Assessment of DNA pooling strategies for mapping of QTLs

The synthesis of DNA pools from segregating populations is an efficient strategy for identifying DNA markers closely linked to genes or genomic regions of interest. To-date, DNA pooling based solely upon phenotypic information, or bulked segregant analysis, has been employed only in the analysis of simply-inherited traits. We have assessed the utility of phenotype-based DNA pools for tagging (e.g., identifying DNA markers closely-linked to) quantitative trait loci (QTLs), segregating in the presence of other such loci, and expressing phenotypes which are influenced by the environment. Theoretical estimates suggest that QTL alleles with phenotypic effects of 0.75–1.0 standard deviations (SD), or larger, should be detectable in back-cross (BC), F₂ and recombinant inbred (RI) or doubled haploid (DH) populations of manageable size (100–200 plants/lines). However, post hoc analysis of three data sets, used in QTL mapping of tomato and rice, indicate that the majority of QTLs identified had allele effects of less than 0.75 SD, and thus could not be easily tagged in DNA pools. Segregation distortion can have a large effect on the allelic composition of DNA pools, necessitating the use of more individuals in the pools to minimize false positive and false negative results. In general, we suggest that use of phenotype-based DNA pools might be successful in tagging QTLs of very large effect, but is unlikely to permit comprehensive identification of the majority of QTLs affecting a complex trait. DNA pools constructed from a priori information should, however, be useful in identifying new DNA markers for regions of the genome known to contain QTLs.     

Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.     

An optimal DNA pooling strategy for progressive fine mapping

We present a cost-effective DNA pooling strategy for fine mapping of a single Mendelian gene in controlled crosses. The theoretical argument suggests that it is potentially possible for a single-stage pooling approach to reduce the overall experimental expense considerably by balancing costs for genotyping and sample collection. Further, the genotyping burden can be reduced through multi-stage pooling. Numerical results are provided for practical guidelines. For example, the genotyping effort can be reduced to only a small fraction of that needed for individual genotyping at a small loss of estimation accuracy or at a cost of increasing sample sizes slightly when recombination rates are 0.5% or less. An optimal two-stage pooling scheme can reduce the amount of genotyping to 19.5%, 14.5% and 6.4% of individual genotyping efforts for identifying a gene within 1, 0.5, and 0.1 cM, respectively. Finally, we use a genetic data set for mapping the rice xl(t) gene to demonstrate the feasibility and efficiency of the DNA pooling strategy. Taken together, the results demonstrate that this DNA pooling strategy can greatly reduce the genotyping burden and the overall cost in fine mapping experiments.     

DNA pooling strategy for saturation mapping in outbred crosses

We present a method to identify molecular markers linked to a genomic interval in outbred pedigrees. Using information from fully informative RFLP markers on a single linkage group containing a quantitative trait locus for wood specific gravity, we constructed four DNA pools from nonrecombinant progeny of a three-generation outbred pedigree. The four pools were screened to identify linked RAPD markers. The phase and zygosity of a linked RAPD marker could be determined directly from the array of RAPD bands present or absent in the four pools. Two hundred fifty-six primers were tested on the four DNA pools, revealing 61 putatively linked loci. Nine RAPD loci were linked to the genomic interval. The approach developed here could be generally applied to saturation mapping in outbred pedigrees where fully informative markers have previously been mapped.     

Linkage mapping and comparative genomics using next-generation RAD sequencing of a non-model organism

Restriction-site associated DNA (RAD) sequencing is a powerful new method for targeted sequencing across the genomes of many individuals. This approach has broad potential for genetic analysis of non-model organisms including genotype-phenotype association mapping, phylogeography, population genetics and scaffolding genome assemblies through linkage mapping. We constructed a RAD library using genomic DNA from a Plutella xylostella (diamondback moth) backcross that segregated for resistance to the insecticide spinosad. Sequencing of 24 individuals was performed on a single Illumina GAIIx lane (51 base paired-end reads). Taking advantage of the lack of crossing over in homologous chromosomes in female Lepidoptera, 3,177 maternally inherited RAD alleles were assigned to the 31 chromosomes, enabling identification of the spinosad resistance and W/Z sex chromosomes. Paired-end reads for each RAD allele were assembled into contigs and compared to the genome of Bombyx mori (n = 28) using BLAST, revealing 28 homologous matches plus 3 expected fusion/breakage events which account for the difference in chromosome number. A genome-wide linkage map (1292 cM) was inferred with 2,878 segregating RAD alleles inherited from the backcross father, producing chromosome and location specific sequenced RAD markers. Here we have used RAD sequencing to construct a genetic linkage map de novo for an organism that has no previous genome data. Comparative analysis of P. xyloxtella linkage groups with B. mori chromosomes shows for the first time, genetic synteny appears common beyond the Macrolepidoptera. RAD sequencing is a powerful system capable of rapidly generating chromosome specific data for non-model organisms.     

Bayesian method for gene detection and mapping, using a case and control design and DNA pooling

Association mapping studies aim to determine the genetic basis of a trait. A common experimental design uses a sample of unrelated individuals classified into 2 groups, for example cases and controls. If the trait has a complex genetic basis, consisting of many quantitative trait loci (QTLs), each group needs to be large. Each group must be genotyped at marker loci covering the region of interest; for dense coverage of a large candidate region, or a whole-genome scan, the number of markers will be very large. The total amount of genotyping required for such a study is formidable. A laboratory effort efficient technique called DNA pooling could reduce the amount of genotyping required, but the data generated are less informative and require novel methods for efficient analysis. In this paper, a Bayesian statistical analysis of the classic model of McPeek and Strahs is proposed. In contrast to previous work on this model, I assume that data are collected using DNA pooling, so individual genotypes are not directly observed, and also account for experimental errors. A complete analysis can be performed using analytical integration, a propagation algorithm for a hidden Markov model, and quadrature. The method developed here is both statistically and computationally efficient. It allows simultaneous detection and mapping of a QTL, in a large-scale association mapping study, using data from pooled DNA. The method is shown to perform well on data sets simulated under a realistic coalescent-with-recombination model, and is shown to outperform classical single-point methods. The method is illustrated on data consisting of 27 markers in an 880-kb region around the CYP2D6 gene.     

Yeast artificial chromosome vectors for efficient clone manipulation and mapping

The yeast artificial chromosome (YAC) cloning system allows the cloning of exogenous DNA several hundred kilobases in length. To enhance the usefulness of this technology, yeast artificial chromosome vectors have been designed for efficient clone characterization, manipulation, and mapping. The vectors contain a polylinker with unique EcoRI, BglII, NotI, EagI, SacII, SalI, NruI, NheI, and ClaI cloning sites and T7 bacteriophage promoters positioned to allow the generation of riboprobes from the exogenous DNA ends. Centric and acentric vector arms were constructed as separate plasmids to allow the recovery of both ends of the YAC insert DNA directly in Escherichia coli. In addition, YACs generated using this vector system contain a yeast gene (SUP 11) that allows visual monitoring of YAC stability and copy number.     

Low-pass sequencing for microbial comparative genomics

Background  

We studied four extremely halophilic archaea by low-pass shotgun sequencing: (1) the metabolically versatile Haloarcula marismortui; (2) the non-pigmented Natrialba asiatica; (3) the psychrophile Halorubrum lacusprofundi and (4) the Dead Sea isolate Halobaculum gomorrense. Approximately one thousand single pass genomic sequences per genome were obtained. The data were analyzed by comparative genomic analyses using the completed Halobacterium sp. NRC-1 genome as a reference. Low-pass shotgun sequencing is a simple, inexpensive, and rapid approach that can readily be performed on any cultured microbe.     

Investigating sample pooling strategies for DIGE experiments to address biological variability

If biological questions are to be answered using quantitative proteomics, it is essential to design experiments which have sufficient power to be able to detect changes in expression. Sample subpooling is a strategy that can be used to reduce the variance but still allow studies to encompass biological variation. Underlying sample pooling strategies is the biological averaging assumption that the measurements taken on the pool are equal to the average of the measurements taken on the individuals. This study finds no evidence of a systematic bias triggered by sample pooling for DIGE and that pooling can be useful in reducing biological variation. For the first time in quantitative proteomics, the two sources of variance were decoupled and it was found that technical variance predominates for mouse brain, while biological variance predominates for human brain. A power analysis found that as the number of individuals pooled increased, then the number of replicates needed declined but the number of biological samples increased. Repeat measures of biological samples decreased the numbers of samples required but increased the number of gels needed. An example cost benefit analysis demonstrates how researchers can optimise their experiments while taking into account the available resources.     

基于DNA池测序法筛选奶牛高信息量SNP标记的可行性

首先选择139个牛SNP标记, 利用DNA池测序法, 根据测序峰图中不同碱基信号峰高的比值确定了92个SNP为高信息量标记(比值>1/2); 为了进一步验证筛选的准确性, 对其中59个标记采用基质辅助激光解析电离飞行时间质谱(Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, MALDI-TOF MS)技术检测了122头荷斯坦牛的基因型。结果显示, 检出率高于85%的标记有56个, 其平均最小等位基因频率(Minor allele frequency, MAF)为0.41, 最小值为0.27, 最大值为0.5; MAF>0.3的标记有54个, 占96.4%(54/56)。文章结果表明, 采用DNA池测序法筛选高信息量SNP标记是可行和可信的。     

Sheep linkage mapping: RFLP markers for comparative mapping studies

Restriction fragment length polymorphisms (RFLPs) detected using cDNA probes for conserved genes provide an important set of markers that anchor or link syntenic groups in a range of divergent mammalian species. DNA probes from sheep, cattle, pig, human and mouse were screened against sheep DNA samples and 24 new RFLP markers for sheep were identified. Among the loci tested, 22 had a homologue that has been mapped in humans. An RFLP for fibronectin (FN1) was linked to α-inhibin (INHA) at a distance of 5cM. The FN1 locus has been assigned to sheep chromosome 2q41–q44 and linkage between FN1 and INHA assigns INHA to the same chromosome in sheep. In addition to the new loci reported here, 28 RFLPs have been published previously by this group and these are collated together with RFLPs published from other laboratories. RFLPs have been reported for 86 loci in sheep. Fifty-four loci have been mapped to 16 different chromosomes.     

Combining DNA pooling with selective recombinant genotyping for increased efficiency in fine mapping

One of the key steps in positional cloning and marker-aided selection is to identify marker(s) tightly linked to the target gene (i.e., fine mapping). Selective genotyping such as selective recombinant genotyping (SRG) is commonly used in fine mapping for cost-saving. To further decrease genotyping effort and rapidly screen for tightly linked markers, we propose here a combined DNA pooling and SRG strategy. A two-stage pooled genotyping can be used for identifying recombinants between a pair of flanking markers more efficiently, and a joint use of bulked DNA analysis and two-stage pooling can also save cost for genotyping recombinants. The combined DNA pooling and SRG strategy can further be extended to fine mapping for polygenic traits. The numerical results based on hypothetical scenarios and an illustrative application to fine mapping of a mutant gene, called xl(t), in rice suggest that the proposed strategy can remarkably reduce genotyping amount compared with the conventional SRG.     

Optical mapping and its potential for large-scale sequencing projects.

Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.     

Resistance gene analogues from rice: cloning, sequencing and mapping

 Degenerate oligonucleotide primers were designed on the basis of nucleotide-binding-site (NBS) motifs conserved between resistance genes of Arabidopsis, flax and tobacco and subsequently used as PCR primers to amplify resistance gene analogues (RGA) in rice. Primers amplified a major band of approximately 500 bp. Restriction analysis of the amplified product revealed that the band was made up of several different fragments. Many of these fragments were cloned. Sixty different cloned fragments were analysed and assigned to 14 categories based on Southern blot analysis. Fourteen clones, each representing one of the 14 categories of RGAs were mapped onto the rice genetic map using a Nipponbare ( japonica)בKasalath’ (indica) mapping population consisting of 186 F₂ lines. Of the 14 clones representing each class 12 could be mapped onto five different chromosomes of rice with a major cluster of 8 RGAs on chromosome 11. Our results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa. Received: 23 September 1998 / Accepted: 28 November 1998