A Restriction Fragment Length Polymorphism Map and Electrophoretic Karyotype of the Fungal Maize Pathogen Cochliobolus Heterostrophus

A restriction fragment length polymorphism (RFLP) map has been constructed of the nuclear genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Tox1, a locus controlling host-specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical length to map distance should make this RFLP map a useful tool for cloning genes.     

Maize tertiary trisomic stocks derived from B-A translocations

Reciprocal translocations between supernumerary B chromosomes and the basic complement of A chromosomes in maize have resulted in a powerful set of tools to manipulate the dosage of chromosomal segments. From 15 B-A reciprocal translocation stocks that have the B-A chromosome genetically marked we have developed tertiary trisomic stocks. Tertiary trisomics are 2n + 1 aneuploids where the extra chromosome is a translocation element, in this case a B-A chromosome. Whereas B-A translocations produce aneuploidy in the sperm, the tertiary trisomic plant efficiently transmits hyperploid gametes maternally. Because the B-A tertiary trisomic stocks and the B-A translocation stocks from which they were derived are introgressed into the W22 inbred line, the effects of maternally and paternally transmitted trisomic B-A chromosomes can be compared. Data are presented on both the male and female transmission rates of the B-A chromosomes in the tertiary trisomic stocks.     

Construction and uses of new compound B-A-A maize chromosome translocations

Maize B-A translocations result from reciprocal interchanges between a supernumerary B chromosome and an arm of an essential A chromosome. Because of the frequent nondisjunction of the B centromere at the second pollen mitosis, B-A translocations have been used to locate genes to chromosome arms and to study the dosage effects of specific A segments. Compound B-A translocations (B-A-A translocations) are created by bringing together a simple B-A translocation with an A-A translocation in which breakpoints in the A-A and B-A translocations are in the same arm. Recombination in the region of shared homology of these A chromosome segments creates a B-A-A translocation. Success in creating and testing for a new B-A-A translocation requires that the B-A translocation be proximal to the A-A translocation and that the A-A translocation be proximal to the tester locus. The breakpoints of most of the A-A translocations have been cytologically defined by earlier investigators. Previous investigators have produced 16 B-A-A translocations and one B-A-A-A translocation, which collectively define 35 A chromosome breakpoints. We have enlarged this group by creating 64 new B-A-A translocations. We present a summary of the total of 81 B-A-A translocations showing their distribution among the chromosome arms and the 163 cytologically defined chromosome segments delimited by them. We also illustrate the method of construction of these B-A-A stocks and their uses.     

Comparative chromosome painting defines the high rate of karyotype changes between pigs and bovids

Human and sheep chromosome-specific probes were used to construct comparative painting maps between the pig (Suiformes), cattle and sheep (Bovidae), and humans. Various yet unknown translocations were observed that would assist in a more complete reconstruction of homology maps of these species. The number of homologous segments that can be identified with sheep probes in the pig karyotype exceeds that described previously by chromosome painting between two non-primate mammals belonging to the same order. Sheep probes painted 62 segments on pig autosomes and delineated not only translocations, but also 9 inversions. All inversions were paracentric and indicate that these rearrangements may be characteristic for chromosomal changes in suiforms. Hybridizations of all sheep painting probes to cattle chromosomes confirmed the chromosome conservation in bovids. In addition, we observed a small translocation that was previously postulated from linkage mapping data, but was not yet described by physical mapping. The chromosome painting data are complemented with a map of available comparative gene mapping data between pig and sheep genomes. A detailed table listing the comparative gene mapping data between pig and cattle genomes is provided. The reanalysis of the pig karyotype with a new generation of human paint probes provides an update of the human/pig comparative genome map and demonstrates two new chromosome homologies. Seven conserved segments not yet identified by chromosome painting are also reported. Received: 2 October 2000 / Accepted: 15 January 2001     

RFLP mapping of the centromere of chromosome 4 in maize using isochromosomes for 4S

 The centromere of maize chromosome 4 was previously localized to a 26-cM interval using molecular markers and B-A translocations. The objective of the present study was to refine the placement of the centromere using secondary trisomics. Two independently isolated secondary trisomics (having an isochromosome plus two normal homologs) for 4S were recovered. RFLP analysis of populations segregating for them placed the centromere of chromosome 4 between bnl15.45 and bnl7.20, two RFLP loci that are 5.4-cM apart on the UMC map and 11.5-cM apart on the BNL map. Received: 19 May 1997 / Accepted: 6 October 1997     

Molecular markers show a complex mosaic pattern of wheat-Thinopyrum intermedium translocations carrying resistance to YDV

Thinopyrum intermedium translocations derived from the wheat (Triticum aestivum L.) substitution line P-29 were previously characterized by RFLP. We have further analyzed these lines and additional related germplasm with publicly available STS and SSRs. Primers which showed a polymorphism between wheat and P-29, were tested in all recombinant and nulli-tetrasomic lines confirming their position on chromosome 7D. The resulting 7D/7E chromosome maps appeared as a mosaic of wheat and Th. intermedium chromatin sections. To verify the composition of the translocation lines suggested by the RFLP-PCR map, F₂ progeny of two crosses (CS/216-1 and CS/260-1) were analyzed with molecular markers. Both populations gave an unexpectedly diverse number of recombinant individuals, suggesting that interstitial translocations occur more frequently than previously thought. This analysis also showed that there is a wide range in the number and position of the interstitial translocations within a given line such as the mosaic chromosome in recombinant line 260-1/CS-26, which has four Th. intermedium chromosome segments. Phenotypic data of the two populations suggested the presence of one gene which we have called Bdv3 to differentiate it from the previously reported orthologous gene Bdv2. Using the PCR-based molecular markers identified in this study, 5 out of 12 elite lines that showed good yields and no YDV symptoms contained Th. intermedium chromatin. Due to the multiple components involved in the YDV disease complex, combining selection for YDV resistance with the molecular markers and maps identified in this study will increase the efficiency of introgressing Th. intermedium chromatin containing YDV resistance or other beneficial traits into elite wheat germplasm.     

Denaturing gradient gel electrophoresis identifies genomic DNA polymorphism with high frequency in maize

Summary We have used denaturing gradient gel electrophoresis (DGGE) to identify genomic DNA polymorphism in maize (Zea mays L.). DGGE probes detect polymorphism in maize at a frequency comparable to the incidence of restriction fragment length polymorphism (RFLP). Probes identifying polymorphism were mapped to maize chromosome arms by utilizing DGGE and maize lines carrying B-A chromosomal translocations. The methods for library construction, probe screening, and genome analysis, described here for maize, can also be applied to the genomic analysis of other organisms.     

Physical mapping of four RAPDs in the B chromosome of maize

 Four DNA fragments were amplified specifically from the B chromosome by PCR using random 10-base oligonucleotides as primers. The location of the fragments in the B chromosome was determined based on whether or not they were amplified from the hypo- ploid DNA generated by four B-A translocations, three of which break in the proximal euchromatic region and the fourth in the distal one-third of the heterochromatic region on the B long arm. Since the hypoploid DNA carries the portion of the B chromosome distal to the breakpoint of a translocation, the presence of a fragment in the hypoploid DNA, but not in the control (which is devoid of any B chromatin), indicates that the fragments is located in the B region distal to the breakpoint in the B long arm. Two fragments were mapped to the euchromatic region and two others to either the distal portion of the euchromatic region or the proximal two-thirds of the heterochromatic region. These fragments in turn mapped three B-A translocations whose breakpoints were located in the euchromatic region. Received: 22 May 1996 / Accepted: 30 August 1996     

Molecular Mapping of Wheat: Major Genes and Rearrangements in Homoeologous Groups 4, 5, and 7

A molecular-marker linkage map of hexaploid wheat (Triticum aestivum L. em. Thell) provides a framework for integration with the classical genetic map and a record of the chromosomal rearrangements involved in the evolution of this crop species. We have constructed restriction fragment length polymorphism (RFLP) maps of the A-, B-, and D-genome chromosomes of homoeologous groups 4, 5, and 7 of wheat using 114 F(7) lines from a synthetic X cultivated wheat cross and clones from 10 DNA libraries. Chromosomal breakpoints for known ancestral reciprocal translocations involving these chromosomes and for a known pericentric inversion on chromosome 4A were localized by linkage and aneuploid analysis. Known genes mapped include the major vernalization genes Vrn1 and Vrn3 on chromosome arms 5AL and 5DL, the red-coleoptile gene Rc1 on 7AS, and presumptively the leaf-rust (Puccinia recondita f.sp. tritici) resistance gene Lr34 on 7DS and the kernel-hardness gene Ha on 5DS. RFLP markers previously obtained for powdery-mildew (Blumeria graminis f.sp. tritici) resistance genes Pm2 and Pm1 were localized on chromosome arms 5DS and 7AL.     

Generation of a Restriction Fragment Length Polymorphism Linkage Map for Toxoplasma Gondii

We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.     

Recombination in the B Chromosome of Maize to Produce a-B-a Chromosomes

The translocations between the supernumerary B chromosomes and the normal A chromosomes of maize provide a valuable tool for gene localizations, dosage studies and characterization of mutants as null, leaky or gain-of-function. A procedure is described, that relies on recombination in the B chromosome, for marking each of the various B-A translocations with a single dominant marker that will allow dosage classifications of individuals at the mature kernel stage. This marker is R-scm3, which conditions anthocyanin pigment in the aleurone of the endosperm and the scutellum of the embryo. A test for recombination in the B chromosome was conducted by crossing together two translocations, that were broken on opposite sides of the B centromere, and in different A chromosome arms, namely TB-1La and TB-10L18. An example was recovered that linked genetic markers on 1L and 10L to the B centromere. Cytological examination at pachytene of meiosis confirmed the new chromosomal linkage. The use of this procedure to produce a comprehensive set of uniformly marked B-A translocations is discussed.     

RFLP maps of potato and their alignment with the homoeologous tomato genome

Summary An RFLP linkage map of the potato is presented which comprises 304 loci derived from 230 DNA probes and one morphological marker (tuber skin color). The self-incompatibility locus of potato was mapped to chromosome I, which is homoeologous to tomato chromosome I. By mapping chromosome-specific tomato RFLP markers in potato and, vice versa, potato markers in tomato, the different potato and tomato RFLP maps were aligned to each other and the similarity of the potato and tomato genome was confirmed. The numbers given to the 12 potato chromosomes are now in accordance with the established tomato nomenclature. Comparisons between potato RFLP maps derived from different genetic backgrounds revealed conservation of marker order but differences in chromosome and total map length. In particular, significant reduction of map length was observed in interspecific compared to intraspecific crosses. The distribution of regions with distorted segregation ratios in the genome was analyzed for four potato parents. The most prominent distortion of recombination was found to be caused by the self-incompatibility locus.     

Detection of chromosomal rearrangements derived from homologous recombination in four mapping populations of Brassica napus L

Genetic maps of Brassica napus were constructed from four segregating populations of doubled haploid lines. Each mapping population had the same male parent and used the same set of RFLP probes, facilitating the construction of a consensus map. Chromosomal rearrangements were identified in each population by molecular marker analysis and were classified as de novo homologous nonreciprocal transpositions (HNRTs), preexisting HNRTs, and homologous reciprocal transpositions (HRTs). Ninety-nine de novo HNRTs were identified by the presence of a few lines having duplication of a chromosomal region and loss of the corresponding homologous region. These de novo HNRTs were more prevalent in one population that had a resynthesized B. napus as a parent. Preexisting HNRTs were identified by fragment duplication or fragment loss in many DH lines due to the segregation of HNRTs preexisting in one of the parents. Nine preexisting HNRTs were identified in the three populations involving natural B. napus parents, which likely originated from previous homologous exchanges. The male parent had a previously described HRT between N7 and N16, which segregated in each population. These data suggest that chromosomal rearrangements caused by homologous recombination are widespread in B. napus. The effects of these rearrangements on allelic and phenotypic diversity are discussed.     

The Effects of Varying Chromosome Arm Dosage on Maize Plant Morphogenesis

Maize is an especially well-suited species for studying the effects of aneuploidy on plant development. We used B-A translocations and testers that were crossed seven times into inbred W22 to generate a dosage series for 14 chromosome arms. This is the first report of dosage effects on maize morphogenesis using inbred B-A stocks and inbred tester stocks. We compared plants containing one dose or three doses of each of the 14 chromosome arms with plants containing two doses for seven measured traits. These were leaf width, leaf length, plant height, ear height, internode length, ear node circumference, and tassel branch number. We observed the typical maize aneuploid syndrome wherein one dose was more widespread and more severe in its effects than three doses. All but two of the one-dose effects were negative, and all of the three-dose effects were negative. The occurrence of positive responses by hyperploid plants in our earlier B-A-A study and the absence of any positive responses among the hyperploids reported for the 14 simple B-A translocations tested for dosage effects in the present study and previously may reflect gene dosage interaction between the two chromosome arm segments present in the B-A-A translocations. The overall congruence of our results with those of previous studies suggests that the traits measured are quantitative traits controlled by multiple genes whose activities provide a balanced regulation that transcends individual inbred lines or diverse genetic backgrounds and that such genes may be especially abundant in chromosome arm 1L.     

A direct comparison between the genetic maps of sorghum and rice

A direct comparison of the genetic linkage maps of sorghum and rice is proposed. It is based on the mapping of a common set of 123 RFLP probes scattered on the genomes of both species. For each species a composite map was established by merging two individual maps comprising many common loci. This enabled us to confirm the global correspondence scheme that had previously been established between the chromosomes of sorghum and rice. It also provided a more detailed insight into the conservation of synteny and colinearity: 69% of the loci mapped on a given rice chromosome mapped to the corresponding homoeologous chromosome in sorghum; among them, 84% formed a colinear arrangement between the two species. Local inversions and translocations were detected. Received: 27 April 2000 / Accepted: 26 May 2000     

Integration of the Aedes aegypti mosquito genetic linkage and physical maps

Two approaches were used to correlate the Aedes aegypti genetic linkage map to the physical map. STS markers were developed for previously mapped RFLP-based genetic markers so that large genomic clones from cosmid libraries could be found and placed to the metaphase chromosome physical maps using standard FISH methods. Eight cosmids were identified that contained eight RFLP marker sequences, and these cosmids were located on the metaphase chromosomes. Twenty-one cDNAs were mapped directly to metaphase chromosomes using a FISH amplification procedure. The chromosome numbering schemes of the genetic linkage and physical maps corresponded directly and the orientations of the genetic linkage maps for chromosomes 2 and 3 were inverted relative to the physical maps. While the chromosome 2 linkage map represented essentially 100% of chromosome 2, approximately 65% of the chromosome 1 linkage map mapped to only 36% of the short p-arm and 83% of the chromosome 3 physical map contained the complete genetic linkage map. Since the genetic linkage map is a RFLP cDNA-based map, these data also provide a minimal estimate for the size of the euchromatic regions. The implications of these findings on positional cloning in A. aegypti are discussed.     

Linkage mapping of starch branching enzyme III in rice (Oryza sativa L.) and prediction of location of orthologous genes in other grasses

 The chromosomal position of Starch Branching Enzyme III (SBEIII) was determined via linkage to RFLP markers on an existing molecular map of rice (Oryza sativa L.). A cDNA of 890 bp was generated using specific PCR primers designed from available SBEIII sequence data and used as a probe in Southern analysis. The SBEIII cDNA hybridized to multiple restriction fragments, but these fragments mapped to a single locus on rice chromosome 2, flanked by CDO718 and RG157. The detection of a multiple-copy hybridization pattern suggested the possibility of a tandemly duplicated gene at this locus. The map location of orthologous SBE genes in maize, wheat, and oat were predicted based on previously published genetic studies and comparative maps of the grass family. Received : 5 August 1996 / Accepted : 13 September 1996     

Comparative Genome Mapping in Brassica

A Brassica nigra genetic linkage map was developed from a highly polymorphic cross analyzed with a set of low copy number Brassica RFLP probes. The Brassica genome is extensively duplicated with eight distinct sets of chromosomal segments, each present in three copies, covering virtually the whole genome. Thus, B. nigra could be descended from a hexaploid ancestor. A comparative analysis of B. nigra, B. oleracea and B. rapa genomes, based on maps developed using a common set of RFLP probes, was also performed. The three genomes have distinct chromosomal structures differentiated by a large number of rearrangements, but collinear regions involving virtually the whole of each the three genomes were identified. The genic contents of B. nigra, B. oleracea and B. rapa were basically equivalent and differences in chromosome number (8, 9 and 10, respectively) are probably the result of chromsome fusions and/or fissions. The strong conservation of overall genic content across the three Brassica genomes mirrors the conservation of genic content observed over a much longer evolutionary span in cereals. However, the rate of chromosomal rearrangement in crucifers is much higher than that observed in cereal genomes.     

Different Frequency in the Recovery of Crossover Products from Male and Female Gametes of Plants Hypoploid for B-a Translocations in Maize

The percentage of crossovers is consistently higher in plants hypoploid for six B-A translocations when crossed as males than when crossed as females; in most instances, this excess of male crossing over exceeds that found in control crosses (involving normal chromosomes). Thus, there seems to be something about the hypoploid condition that is responsible for the higher percentage of male crossovers. Several explanations for this "hypoploid effect" are possible, but none has been demonstrated to be responsible for the phenomenon.     

Mapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping

By combining the amplified fragment length polymorphism (AFLP) technique with selective genotyping, we constructed a linkage map for rice and assigned each linkage group to a corresponding chromosome. The AFLP map, consisting of 202 AFLP markers, was generated from 74 recombinant inbred lines (RIL) which were selected from both extremes of the population (250 lines) with respect to the response to complete submergence. Map length was 1756 cM, with an average interval size of 8.5 cM. To assign linkage groups to chromosomes, we used 50 previously mapped AFLP markers as anchor markers distributed over the 12 chromosomes. Other AFLP markers were then assigned to specific chromosomes based on their linkage to anchor markers. This AFLP map is equivalent to the RFLP/AFLP map constructed previously as the anchors were in the same order in both maps. Furthermore, tests with two restriction fragment length polymorphism (RFLP) markers and two sequence-tagged site (STS) markers showed that they mapped in the expected positions. Using this AFLP map, a major gene for submergence tolerance was localized on chromosome 9. Quantitative trait loci (QTL) associated with submergence tolerance were detected on chromosomes 6, 7, 11, and 12. We conclude that the combination of AFLP mapping and selective genotyping provides a much faster and easier approach to QTL identification than the use of RFLP markers. Received: 20 December 1996 / Accepted: 21 January 1997