Comparative Gene Mapping Research Articles

Comparative QTL mapping of resistance to sugarcane mosaic virus in maize based on bioinformatics

The development of genomics and bioinformatics offers new tools for comparative gene mapping. In this paper, an integrated QTL map for Sugarcane mosaic virus (SCMV) resistance in maize was constructed by compiling a total of 81 QTL loci available with the Genetic Map IBM2 2005 Neighbors as reference. These 81 QTL loci were scattered on 7 chromosomes of maize, and most of them was clustered on chromosome 3 and 6. By using meta- analysis method, we identified one and two "consensus QTLs" on chromosomes 3 and 6, respectively. These three QTLs cover the genetic distances of 6.44 cM, 6.16 cM and 27.48 cM on the genetic map IBM2 2005 Neighbors, respectively. Four positional candidate resistant genes were identified within the "consensus QTL" on chromosome 3 via comparative genomics strategy. These results suggested that application of the combined meta-analysis within a species with sequence homologous comparison in a related model plant is an efficient approach to identify the major QTL and its candidate gene(s) for the target traits. The results of this study provided useful information for identifying and cloning of the major gene(s) conferring resistance to SCMV in maize.

Designing and optimizing comparative anchor primers for comparative gene mapping and phylogenetic inference

Here we describe protocols for designing, optimizing and implementing conserved anchor primers for use in genome mapping or phylogenetic applications, with particular emphasis on homologous gene sequences among mammals. The increasing number of whole genome sequences in public databases makes this approach applicable across a wide range of taxa. Genome sequences from representatives of two or more divergent subclades within a taxonomic group of interest are used to identify candidate local alignments (i.e., exons, exons spanning introns or conserved 5'- or 3'-untranslated regions) that contain sequences with appropriate variability for the chosen downstream application. PCR primers are designed to maximize amplification success across a broad range of taxa, and are optimized under a touchdown thermocycling protocol. Based on the initial optimization results, primers are selected for application in a diverse sampling of species, or for mapping the genome of a target species of interest. We discuss factors that have to be considered for experimental design of broad-scope phylogenetic studies. With this protocol, primers can be designed, optimized and implemented within as little as 1-2 weeks.

Mapping barley genes to chromosome arms by transcript profiling of wheat–barley ditelosomic chromosome addition lines

Wheat-barley disomic and ditelosomic chromosome addition lines have been used as genetic tools for a range of applications since their development in the 1980s. In the present study, we used the Affymetrix Barley1 GeneChip for comparative transcript analysis of the barley cultivar Betzes, the wheat cultivar Chinese Spring, and Chinese Spring - Betzes ditelosomic chromosome addition lines to physically map barley genes to their respective chromosome arm locations. We mapped 1257 barley genes to chromosome arms 1HS, 2HS, 2HL, 3HS, 3HL, 4HS, 4HL, 5HS, 5HL, 7HS, and 7HL based on their transcript levels in the ditelosomic addition lines. The number of genes assigned to individual chromosome arms ranged from 24 to 197. We validated the physical locations of the genes through comparison with our previous chromosome-based physical mapping, comparative in silico mapping with rice and wheat, and single feature polymorphism (SFP) analysis. We found our physical mapping of barley genes to chromosome arms to be consistent with our previous physical mapping to whole chromosomes. In silico comparative mapping of barley genes assigned to chromosome arms revealed that the average genomic synteny to wheat and rice chromosome arms was 63.2% and 65.5%, respectively. In the 1257 mapped genes, we identified SFPs in 924 genes between the appropriate ditelosomic line and Chinese Spring that supported physical map placements. We also identified a single small rearrangement event between rice chromosome 9 and barley chromosome 4H that accounts for the loss of synteny for several genes.

Comparative genetic analysis and molecular mapping of fertility restoration genes for WA, Dissi, and Gambiaca cytoplasmic male sterility systems in rice

The genetic relationship among three cytoplasmic male sterility (CMS) systems, consisting of WA, Dissi, and Gambiaca, was studied. The results showed that the maintainers of one CMS system can also maintain sterility in other cytoplasmic backgrounds. The F1 plants derived from crosses involving A and R lines of the respective cytoplasm and their cross-combination with other CMS systems showed similar pollen and spikelet fertility values, indicating that similar biological processes govern fertility restoration in these three CMS systems. The results from an inheritance study showed that the pollen fertility restoration in all three CMS systems was governed by two independent and dominant genes with classical duplicate gene action. Three F2 populations, generated from the crosses between the parents of good-performing rice hybrids, that possess WA, Dissi, and Gambiaca CMS cytoplasm, were used to map the Rf genes. For the WA-CMS system, Rf3 was located at a distance of 2.8 cM from RM490 on chromosome 1 and Rf4 was located at 1.6 cM from RM1108 on chromosome 10. For the Dissi-CMS system, Rf3 was located on chromosome 1 at 1.9 cM from RM7466 and Rf4 on chromosome 10 was located at 2.3 cM from RM6100. The effect of Rf3 on pollen fertility appeared to be stronger than the effect of Rf4. In the Gambiaca-CMS system, only one major locus was mapped on chromosome 1 at 2.1 cM from RM576. These studies have led to the development of marker-assisted selection (MAS) for selecting putative restorer lines, new approaches to alloplasmic line breeding, and the transfer of Rf genes into adapted cultivars through a backcrossing program in an active hybrid rice breeding program.

Different origins of bird and reptile sex chromosomes inferred from comparative mapping of chicken Z-linked genes

Recent progress of chicken genome projects has revealed that bird ZW and mammalian XY sex chromosomes were derived from different autosomal pairs of the common ancestor; however, the evolutionary relationship between bird and reptilian sex chromosomes is still unclear. The Chinese soft-shelled turtle (Pelodiscus sinensis) exhibits genetic sex determination, but no distinguishable (heteromorphic) sex chromosomes have been identified. In order to investigate this further, we performed molecular cytogenetic analyses of this species, and thereby identified ZZ/ZW-type micro-sex chromosomes. In addition, we cloned reptile homologues of chicken Z-linked genes from three reptilian species, the Chinese soft-shelled turtle and the Japanese four-striped rat snake (Elaphe quadrivirgata), which have heteromorphic sex chromosomes, and the Siam crocodile (Crocodylus siamensis), which exhibits temperature-dependent sex determination and lacks sex chromosomes. We then mapped them to chromosomes of each species using FISH. The linkage of the genes has been highly conserved in all species: the chicken Z chromosome corresponded to the turtle chromosome 6q, snake chromosome 2p and crocodile chromosome 3. The order of the genes was identical among the three species. The absence of homology between the bird Z chromosome and the snake and turtle Z sex chromosomes suggests that the origin of the sex chromosomes and the causative genes of sex determination are different between birds and reptiles.

Evaluation of Compass as a comparative mapping tool for ESTs using horse radiation hybrid maps

Loci for 9322 equine expressed sequence tags (ESTs) were predicted using the Comparative Mapping by Annotation and Sequence Similarity (Compass) strategy in order to evaluate the programme's ability to make accurate locus predictions in species with comparative gene maps. Using human genome sequence information from Build 35 (May 2004) and published marker information from the radiation hybrid (RH) maps for equine chromosomes (ECA) 17 and X, 162 ESTs were predicted to locations on ECA17 and 328 ESTs to locations on ECAX by selection of the 'top blast hit'. The locations of 30 ESTs were assessed experimentally by RH mapping analysis to evaluate the accuracy of the Compass predictions. The data revealed that 53% (16 of 30) of the ESTs predicted on ECA17 and ECAX mapped to those chromosomes. Analysis of the results suggested the need to identify expressed orthologous sequences in order to generate more accurate predictions for ESTs. Locus predictions were reassessed with three modifications to the Compass strategy's orthologue selection parameters. Selection of the 'top gene hit' improved accuracy to 72% (21 of 29), while selection of the 'top expressed gene hit' improved accuracy to 86% (24 of 28). Using the default Compass parameters with the UniGene database improved prediction accuracy to 96% (22 of 23); however, this level of accuracy came with a substantial decrease in the total number of predictions. When used with optimized prediction parameters, the Compass strategy can be a practical in silico map location prediction tool for large EST sample sets from unsequenced animal genomes.

The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z

A comparative study of the karyotype of the short-beaked echidna shows that monotremes appear to have a unique XY sex chromosome system that shares some homology with the avian Z.

Reconstruction of the ancestral ferungulate karyotype by electronic chromosome painting (E-painting)

By comparing high-coverage and high-quality whole genome sequence assemblies it is now possible to reconstruct putative ancestral progenitor karyotypes, here called protokaryotypes. For this study we used the recently described electronic chromosome painting technique (E-painting) to reconstruct the karyotype of the 85 million-year-old (MYA) ferungulate ancestor. This model is primarily based on dog (Canis familiaris) and cattle (Bos taurus) genome data and is highly consistent with comparative gene mapping and chromosome painting data. The protokaryotype bears 23 autosomal chromosome pairs and the sex chromosomes and preserves most of the chromosomal associations described previously for the boreo-eutherian protokaryotype. The model indicates that five interchromosomal rearrangements occurred during the transition from the boreo-eutherian to the ferungulate ancestor. From there on 66 further interchromosomal rearrangements took place in the lineage leading to cattle and 61 further interchromosomal rearrangements in the lineage to dog.

High-resolution gene maps of horse chromosomes 14 and 21: Additional insights into evolution and rearrangements of HSA5 homologs in mammals

High-resolution physically ordered gene maps for equine homologs of human chromosome 5 (HSA5), viz., horse chromosomes 14 and 21 (ECA14 and ECA21), were generated by adding 179 new loci (131 gene-specific and 48 microsatellites) to the existing maps of the two chromosomes. The loci were mapped primarily by genotyping on a 5000-rad horse × hamster radiation hybrid panel, of which 28 were mapped by fluorescence in situ hybridization. The approximately fivefold increase in the number of mapped markers on the two chromosomes improves the average resolution of the map to 1 marker/0.9 Mb. The improved resolution is vital for rapid chromosomal localization of traits of interest on these chromosomes and for facilitating candidate gene searches. The comparative gene mapping data on ECA14 and ECA21 finely align the chromosomes to sequence/gene maps of a range of evolutionarily distantly related species. It also demonstrates that compared to ECA14, the ECA21 segment corresponding to HSA5 is a more conserved region because of preserved gene order in a larger number of and more diverse species. Further, comparison of ECA14 and the distal three-quarters region of ECA21 with corresponding chromosomal segments in 50 species belonging to 11 mammalian orders provides a broad overview of the evolution of these segments in individual orders from the putative ancestral chromosomal configuration. Of particular interest is the identification and precise demarcation of equid/Perissodactyl-specific features that for the first time clearly distinguish the origins of ECA14 and ECA21 from similar-looking status in the Cetartiodactyls.

Comparative mapping of genes for glume colouration and pubescence in hexaploid wheat (Triticum aestivum L.)

Microsatellite markers were used to map the major genes Bg (determining black glume colour), Rg1 and Rg3 (red glume), and a locus determining smokey-grey coloured glume to the distal ends of the short arms of the homoeologous group 1 chromosomes, proximally (or closely linked) to Xgwm1223 and distal to Xgwm0033. On this basis, we propose that these genes represent a set of homoeoloci, designated Rg-A1, Rg-B1, and Rg-D1. Rg3 and Bg appear to be variant alleles of Rg-A1. Both Rg3 and Bg are closely linked with the major glume pubescence gene Hg. Similarly, the hexaploid wheat smokey-grey glume gene and Rg2 represent alleles at Rg-D1. The microsatellite markers linked to the Rg genes were used to analyse a phenotypically and genotypically characterized set of Siberian spring wheats. A coincidence between the presence of the 264-bp allele of Xgwm0136 and Rg-A1b (Rg3) was observed; so Xgwm0136 can probably be used as a diagnostic marker for this gene.

Assignment of zebrafish genetic linkage groups to chromosomes

In this report the zebrafish genetic linkage groups are assigned to specific chromosomes using fluorescence in situ hybridization (FISH) with BAC probes containing genes mapped to each linkage group (LG). Chromosomes were identified using a combination of relative size and arm ratios. The largest genetic maps generally corresponded to the largest chromosomes, but genetic recombination tended to be elevated in the smaller chromosomes and near telomeres. Large insert clones containing genes near telomeres often hybridized to telomeres of multiple chromosome pairs, suggesting the presence of shared subtelomeric repetitive DNAs near telomeres. Evidence from comparative gene mapping in medaka, zebrafish, pufferfish, and humans suggests that the linkage groups of these species have the content of duplicate proto-chromosomes. However, these duplicate linkage groups are not associated with chromosomes of similar size or morphology. This suggests that considerable chromosome restructuring occurred subsequent to the genome duplication in teleosts.

Comparative gene and QTL mapping in aquaculture species

Mapping genes and quantitative trait loci (QTLs) is of fundamental and applied interest in aquaculture species. Such studies can pro- vide essential information about rates and patterns of evolutionary change and may pro- vide tools which can be used in marker-assist- ed selection (MAS; e.g., Groenen et al., 2000). Mapping typically involves producing divergent crosses and analyzing segregation patterns in the offspring of hybrids between the divergent crosses. The more divergent the cross is, the greater the potential for finding useful marker and trait differences that can be mapped. The basic goal of mapping is to understand the position of loci on chromo- somes and how the positions change during evolution. The applied goal of mapping is to understand the position of loci associated with phenotypic traits which could be important in breeding programs. Correspondingly, QTL mapping seeks to identify the number and location of loci associated with phenotypic traits (e.g., Mackay, 2001; Mauricio, 2001; Burt and Hocking, 2002; Erickson et al., 2004) while applied QTL studies seek to identify candidate genes or markers for use in MAS.

A 12,000-rad porcine radiation hybrid (IMNpRH2) panel refines the conserved synteny between SSC12 and HSA17

Reverse or bidirectional Zoo-FISH suggests that synteny between porcine chromosome 12 (SSC12) and human chromosome 17 (HSA17) is completely conserved. The construction of a high-resolution radiation hybrid (RH) map for SSC12 provides a unique opportunity to determine whether chromosomal synteny is reflected at the molecular level by comparative gene mapping of SSC12 and HSA17. We report an initial, high-resolution RH map of SSC12 on the 12,000-rad IMNpRH2 panel using CarthaGene software. This map contains a total of 320 markers, including 20 microsatellites and 300 ESTs/genes, covering ∼4836.9 cR 12,000. The markers were ordered in 16 linkage groups at LOD 6.0 using framework markers previously mapped on the IMpRH 7000-rad SSC12 and porcine genetic maps. Ten linkage groups ordered more than 10 markers, with the largest containing 101 STSs. The resolution of the current RH map is ∼15.3 kb/cR on SSC12, a significant improvement over the second-generation EST SSC12 RH 7000-rad map of 103 ESTs and 15 framework markers covering ∼2287.2 cR 7000. Compared to HSA17, six distinct segments were identified, revealing macro-rearrangements within the apparently complete synteny between SSC12 and HSA17. Further analysis of the order of 245 genes (ESTs) on HSA17 and SSC12 also revealed several micro-rearrangements within a synteny segment. A high-resolution SSC12 RH 12,000-rad map will be useful in fine-mapping QTL and as a scaffold for sequencing this chromosome.

(13) Comparative Mapping of Early Blight Resistance QTLs and Candidate Resistance Genes in F2, F3, F4 and a RIL Population of Tomato

Most cultivars of tomato (Lycopersiconesculentum) are susceptible to early blight (EB), a devastating fungal (Alternariasolani) disease of tomato in the northeast and eastern part of the U.S. The most economic and durable measure of disease control is by using genetic resistance. There is limited EB resistance within the cultivated tomato. However, genetic sources of resistance exist within the tomato-related wild species L. hirsutum and L. pimpinellifolium. Early blight resistance does not follow the gene-for-gene model of host–pathogen interaction. Mapping QTLs conferring horizontal resistance is an effective approach for studying complex resistance traits such as EB. We have developed F2, F3, F4, and an F2:7 derived RIL population of a L. esculentum× L. pimpinellifolium cross and evaluated them for EB resistance under field conditions. Genetic maps were constructed based on the F2 (including 256 RFLP, EST and RGA markers) as well as the RIL population (including over 220 RFLP and EST markers). In each of the F2, F3, and F4 population, an average of seven QTLs were identified for resistance, which were highly consistent across populations. Mapping of EB resistance QTLs in the RILs is underway. Co-localizations of QTLs with several ESTs and RGAs were observed, suggesting potential involvement of the latter markers with EB resistance. Furthermore, co-localizations were observed among QTLs, ESTs, and RGAs and several known tomato vertical disease resistance genes. Possible occurrence of such co-localization in the RIL population will be reported. It is speculated that candidate-gene approach is an effective way of identifying and mapping new R genes in tomato. This study may lead to the identification of genes underlying EB resistance in tomato.

Comparative Mapping of Genes for Brittle Rachis in Triticum

The brittle rachis phenotype is of adaptive value in wild grass species because it causes spontaneous spike shattering. The genes on the homoeologous group 3 chromosomes determine the brittle rachis in Triticeae. A few genotypes with brittle rachis have also been found in the cultivated Triticum. Using microsatellite markers, the homoeologous genes for brittle rachis were mapped in hexaploid wheat (Triticum aestivum L.), durum wheat (Triticum turgidum L. conv. durum /Desf./) and Aegilops tauschii Coss. On chromosome 3AS, the gene for brittle rachis, Br<sub>2</sub>, was linked with the centromeric marker, Xgwm32, at the distance of 13.3 cM. Br<sub>3 </sub>was located on chromosome 3BS and linked with the centromeric marker,<br />Xgwm72 (14.2 cM). Br<sub>1 </sub>was located on chromosome 3DS. The distance from the centromeric marker Xgdm72 was 23.6 cM. The loci Br<sub>1</sub>, Br<sub>2</sub> and Br<sub>3</sub> determine disarticulation of rachides above the junction of the rachilla with the rachis so that a fragment of rachis is attached below each spikelet. The rachides of Ae. tauschii are brittle at every joint, so that the mature spike disarticulates into barrel type. The brittle rachis was determined by a dominant gene, Br<sup>t</sup>, which was linked to the centromeric marker, Xgdm72 (19.7 cM), on chromosome 3DS. A D-genome introgression line, R-61, was derived from the cross Bet Hashita/Ae. tauschii, whose rachis disarticulated as a wedge type. The gene for brittle rachis of R-61, tentatively designated as Br<sup>61</sup>, was distally located on chromosome 3DS, and was linked with the centromeric marker, Xgdm72 (27.5 cM). We discussed how the brittle rachis of R-61 originated genetically.    

A 6-Mb contig-based comparative gene and linkage map of the rat schwannoma tumor suppressor region at 10q32.3

Frequent genetic aberrations of malignant schwannomas induced by the alkylating agent N-ethyl- N-nitrosourea in hybrids from inbred BD rat strains include allelic imbalances of the telomeric 20 Mb of chromosome 5 ( Dis-2) and of the telomeric 5 Mb of chromosome 10q32 ( Dis-1) in 59 and 94% of the tumors, respectively. The Dis-1 minimal loss of heterozygosity consensus region extends from D10Rat4 to the telomere and harbors a putative tumor suppressor gene(s). We constructed a 6-Mb BAC/PAC contig containing more than 70 known genes, 18 mapped microsatellites, and further ESTs/reference RNAs. A continuous block of strongly conserved synteny with mouse chromosome 11E2 and human chromosome 17q25.3 was found. Combining the sequence information from the rat and closely related syntenic regions of different mammalian species produces nearly complete gene maps as a basis for a positional candidate approach and gives insight into mammalian genomic evolution.

RatMap--rat genome tools and data

The rat genome database RatMap (http://ratmap.org or http://ratmap.gen.gu.se) has been one of the main resources for rat genome information since 1994. The database is maintained by CMB–Genetics at Göteborg University in Sweden and provides information on rat genes, polymorphic rat DNA-markers and rat quantitative trait loci (QTLs), all curated at RatMap. The database is under the supervision of the Rat Gene and Nomenclature Committee (RGNC); thus much attention is paid to rat gene nomenclature. RatMap presents information on rat idiograms, karyotypes and provides a unified presentation of the rat genome sequence and integrated rat linkage maps. A set of tools is also available to facilitate the identification and characterization of rat QTLs, as well as the estimation of exon/intron number and sizes in individual rat genes. Furthermore, comparative gene maps of rat in regard to mouse and human are provided.

A dense comparative gene map between human chromosome 19q13.3→q13.4 and a homologous segment of swine chromosome 6

The human chromosome (HSA)19q region has been shown to correspond to swine chromosome (SSC) 6q11→q21 by bi-directional chromosomal painting and gene mapping. However, since the precise correspondence has not been determined, 26 genes localized in HSA19q13.3→q13.4 were assigned to the SSC6 region mainly by radiation hybrid (RH) mapping, and additionally, by somatic cell hybrid panel (SCHP) mapping, and fluorescent in situ hybridization (FISH). Out of the 26 genes, 24 were assigned to a swine RH map with LOD scores greater than 6 (threshold of significance). The most likely order of the 24 genes along SSC6 was calculated by CarthaGene, revealing that the order is essentially the same as that in HSA19q13.3→q13.4. For AURKC and RPS5 giving LOD scores not greater than 6, SCHP mapping and FISH were additionally performed; SCHP mapping assigned AURKC and RPS5 to SSC6q22→q23 and SSC6q21, respectively, which is consistent with the observation of FISH. Consequently, all the genes (26 genes) examined in the present study were shown to localize in SSC6q12→q23, and the order of the genes along the chromosomes was shown to be essentially the same in swine and human, though several intrachromosomal rearrangements were observed between the species.

Comparative map between the domestic pig and dog

Cross-species chromosome painting with probes derived from flow-sorted dog and human chromosomes was used to construct a high-resolution comparative map for the pig. In total 98 conserved autosomal segments between pig and dog were detected by probes specific for the 38 autosomes and X Chromosome of the dog. Further integration of our results with the published human--dog and cat--dog comparative maps, and with data from comparative gene mapping, increases the resolution of the current pig--human comparative map. It allows for the conserved syntenies detected in the pig, human, and cat to be aligned against the putative ancestral karyotype of eutherian mammals and for the history of karyotype evolution of the pig lineage to be reconstructed. Fifteen fusions, 17 fissions, and 23 inversions are required to convert the ancestral mammalian karyotype into the extant karyotype of the pig.

Chromosomal patterns of major and 5S ribosomal DNA in six icefish species (Perciformes, Notothenioidei, Channichthyidae)

Comparative chromosomal mapping of major and 5S ribosomal genes in six species of the family Channichthyidae, namely Champsocephalus gunnari, Channichthys rhinoceratus, Chionodraco hamatus, Cryodraco atkinsoni, Pagetopsis macropterus and Neopagetopsis ionah, was performed by fluorescence in-situ hybridization, and using 28S and 5S ribosomal gene (rDNA) sequences as probes. Clusters of major and 5S ribosomal genes co-localize and likely compose the entire arm of a single pair of submetacentric chromosomes in all the species. In one species, P. macropterus, a second pair of chromosomes bears an additional common locus for both the two families of ribosomal genes. In all species, except N. ionah, additional copies of 5S rDNA sequences are also present on two other chromosome pairs, including the Y-chromosome in the males of Chionodraco hamatus. The pattern of ribosomal DNAs contributes to species-specific characterization in this fish family, and to our general knowledge and understanding of the chromosomal organization and evolution of the icefish genome.