Sol Genomics Network Marker Search Tutorial

Hello, my name is Heather Merk. I am the content coordinator for the plant breeding and genomics community of practice on e-extension. Today, I will demonstrate how to find the marker search tool on the Sol Genomics Network and also how to use this tool. The Sol Genomics Network, also referred to as SGN, can be found at http ://solgenomics.net SGN houses numerous databases with a wealth of Solanaceae related information, from molecular markers, and genetic and physical maps, to genes, metabolic pathways, and entire genomes. Although SGN was designed with basic researchers in mind, there are tools on SGN that are relevant to breeders. These are housed in the Breeders Toolbox. The Breeders Toolbox provides direct links to breeder relevant tools and tutorials. There are currently four tools in the Breeders Toolbox. a trait search, a marker search, a QTL analysis tool, and a SNP tool. In addition, if we scroll down the page, we can find the SGN related tutorials that have been developed specifically for breeders. When the current tutorial was developed, a webinar tutorial for the tomato genome browser was available, as was a slideshare tutorial for the CAPS designer tool. Additional tutorials are being developed. This tutorial focuses on the marker search tool, called “markers” in the Breeders Toolbox. Using the markers tool, you can search for more information about a specific marker or you can search for markers based on a number of selection criteria. This tutorial provides examples of both cases. Note that you can not currently use this tool to identify markers associated with a trait. The search tool is loaded after clicking on the markers button in the Breeders Toolbox. Alternatively, you can get to the marker search tool directly by copying and pasting the link provided at the bottom of the screen. The remainder of the tutorial will consist of examples of searches you may want to conduct using the SGN marker search tool. I will first demonstrate searches by marker name. If you already know how to search by marker name, you may want to skip ahead to slide 12, when I demonstrate how to search by marker criteria. To search for a marker by name, type the name of the marker in the text box and click search. In this example, I am interested in finding more about the marker SSR74. To perform the search, I type SSR74 circled in red, in the search box. I am using the default settings. The marker name starts with the text in the search box and I only want to find markers that are mapped. The marker search results are shown at the top of the page. In this example, there is one result for SSR74. The result provides five pieces of information about the marker: the protocol, map, chromosome, position, and confidence. The protocol is the marker assay used. In this case, SSR74 was assayed as an SSR marker. The map is the map onto which the marker has been positioned. In this case, SSR74 was positioned on the tomato EX-PEN 2000 map. This is a high density tomato map with over 2000 markers. It is an F2 map developed using 80 individuals generated in a cross between Solanum lycopersicum accession LA925 and Solanum pennellii accession LA716. Chromsome is the chromosome to which the marker has been mapped. In this example, SSR74 was mapped to chromosome 10. Position is the chromosome position the marker has been mapped to. In this case, SSR74 was mapped to position 74. Confidence is the level of statistical certainty with which the marker has been mapped. FLOD3 is the highest confidence level, while one and uncalculated are the lowest confidence levels. In this example, the confidence level is one LOD two. If desired, you can download the results as a text file by clicking on the link “download these results as a text file.” Clicking on the SSR74 link will give even more information about SSR74 as well see in the next slide. The extent of the information provided in the detailed search results depends on what is known about a particular marker. I am using SSR74 as an example of a typical marker on SGN. At the top of the search results we find information relevant for genotyping SSR74. For example, low and high annealing temperatures, forward and reverse primers, and predicted product sizes are listed. The approximate map position of SSR74 is visually displayed. We can also learn about the sequence SSR74 was derived from by clicking on the EST read SGN-E157993 link. We can also learn more about the map by clicking on the tomato EX-PEN 2000 v52 link. If we scroll down the search results page we will find information about SSR74 obtained from analysis of the tomato genome sequence in a genome browser, or GBROWSE, format. In the case of SSR74, there is information from three releases of the tomato genome: ITAG1, ITAG2, and ITAG2.3. ITAG is an abbreviation for the International Tomato Annotation Group. It is typically best to consult the most up to date information, so we will focus on the ITAG2.3 release. On the next slide we will delve into the information from the ITAG2.3 release using the tomato genome browser. When using the tomato genome browser, it is often helpful to work from the top down. The first thing to note is that SSR74 falls within a gene. Knowing that SSR74 falls within a gene, a next logical question is “what is the function of that gene?” The gene models information is a prediction of genes based on analysis of the DNA sequence and its similar to the sequence of known genes and gene families. Based on gene models, SSR74 is predicted to be a part of a glycosyl transferase gene. Looking further down the browser, the SGN unigenes are gene sequences determined from multiple expressed sequence tag, or EST, reads. The unigene information also indicates that SSR74 is part of a gene. Knowledge of the gene within which a marker is located can be highly informative if for example, the marker is highly correlated with a phenotype. To find out more information about a gene model or unigene, such as the DNA sequence, mRNA sequence, location of introns and exons, and gene ontology terms, simply click on the gene name. You can learn more about using the tomato genome browser by clicking on the tutorial link of the main page of the Breeders Toolbox. To search by marker criteria, we must return to the marker search page and use the advanced search options. SGN has provided a help page that help users understand the search criteria for more effective searching. Descriptions are provided for each of the items that are numbered and circled. These descriptions can be found by scrolling down the help page. In some cases these descriptions will be more in-depth and technical than those provided in this tutorial. Let’s now return to the marker search tool. There are two categories of advanced search criteria: marker options and map locations. We will explore each of these categories. The first set of criteria in the marker options has to do with BACs, which are bacterial artificial chromosomes. BACs have segments of DNA that was inserted using molecular biology techniques. On SGN, the inserted DNA segments are from Solanaceous species. We can choose to search for markers associated with BACs by checking the box beside the red arrow. When this tutorial was developed, the search identified 19, 443 markers. We can narrow our search of markers associated with BACs to search for markers that have been associated with BACs according to three criteria: as overgos, computationally, and manually. In the case of association as an overgo, a marker has been experimentally associated with a BAC. In the case of computational association, both the marker and the BAC have been at least partially sequenced and the sequences aligned. In the case of manual association, the marker and BAC have been associated using some other method. In addition to searching for markers associated with BACs, we can search for markers mapped in a particular species, markers genotyped using a specific assay, or markers that belong to a group designated by SGN. These groups include conserved orthologous sequences (COSI and COSII) that are present in two or more species, like Arabidopsis and tomato. We can select multiple options within a category by holding the control key on a PC or the command key on a Mac, while clicking. Using the map locations search options, we can limit our search to markers mapped to a specific chromosome, a range of map positions on a specific chromosome in centimorgan units, the confidence with which markers have been mapped, and markers localized to specific maps. When conducting these searches, we can specify as many search criteria as we wish. Any search criteria that we haven’t specific will not be included in our search. As an example, we will search for CAPS markers that have been mapped in potato or pepper. This search will not take into consideration the BAC association criteria, marker group, or any of the map locations criteria. Our search for CAPS markers that have been mapped in potato or pepper brings up 4,827 results. The first 30 results are shown on the first page. The remaining results are shown on subsequent pages. Like the results when we searched by marker name, the marker name, protocol, map, chromosome, position, and confidence are shown. More detailed information can be seen by clicking on a marker. As well, the results can be downloaded as a text file. In summary, we can use the marker search tool of the SGN Breeders Toolbox to search for markers by their name or other criteria, such as the marker location.