microsatellite information exchange

for Brassica


Curated by Graham King, Rothamsted Research, 2006-2010
Originally prepared by Charlotte Allender, HRI, Wellesbourne; Jan 2004

Introduction

As part of the Brassica GeneFlow Consortium project link funded by the BBSRC link and NERC link under the 'Gene Flow in Plants and Microorganisms' initiative, primer and associated information was originally collated by Charlotte Allender for 618 Brassica microsatellites (including those developed and tested in Arabidopsis) currently in the public domain. A summary of available map locations and polymorphism data is also presented. This information is given below together with previously unpublished screening and diversity data collected at HRI. Addititional sets of public domain SSRs are being added as they appear or we are notified.

Contents

An Introduction to Microsatellites

Synonyms : SSR (Simple Sequence Repeat) ; STR (Short Tandem Repeat)

Microsatellites are sections of DNA composed of repeats of short motifs (e.g. CA, GTG, TGCT etc) arranged in tandem (see below). The sequence surrounding the repeat region is usually conserved, allowing PCR primers to be designed so that the repeat region and a short flanking sequence can be amplified. Individuals may differ in the number of repeats present, meaning that the length of the PCR product varies. Products can be scored using various forms of high resolution electrophoresis (polyacrylamide or semi automated sequencers). Used as molecular markers, they are co-dominant, often highly polymorphic and relatively easy to score. The high polymorphism of microsatellites is attributed to relatively high rates of error during DNA replication (slippage) and during recombination (unequal crossover).


Figure 1. Representation of a CTT (trinucleotide) microsatellite and flanking region. The position of the PCR primers is indicated. Three length variants are shown (A, B and C).

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Purpose of this Web Page

The purpose of this web page is to provide a summary of available information for all known Brassica microsatellites, and to provide a means for information on Brassica microsatellites to be exchanged between researchers.

There have been numerous attempts to isolate microsatellites from various Brassica species, ranging from large consortium projects to small experimental investigations that have located microsatellites as a by-product of other studies. Very few Brassica microsatellite sequences have been deposited in databases such as Genbank, meaning that there is no simple way of finding details of microsatellites. Most projects have released details of primer sequences into the public domain (either through publication or via the internet), however several projects have not done so. Details of these projects are included below.

The microsatellites described below have been subjected to various levels of characterisation. This ranges from ascertaining that reproducible fragments are amplified using the PCR primers, testing the primers in single or multiple Brassica species to determine the number and sizes of the fragments produced and finally locating the position(s) of the microsatellite on a genetic map.

Microsatellites are used as DNA markers for a variety of purposes. They can be used to construct genetic maps, for example to investigate QTLs for various traits. They may also be used to investigate genetic diversity, both within and between species. Microsatellites are also suitable markers to investigate intra-specific gene-flow and demographic parameters such as population size fluctuation. They may also be suitable for phylogenetic studies. However, not all microsatellites are useful for all types of investigation. Some may not show enough polymorphism, either within or between species, to be useful in any of the above types of study. Most statistical analyses for studies of evolutionary history and/or population demography require that each primer pair amplify a single, distinct locus. However, due to the duplications that have occurred during the evolutionary history of Brassicas, and the amphidiploid nature of species such as B. napus and B. carinata, a significant number of primer pairs amplify two or more loci. If the allele size ranges of these loci overlap then it becomes very difficult to assign a fragment to a particular locus. In addition, the design of the PCR primers may allow the amplification of multiple fragments, either through mis-annealing at a single locus or by annealing to multiple sites and producing non-specific products containing no microsatellite. However primer pairs that amplify multiple loci may be useful for other purposes such as individual, variety or species identification as more unique alleles may be identified in this way. It is therefore important to select microsatellite primer pairs that result in data compatible with the problem under investigation.

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Brassica microsatellites

Summary details (including primer sequences and repeat classes) of the various sets of Brassica microsatellite markers are given below, together with details of original references. A brief description to the background of each set is provided, along with an indication of the number of microsatellites contained. In some publications, details of primer pairs that failed to yield scorable PCR products were included - these have not been included on the lists below. In all cases, for more detailed information please refer to the original paper as this web page is intended as an indication of the markers currently available.

Microsatellite Set Number in Public Domain
BBSRC Microsatellite Programme 397
HRI set 6
Kresovich and Szewc-McFadden 24
Lagercrantz et al (1993) 5
AAFC Consortium (commercial) 80
Plieske and Struss (2001) 0
Suwabe et al (2002) and (2003) 38
Uzanova and Ecke (1999) 8
Bell and Ecker (1994) (Arabidopsis microsatellites) 30
INRA Versailles (Arabidopsis microsatellites) 120
Celera Consortium (commercial) 171
Total 799

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BBSRC Microsatellite Programme

This project resulted in the isolation and description of 397 microsatellites from B. oleracea, B. rapa, B. napus and B. nigra. Details are held in the online database BrassicaDB link. A small number of these are characterised in Lowe et al (2002) word. Further details of each microsatellite in this set were published more recently by Lowe et al (2003) word.

Microsatellites developed at HRI

This group of six microsatellites was developed at HRI by different researchers (Guy Barker, Neil Periam, Lee Smith).

Kresovich word and Szewc-McFadden word

This set contains 24 microsatellites that were isolated from B. napus and are described in two papers; Kresovich et al (1995) and Szewc-McFadden et al (1996).

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Lagercrantz et al (1993) word

This group of 5 microsatellites was isolated from B. napus

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AAFC Consortium

This commercial consortium is releasing 80 microsatellites into the public domain in January 2003 link. They will be free to use for academic research (subject to a Material Transfer Agreement). A link to the AAFC Brassica/Arabidopsis Genomics Initiative is given below.

Plieske and Struss (2001) word and Saal et al (2001) word

This group consists of 81 microsatellites originally isolated from B. napus. Plieske and Struss (2001) provide the original description, and amplification and polymorphism tests on various Brassica and related species. A subset of these markers were mapped in B. oleracea (Saal et al, 2001). However, no details of specific primer sequences have been released.

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Suwabe et al (2002) word

This group of 38 microsatellites was isolated from B. rapa. A further two microsatellites were published in Suwabe et al (2003). word More information on the whole set is presented as a poster at the PAGXII conference (click here link for details).

Uzanova and Ecke (1999) word

These 8 microsatellites were isolated from B. napus.

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Bell and Ecker (1994) word

These microsatellites were originally isolated from Arabidopsis thaliana. Some primers were designed to microsatellites located in genomic sequences published in Genbank, others were isolated from an A. thaliana library. There is a total of 30 microsatellites in this set. These Arabidopsis microsatellites are included here as many of the primer pairs will amplify products in Brassica spp but a more detailed investigation (Westman and Kresovich, 1998) revealed that many of the products contained no repeat region, so care must be taken if using any of these.

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INRA Versailles link have developed and tested over 100 microsatellites for Arabidopsis as well as some of the Bell and Ecker markers. These have been tested for polymorphism in various Arabidopsis ecotypes. Details of map location, primer sequence and polymorphism may be found here. Other than the Bell and Ecker microsatellites, none of these have been tested on Brassica species.

Celera AgGen Brassica Consortium

This was an industrial consortium, with results appearing in 2005 in publication by Piquemal et al. link

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Map Locations of Brassica Microsatellites

Some of the markers described above have been located on genetic maps of different Brassica species. A summary table of map information is given below, including references. Linkage group nomenclature follows that of Sharpe et al (1995), word where B. rapa groups R1 to R10 are equivalent to B. napus linkage groups N1 to N10, and B. oleracea O1 to O9 are equivalent to B. napus N11 to N19.

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Inter-Species Testing

Many of the microsatellites available in the public domain have been used successfully or tested on species other than that from which they were originally isolated. The spreadsheet below gives a summary of current information regarding inter-species amplification and data on polymorphism (where available). This information has been collated from various publications, and also includes unpublished screening data from HRI, as this provides further useful information. However, for the latter it should be remembered that the screening took place under a single set of PCR conditions, with no optimisation for particular primer pairs. Therefore, different results may be obtained using different conditions.

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Evaluation of Brassica Microsatellites

In order to select microsatellites suitable for measuring gene flow as well as for genetic marker and diversity studies, a selection of available primer pairs were screened against a panel of B. oleracea, B. rapa and B. napus lines. PCR conditions were kept uniform to facilitate high throughput genotyping. Initial screening comprised of separation of PCR products on small non-denaturing acrylamide gels and visualisation by staining with ethidium bromide. The PCR products were assessed on the basis of polymorphism and number of fragments amplified. Those with a small number of fragments (1-4) that showed polymorphism either within or between species were then screened again using a larger panel of lines. One of the primers was labelled with FAM, HEX or NED for separation and visualisation using an ABI 3100 capillary sequencer. A summary of results is given below.

HRI microsatellite evaluation summary Excel

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Useful Links

Below are some links to other internet resources that may be of interest to Brassica microsatellite users. Suggestions for other links are always welcome - please use email address given below.

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Contribution of Information to this Page

We hope that other researchers will use this page as a means of disseminating information on Brassica microsatellites, for example describing new microsatellites or adding data on polymorphism or map position. Please email Graham King with any enquiries, contributions or suggestions. We will also keep this page updated with regard to the ongoing microsatellite screening programme at HRI, and also with any new microsatellite publications. Please check back regularly for updates!

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This Page Was Last Edited: 08-Jan-2015