75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

349 Complexity Reduction of Polymorphic Sequences (CRoPS): A Novel Approach For High
Throughput Polymorphism Discovery
Michiel van Eijk, Nathalie van Orsouw, Jan van Oeveren, Rene Hogers, Antoine Janssen, Sandor Snoeijers, Esther
Verstege
Keygene N.V.
Discovery of polymorphisms, including single nucleotide polymorphisms (SNP) and simple sequence repeats
(SSR), in low polymorphic species is still a challenging and costly endeavor, despite widespread availability of capillary
electrophoresis sequence technology. We present a novel approach for polymorphism discovery (CRoPS) in plants by
combining the power of reproducible genome complexity reduction of AFLP with recently developed breakthrough
sequencing by synthesis (pyrosequencing) technology of 454 Life Sciences (Margulies et al., 2005. Nature 437, 376-
380), commerialized as Genome Sequencer 20 (GS 20) by Roche Diagnostics.
The principle or CRoPS is that tagged complexity-reduced libraries of two or more genetically diverse samples are
prepared by AFLP and sequenced at 5-10-fold redundancy in microfabricated high-density picolitre reactions. A typical
sequence-run yields over 200,000 sequence reads with a median length of 100 bases. Resulting sequences are clustered
and sequence contigs inspected for sequence differences using bio-informatics tools. Rigorous quality measures are
applied to separate sequence errors from true polymorphisms, based on redundant sequencing, sample origin information
and allele frequencies. Using CRoPS, low cost polymorphism discovery will become affordable in organisms with low
levels of germplasm polymorphism and / or highly repeated genomes, which includes many crop species. CRoPS results
of SNP and SSR mining and validation in pepper and maize will be presented. Widespread availability of SNP markers
will expedite the use of novel high-throughput SNP detection platforms to drive down the cost of marker-assisted breeding
approaches. Alternative applications of the CRoPS technology currently in development are sequence-based transcript
profiling and genotyping.
The AFLP and CRoPS technologies are covered by patents and patent applications owned by Keygene N.V. AFLP® is a registered trademark of
Keygene N.V.
350 Fine mapping of flowering Arabidopsis QTLs of small effect
Eduardo Sanchez-Bermejo, Belen Mendez-Vigo, Jose Martinez-Zapater, Carlos Alonso-Blanco
Departamento de Genetica Molecular de Plantas, Centro Nacional de Biotecnologia, Consejo Superior de
Investigaciones Cientificas (CNB-CSIC), Darwin 3, Cantoblanco, Madrid-28049, Spain.
Wild genotypes of Arabidopsis collected from different natural populations differ strongly in their flowering time
behaviour and, presumably, this variation is involved in the adaptation to different environments (Koornneef et al. 2004).
Quantitative genetic analyses of the naturally occurring variation for flowering initiation in Arabidopsis have identified five
major effect QTLs that have been already isolated, and a higher number of small effect ones (Alonso-Blanco et al., 2005).
To understand the genetic and molecular bases of this variation we have systematically developed single introgression
lines that carry either early or late flowering alleles from 5 different wild accessions in the reference Landsberg erecta
(Ler) genetic background. Based on those introgression lines, we have identified 14 different genomic regions affecting
flowering time located in all five chromosomes, which we have named as Flowering Arabidopsis QTLs (FAQ). Currently,
we have begun the fine mapping of several FAQ regions identified in several crosses as carrying alleles of small effect
under long-day photoperiod, such as FAQ2 located on top of chromosome 1 and FAQ3 located on chromosome 3. For
that, we have developed new sets of Ler background introgression lines where each line carries a smaller introgression
segment of different size, but the various introgressions of the set overlap like a stair (Komproglou et al., 2002). Using
this approach that we refer to as stairs of mini-introgression lines, we have found that FAQ2 is a complex QTL consisting
of at least two closely linked loci, while FAQ3 seems to be a single locus.
References
Alonso-Blanco et al., (2005) International Journal of Developmental Biology 49: 717-732.
Komproglou et al., (2002) Plant Journal 31: 355-364.
Koornneef et al., (2004) Annu. Rev. Plant Biol. 55: 141-172