http://crop.scijournals.org/cgi/content/abstract/49/2/381
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY
CEL I Nuclease Digestion for SNP Discovery and Marker Development in Common Bean (Phaseolus vulgaris L.)
Carlos H. Galeano, Marcela Gomez, Lina M. Rodriguez and Matthew W. Blair*
CIAT- International Center for Tropical Agriculture, A.A. 6713, Cali, Colombia, South America

* Corresponding author (m.blair@cgiar.org).

Single nucleotide polymorphisms (SNPs) are the most common sequence difference found in plant genomes, yet they have not been widely exploited for producing molecular markers in common bean (Phaseolus vulgaris L.). The objective of this study was to develop a SNP assay based on a type of heteroduplex mismatch cleavage called EcoTILLING for molecular marker development in this important legume, and apply the assay (i) to the conversion of a sequence-characterized amplified region (SCAR) marker useful for selecting virus resistance (SR2) and (ii) to the screening of SNP polymorphisms in newly developed expressed sequence tag (EST)–based markers. The SNP assay involved heteroduplex mismatch cleavage by a single-strand specific nuclease ‘CEL I’ which was used to uncover two SNPs in the SR2 fragment and 22 SNPs in 37 candidate ESTs, some of which were used in segregation analysis. While developing the SNP techniques we tested several platforms, including LI-COR, nondenaturing polyacrylamide, and agarose gel detection. The agarose gel system was used for SNP genetic mapping in two common bean mapping populations, showing that heteroduplex cleavage is a useful technique for increasing molecular marker number for the crop. Examples are given of mapped SNP markers for the phytic acid pathway gene for myo-inositol-1-phosphate synthase and a drought tolerance–related gene, S-adenosylmethionine decarboxylase.


Abbreviations: ARMS, amplification refractory mutation system • BGYMV, bean golden yellow mosaic virus • CAPS, cleaved amplified polymorphic sequence • COS, conserved ortholog set • dHPLC, denaturing high performance liquid chromatography • EST, expressed sequence tag • SCAR • sequence-characterized amplified region • SNP, single nucleotide polymorphism • SSCP, single strand conformational polymorphism • RIL, recombinant inbred line • TILLING, target induced local lesion in genomes



J. Li, R. Berbeco, R. J. Distel, P. A. Janne, L. Wang, and G. M. Makrigiorgos
s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis
Nucleic Acids Res., June 9, 2007; 35(12): e84 - e84.

http://bfgp.oxfordjournals.org/cgi/content/abstract/eln046v1
http://bfgp.oxfordjournals.org/cgi/content/abstract/eln046v1
Reverse genetics in zebrafish by TILLING
Cecilia B. Moens, Thomas M. Donn, Emma R. Wolf-Saxon and Taylur P. Ma

Corresponding author. Cecilia B. Moens, HHMI and Division of Basic Science, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Ave. N., Seattle, WA 98109, USA. Tel: +1 206 667 5627; Fax: +1 206 667 5939; E-mail: cmoens@fhcrc.org
TILLING, for Targeting Induced Local Lesions in Genomes, is a reverse genetics strategy that identifies mutations in specific genes of interest in chemically mutagenized populations. First described in 2000 for mutation detection in Arabidopsis, TILLING is now used in a wide range of plants including soybean, rice, barley and maize as well as for animal model systems, including Arabidopsis, Drosophila, Caenorhabditis elegans, rat, medaka and zebrafish and for the discovery of naturally occurring polymorphisms in humans. This review summarizes current TILLING methodologies as they have been applied to the zebrafish, ongoing TILLING projects and resources in the zebrafish community, and the future of zebrafish TILLING.