Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations.

Abstract

We developed bulked segregant analysis as a method for rapidly identifying markers linked to any specific gene or genomic region. Two bulked DNA samples are generated from a segregating population from a single cross. Each pool, or bulk, contains individuals that are identical for a particular trait or genomic region but arbitrary at all unlinked regions. The two bulks are therefore genetically dissimilar in the selected region but seemingly heterozygous at all other regions. The two bulks can be made for any genomic region and from any segregating population. The bulks are screened for differences using restriction fragment length polymorphism probes or random amplified polymorphic DNA primers. We have used bulked segregant analysis to identify three random amplified polymorphic DNA markers in lettuce linked to a gene for resistance to downy mildew. We showed that markers can be reliably identified in a 25-centimorgan window on either side of the targeted locus. Bulked segregant analysis has several advantages over the use of near-isogenic lines to identify markers in specific regions of the genome. Genetic walking will be possible by multiple rounds of bulked segregation analysis; each new pair of bulks will differ at a locus identified in the previous round of analysis. This approach will have widespread application both in those species where selfing is possible and in those that are obligatorily outbreeding.We have developed a method, bulked segregant analysis, as a rapid procedure for identifying markers in specific regions of the genome. The method involves comparing two pooled DNA samples of individuals from a segregating population originating from a single cross. Within each pool, or bulk, the individuals are identical for the trait or gene of interest but are arbitrary for all other genes. Two pools contrasting for a trait (e.g., resistant and susceptible to a particular disease) are analyzed to identify markers that distinguish them. Markers that are polymorphic between the pools will be genetically linked to the loci determining the trait used to construct the pools. Bulked segregant analysis has two immediate applications in developing genetic maps. Detailed genetic maps for many species (1) are being developed by analyzing the segregation of randomly selected molecular markers in single populations. As a genetic map approaches saturation, the continued mapping of polymorphisms detected by arbitrarily selected markers becomes progressively less efficient (2). Bulked segregant analysis provides a method to focus on regions of interest or areas sparsely populated with markers. Also, bulked segregant analysis is a method for rapidly locating genes that do not segregate in populations initially used to generate the genetic map.Two types of molecular markers have been used to develop detailed genetic maps, restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs). The majority of studies over the last 7 years have employed RFLPs. They are often codominant bu...

The novel, major locus Rpi-phu1 for late blight resistance maps to potato chromosome IX and is not correlated with long vegetation period

“…In bulked segregant analysis (Michelmore et al 1991), the following were tested: 100 random ampliWed polymorphic DNA (RAPD) primers, sets A-E, received from Operon Technologies (Alameda, CA, USA), 21 RAPD primers with known map positions in tomato (McNally and Mutschler 1997) and 100 ISSR (intersimple sequence repeat) primers (set #9, received from the Biotechnology Laboratory, University of British Columbia, Vancouver, Canada). The bulks contained equal amounts of DNA from eight resistant (resistant bulk) or eight susceptible (susceptible bulk) individuals.…”

Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k-mers

“…Identification of causal mutations typically begins with genetic mapping, followed by candidate gene sequencing and complementation studies using transformation. Advances in DNA sequencing technologies have tremendously accelerated genetic mapping by combining bulk segregant analysis, that is, pooling recombinant genomes, with whole-genome sequencing, usually referred to as mapping by sequencing 2,3 . This approach is now becoming standard for mutation mapping and identification in many model species [3][4][5][6][7][8][9][10][11][12] and has even been applied to decipher quantitative traits with complex genetic architectures 13,14 .…”

Molecular mapping of soybean rust resistance in soybean accession PI 561356 and SNP haplotype analysis of the Rpp1 region in diverse germplasm

“…To Wnd the position(s) of the resistance gene(s) in PI 561356, bulked segregant analysis (BSA) was used (Michelmore et al 1991). A resistant bulk was formed by pooling an equal amount of DNA from ten lines with RB reactions and a susceptible bulk was formed by pooling DNA from ten lines with TAN reactions.…”