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 Arabidopsis genome contains ‫ف‬ 200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Through a reiterative process of sequence analysis and reannotation, we identified 149 NBS-LRR-encoding genes in the Arabidopsis (ecotype Columbia) genomic sequence. Fifty-six of these genes were corrected from earlier annotations. At least 12 are predicted to be pseudogenes. As described previously, two distinct groups of sequences were identified: those that encoded an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS-LRR, or TNL), and those that encoded an N-terminal coiled-coil motif (CC-NBS-LRR, or CNL). The encoded proteins are distinct from the 58 predicted adapter proteins in the previously described TIR-X, TIR-NBS, and CC-NBS groups. Classification based on protein domains, intron positions, sequence conservation, and genome distribution defined four subgroups of CNL proteins, eight subgroups of TNL proteins, and a pair of divergent NL proteins that lack a defined N-terminal motif. CNL proteins generally were encoded in single exons, although two subclasses were identified that contained introns in unique positions. TNL proteins were encoded in modular exons, with conserved intron positions separating distinct protein domains. Conserved motifs were identified in the LRRs of both CNL and TNL proteins. In contrast to CNL proteins, TNL proteins contained large and variable C-terminal domains. The extant distribution and diversity of the NBS-LRR sequences has been generated by extensive duplication and ectopic rearrangements that involved segmental duplications as well as microscale events. The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.

Cultivated peanut (Arachis hypogaea) is an allotetraploid with closely related subgenomes of a total size of ~2.7 Gb. This makes the assembly of chromosomal pseudomolecules very challenging. As a foundation to understanding the genome of cultivated peanut, we report the genome sequences of its diploid ancestors (Arachis duranensis and Arachis ipaensis). We show that these genomes are similar to cultivated peanut's A and B subgenomes and use them to identify candidate disease resistance genes, to guide tetraploid transcript assemblies and to detect genetic exchange between cultivated peanut's subgenomes. On the basis of remarkably high DNA identity of the A. ipaensis genome and the B subgenome of cultivated peanut and biogeographic evidence, we conclude that A. ipaensis may be a direct descendant of the same population that contributed the B subgenome to cultivated peanut. A r t i c l e s npg © 2016 Nature America, Inc. All rights reserved.Nature GeNetics VOLUME 48 | NUMBER 4 | APRIL 2016 4 3 9 subgenomes of A. hypogaea. Progeny are vigorous, phenotypically normal and fertile and showed lower segregation distortion 16,17 than has been observed for some populations derived from A. hypogaea intraspecific crosses [18][19][20][21] . Therefore, as a first step to characterizing the genome of cultivated peanut, we sequenced and analyzed the genomes of the two diploid ancestors of cultivated peanut. RESULTS Sequencing and assembly of the diploid A and B genomesConsidering that A. duranensis V14167 and A. ipaensis K30076 are likely good representatives of the ancestral species of A. hypogaea, we sequenced their genomes. After filtering, the data generated from the seven paired-end libraries corresponded to an estimated 154× and 163× base-pair coverage for A. duranensis and A. ipaensis, respectively (Supplementary Tables 1-6). The total assembly sizes were 1,211 and 1,512 Mb for A. duranensis and A. ipaensis, respectively, of which 1,081 and 1,371 Mb were represented in scaffolds of 10 kb or greater in size (Supplementary Table 7). Ultradense genetic maps were generated through genotyping by sequencing (GBS) of two diploid recombinant inbred line (RIL) populations (Supplementary Data Set 1). SNPs within scaffolds were used to validate the assemblies and confirmed their high quality; 190 of 1,297 initial scaffolds of A. duranensis and 49 of 353 initial scaffolds of A. ipaensis were identified as chimeric, on the basis of the presence of diagnostic population-wide switches in genotype calls occurring at the point of misjoin. Chimeric scaffolds were split, and their components were remapped. Thus, approximate chromosomal placements were obtained for 1,692 and 459 genetically verified scaffolds, respectively. Conventional molecular marker maps (Supplementary Data Set 2) and syntenic inferences were then used to refine the ordering of scaffolds within the initial genetic bins. Generally, agreement was good for maps in euchromatic arms and poorer in pericentromeric regions (although one map 22 showed large inversions in two lin...

Classical genetic and molecular data show that genes determining disease resistance in plants are frequently clustered in the genome. Genes for resistance (R genes) to diverse pathogens cloned from several species encode proteins that have motifs in common. These motifs indicate that R genes are part of signal-transduction systems. Most of these R genes encode a leucine-rich repeat (LRR) region. Sequences encoding putative solvent-exposed residues in this region are hypervariable and have elevated ratios of nonsynonymous to synonymous substitutions; this suggests that they have evolved to detect variation in pathogen-derived ligands. Generation of new resistance specificities previously had been thought to involve frequent unequal crossing-over and gene conversions. However, comparisons between resistance haplotypes reveal that orthologs are more similar than paralogs implying a low rate of sequence homogenization from unequal crossing-over and gene conversion. We propose a new model adapted and expanded from one proposed for the evolution of vertebrate major histocompatibility complex and immunoglobulin gene families. Our model emphasizes divergent selection acting on arrays of solvent-exposed residues in the LRR resulting in evolution of individual R genes within a haplotype. Intergenic unequal crossing-over and gene conversions are important but are not the primary mechanisms generating variation.Plants, like animals, are continually challenged by a myriad of potential pathogens. There is increasing evidence that defense systems of plants are at least as complex as vertebrate defense systems. Unlike animals, however, plants do not have a circulatory system and therefore cannot rely on a specialized, proliferative immune system. Each plant cell has to be capable of defense, even though this defense is coordinated locally and systemically between cells. There are a variety of types of resistance genes and mechanisms, some induced and some constitutive (for review, see Godiard et al. 1994;Michelmore 1995;Hammond-Kosack and Jones 1997). Often, although not always, disease resistance in plants is determined by single, usually dominant, genes. The recent cloning of several such resistance genes (R genes) is providing insight into their function and evolution. The defense system of plants may be ancient and predate the evolution of the immune system; genes similar to plant R genes have been identified in mammals van der Biezen and Jones 1998).In this review we consider what is known about the genomic organization and evolution of disease resistance genes in plants. The picture that is emerging for the organization and evolution of plant R genes is similar to that of the vertebrate major histocompatibility complex (MHC), T-cell receptor (TCR), and immunoglobulin genes. Therefore, although the specific types of genes involved are different, the evolutionary forces shaping the plant and vertebrate defense systems may be similar. We propose a model for the evolution of plant R genes that is adapted and expanded from a m...

Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.