4 = 384) more than that obtained with a single dye (96 samples) per run. To be accurate in determining allele size, an internal molecular sizing marker labeled with LIZ was co-separated with samples through each capillary tube. GeneMapper software identifies