Direct amplification of minisatellite-region DNA with VNTR core sequences in the genus Oryza

 A polymerase chain reaction (PCR) application, involving the directed amplification of minisatellite-region DNA (DAMD) with several minisatellite core sequences as primers, was used to detect genetic variation in 17 species of the genus Oryza and several rice cultivars (O. sativa L.). The electrophoretic analysis of DAMD-PCR products showed high levels of variation between different species and little variation between different cultivars of O. sativa. Polymorphisms were also found between accessions within a species, and between individual plants within an accession of several wild species. The DAMD-PCR yielded genome-specific banding patterns for the species studied. Several DAMD-PCR-generated DNA fragments were cloned and characterized. One clone was capable of detecting multiple fragments and revealed individual-specific hybridization banding patterns using genomic DNA from wild species as well as rice cultivars. A second clone detected only a single polymorphic locus, while a third clone expressed a strong genome specificity by Southern analysis. The results demonstrated that DAMD-PCR is potentially useful for species and genome identification in Oryza. The DAMD-PCR technique also allows for the isolation of informative molecular probes to be utilized in DNA fingerprinting and genome identification in rice. Received: 1 October 1996 / Accepted: 25 April 1997     

Comparative genomic in situ hybridization (cGISH) analysis of the genomic relationships among Sinapis arvensis, Brassica rapa and Brassica nigra

To further understand the relationships between the SS genome of Sinapis arvensis and the AA, BB genomes in Brassica, genomic DNA of Sinapis arvensis was hybridized to the metaphase chromosomes of Brassica nigra (BB genome), and the metaphase chromosomes and interphase nucleus of Brassica rapa (AA genome) by comparative genomic in situ hybridization (cGISH). As a result, every chromosome of B. nigra had signals along the whole chromosomal length. However, only half of the condensed heterochromatic areas in the interphase nucleus and the chromosomes showed rich signals in Brassica rapa. Interphase nucleus and the metaphase chromosomes of S. arvensis were simultaneously hybridized with digoxigenin-labeled genomic DNA of B. nigra and biotin-labeled genomic DNA of B. rapa. Signals of genomic DNA of B. nigra hybridized throughout the length of all chromosomes and all the condensed heterochromatic areas in the interphase nucleus, except chromosome 4, of which signals were weak in centromeric regions. Signals of the genomic DNA of B. rapa patterned the most areas of ten chromosomes and ten condensed heterochromatic areas, others had less signals. The results showed that the SS genome had homology with AA and BB genomes, but the homology between SS genome and AA genome was clearly lower than that between the SS genome and BB genome.     

Repetitive, genome-specific probes in wheat (Triticum aestivum L. em Thell) amplified with minisatellite core sequences

The detection and analysis of DNA polymorphisms in crops is an essential component of marker-assisted selection and cultivar identification in plant breeding. We have explored the direct amplification of minisatellite DNA by PCR (DAMD-PCR) as a means for generating DNA probes that are useful for detecting DNA polymorphisms and DNA fingerprinting in wheat. This technique was facilitated by high-stringency PCR with known plant and animal minisatellite core sequences as primers on wheat genomic DNA. The products of DAMD-PCR from Triticum aestivum, T. durum, T. monococcum, T. speltoides and T. tauschii showed a high degree of polymorphism and the various genomes could be identified. Cloning of the DAMD-PCR products and subsequent Southern hybridization frequently revealed polymorphic probes showing a good degree of genome specificity. In addition, polymorphic, single locus, and moderately dispersed PCR products were cloned that may have a potential for DNA fingerprinting. Our experiments were limited primarily to diploid wheats and the results indicated that DAMD-PCR may isolate genome-specific probes from wild diploid wheat species that could be used to monitor genome introgression into hexaploid wheat.This paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the University of Missouri, the Agricultural Experimental Station and U.S. Department of Agriculture-Agricultural Research Service, Plant Genetics Research Unit, journal series No. 12523     

PCR primed with minisatellite core sequences yields DNA fingerprinting probes in wheat

 Four minisatellite core sequences were used as primers in a polymerase chain reaction (PCR) technique, known as the directed amplification of minisatellite-region DNA (DAMD), to detect polymorphisms in three pairs of hexaploid/tetraploid wheat cultivars. In each pair, the tetraploid cultivar (genomic formula AABB) was extracted from its corresponding hexaploid (genomic formula AABBDD) parent. Reproducible profiles of the amplified products revealed characteristic bands that were present only in the hexaploid wheats but not in their extracted tetraploids. Some polymorphisms were observed among the hexaploid cultivars. Twenty-three DAMD-PCR amplified fragments were isolated and screened as molecular probes on the genomic DNA of wild wheat species, hexaploid wheat and triticale cultivars. Subsequently, 8 of the fragments were cloned and sequenced. The DAMD-PCR clones revealed various degrees of polymorphism among different wild and cultivated wheats. Two clones yielded individual-specific DNA fingerprinting patterns which could be used for species differentiation and cultivar identification. The results demonstrated the use of DAMD-PCR as a tool for the isolation of informative molecular probes for DNA fingerprinting in wheat cultivars and species. Received: 13 May 1996/Accepted: 11 October 1996     

Isolation of A/D and C genome specific dispersed and clustered repetitive DNA sequences from Avena sativa.

DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.     

Comparative fluorescence in situ hybridization mapping of a 431-kb Arabidopsis thaliana bacterial artificial chromosome contig reveals the role of chromosomal duplications in the expansion of the Brassica rapa genome

Comparative genome studies are important contributors to our understanding of genome evolution. Most comparative genome studies in plants have been based on genetic mapping of homologous DNA loci in different genomes. Large-scale comparative physical mapping has been hindered by the lack of efficient and affordable techniques. We report here the adaptation of fluorescence in situ hybridization (FISH) techniques for comparative physical mapping between Arabidopsis thaliana and Brassica rapa. A set of six bacterial artificial chromosomes (BACs) representing a 431-kb contiguous region of chromosome 2 of A. thaliana was mapped on both chromosomes and DNA fibers of B. rapa. This DNA fragment has a single location in the A. thaliana genome, but hybridized to four to six B. rapa chromosomes, indicating multiple duplications in the B. rapa genome. The sizes of the fiber-FISH signals from the same BACs were not longer in B. rapa than those in A. thaliana, suggesting that this genomic region is duplicated but not expanded in the B. rapa genome. The comparative fiber-FISH mapping results support that chromosomal duplications, rather than regional expansion due to accumulation of repetitive sequences in the intergenic regions, played the major role in the evolution of the B. rapa genome.     

Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors

Investigating recombination of homoeologous chromosomes in allopolyploid species is central to understanding plant breeding and evolution. However, examining chromosome pairing in the allotetraploid Brassica napus has been hampered by the lack of chromosome-specific molecular probes. In this study, we establish the identification of all homoeologous chromosomes of allopolyploid B. napus by using robust molecular cytogenetic karyotypes developed for the progenitor species Brassica rapa (A genome) and Brassica oleracea (C genome). The identification of every chromosome among these three Brassica species utilized genetically mapped bacterial artificial chromosomes (BACs) from B. rapa as probes for fluorescent in situ hybridization (FISH). With this BAC-FISH data, a second karyotype was developed using two BACs that contained repetitive DNA sequences and the ubiquitous ribosomal and pericentromere repeats. Using this diagnostic probe mix and a BAC that contained a C-genome repeat in two successive hybridizations allowed for routine identification of the corresponding homoeologous chromosomes between the A and C genomes of B. napus. When applied to the B. napus cultivar Stellar, we detected one chromosomal rearrangement relative to the parental karyotypes. This robust novel chromosomal painting technique will have biological applications for the understanding of chromosome pairing, homoeologous recombination, and genome evolution in the genus Brassica and will facilitate new applied breeding technologies that rely upon identification of chromosomes.     

Relationships betweenOryza species (Poaceae) based on 5S DNA sequences

Relationships between 9Oryza species, covering 6 different genomes, have been studied using hybridization and nucleotide sequence information from the5S Dna locus. Four to five units of the major size class of 5S DNA in each species, 55 units in all, were cloned and sequenced. Both hybridization and sequence data confirmed the basic differences between the A and B, C, D genome species suggested by morphological and cytological data. The 5S DNA units of the A genome species were very similar, as were the ones from the B, C, and D genome-containing species. The 5S DNA ofO. australiensis (E genome) grouped with the B, C, D cluster, while the units ofO. brachyantha (F genome) were quite different and grouped away from all other species. 5S DNA units fromO. minuta, O. latifolia, O. australiensis, andO. brachyantha hybridized strongly, and preferentially, to the genomic DNA from which the units were isolated and hence could be useful as species/genome specific probes. The 5S DNA units fromO. sativa, O. nivara, andO. rufipogon provided A genome-specific probes as they hybridized preferentially to A genome DNA. The units fromO. punctata andO. officinalis displayed weaker preferential hybridization toO. punctata DNA, possibly reflecting their shared genome (C genome).     

The CACTA transposon Bot1 played a major role in Brassica genome divergence and gene proliferation

We isolated and characterized a Brassica C genome-specific CACTA element, which was designated Bot1 (Brassica oleracea transposon 1). After analysing phylogenetic relationships, copy numbers and sequence similarity of Bot1 and Bot1 analogues in B. oleracea (C genome) versus Brassica rapa (A genome), we concluded that Bot1 has encountered several rounds of amplification in the oleracea genome only, and has played a major role in the recent rapa and oleracea genome divergence. We performed in silico analyses of the genomic organization and internal structure of Bot1, and established which segment of Bot1 is C-genome specific. Our work reports a fully characterized Brassica repetitive sequence that can distinguish the Brassica A and C chromosomes in the allotetraploid Brassica napus, by fluorescent in situ hybridization. We demonstrated that Bot1 carries a host S locus-associated SLL3 gene copy. We speculate that Bot1 was involved in the proliferation of SLL3 around the Brassica genome. The present study reinforces the assumption that transposons are a major driver of genome and gene evolution in higher plants.     

A Modified Enrichment Method to Construct Microsatellite Library from Plateau Pika Genome （Ochotona curzoniae）

A microsatellite-enriched library of plateau pika(Ochotona curzoniae)was constructed according to the strong affinity between biotin and streptavidin.Firstly,genomic DNA was fragmented by ultrasonication,which is a major improvement over traditional methods.Linker-ligated DNA fragments were hybridized with biotinylated microsatellite probes,and then were subjected to streptavidin-coated magnetic beads.PCR amplification was performed to obtain double-stranded DNA fragments containing microsatellites.Ligation and transformation were carried out by using the pGEM-T Vector System Ⅰ and Escherichia coli DH10B competent cells.Sequencing results showed that 80.2% of clones contained microsatellite repeat motif.Several modifications make this protocol time-efficient and technically easier than the traditional ones; particularly,composition and relative abundance of microsatellite repeats in plateau pika genome were truly represented through the optimized PCR conditions.This method has also been successfully applied to construct microsatellite-enriched genomic libraries of Chinese hamster(Cricetulus griseus)and small abalone[Haliotis diversicolor(Reeve)]with high rates of positive clones,demonstrating its feasibility and stability.     

PCR primed with VNTR core sequences yields species specific patterns and hypervariable probes.

The use of genomic DNA-based techniques in ecological and evolutionary studies has been limited by the availability of suitable probes for species of interest due to the technical difficulty of isolating and applying such probes. We have developed a simple technique that directs polymerase chain reaction (PCR) amplification to regions rich in variable number of tandem repeats (VNTRs). By using published VNTR core sequences as primers in PCRs, fragments were amplified that showed little variation within a species, but did show differences between species. When the amplified fragments were used as probes with genomic DNA Southern blots they produced hypervariable single-locus or few-locus patterns in fish, birds, and humans. We have named this procedure as Directed Amplification of Minisatellite-region DNA (DAMD).     

Synthesis of intergeneric hybrids and establishment of genomic affinity between <Emphasis Type="Italic">Diplotaxis catholica</Emphasis> and crop <Emphasis Type="Italic">Brassica</Emphasis> species

Intergeneric hybrids of the wild crucifer Diplotaxis catholica (2n = 18, D(C)D(C)) as female with two crop Brassica species, namely Brassica rapa (2n = 20; AA) and Brassica juncea (2n = 36; AABB) as male, were developed, using ovary and sequential culture. Reciprocal crosses were not successful, suggesting unilateral cross incompatibility. Morphologically, the hybrid plants resembled the crop brassica parents, but were nearly male- as well as female-sterile. Induction of amphiploidy helped to improve pollen fertility for the D. catholica x B. rapa cross (73%), but less so for the D. catholica x B. juncea cross (35-40%). Female fertility was also higher in both the amphiploids. Cytological analysis of the F(1) hybrids revealed aberrant meiosis with predominant occurrence of the univalents. Partial genomic homoeology between the A genome of B. rapa and the D(C) genome of D. catholica was indicated by the presence of up to five bivalents in 14.7% of the PMCs in the D. catholica x B. rapa hybrid, and 1-2 trivalents or a quadrivalent in nearly 44% of the PMCs in the derived amphiploid. In the second cross, D. catholica x B. juncea, up to six bivalents and one trivalent were observed indicating homoeology between the A/B genomes of B. juncea and the D(C) genome of D. catholica. The possibility of introgression of desirable genes from D. catholica into crop Brassica species exists in view of significant affinity between the D(C) and A/B genomes.     

Genome doubling and chromosome elimination with fragment recombination leading to the formation of Brassica rapa-type plants with genomic alterations in crosses with Orychophragmus violaceus.

In distant hybridization of plants, nonclassical hybrids with unexpected chromosome complements, chromosome elimination, and genetic introgression have been well documented. We obtained intergeneric hybrids between Brassica rapa, B. rapa var. chinensis, and another cruciferous species, Orychophragmus violaceus, following embryo rescue. Hybrids mainly displayed phenotypes of B. rapa, although certain O. violaceus or novel characteristics also appeared. Variable numbers of chromosomes were observed in somatic cells in the roots of plantlets on medium and in ovaries and pollen mother cells (PMCs). However, higher numbers were recorded in the roots. GISH revealed that the majority of ovary cells and PMCs contained 20 chromosomes of B. rapa with or without individual O. violaceus chromosomes or fragments added or introgressed. AFLP analysis showed that fragments deleted from the B. rapa genome were much more frequent than novel and O. violaceus fragments. The mechanisms involved genome doubling and successive elimination of O. violaceus chromosomes accompanied by fragment recombination and introgression, producing B. rapa-type plants with modified genetic constitutions and phenotypes.     

Characterization of seedling proteomes and development of markers to distinguish the Brassica A and C genomes

The diploid species Brassica rapa(genome AA)and B.oleracea(genome CC)were compared by fuU-seale proteome analyses of seedling.A total of 28.2% of the proteins was common to both species,indicating the existence of a basal or ubiquitous proteome.How-ever,a number of discriminating proteins(32.0%)and specific proteins(39.8%)of the Brassica A and C genomes,respectively,were identified,which could represent potentially species-specific functions.Based on these A or C genome-specific proteins,a number of PCR-based markers to distinguish B.rapa and B.oleracea species were also developed.     

The mosaic of ancestral karyotype blocks in the Sinapis alba L. genome

The organisation of the Sinapis alba genome, comprising 12 linkage groups (n = 12), was compared with the Brassicaceae ancestral karyotype (AK) genomic blocks previously described in other crucifer species. Most of the S. alba genome falls into conserved triplicated genomic blocks that closely match the AK-defined genomic blocks found in other crucifer species including the A, B, and C genomes of closely related Brassica species. In one instance, an S. alba linkage group (S05) was completely collinear with one AK chromosome (AK1), the first time this has been observed in a member of the Brassiceae tribe. However, as observed for other members of the Brassiceae tribe, ancestral genomic blocks were fragmented in the S. alba genome, supporting previously reported comparative chromosome painting describing rearrangements of the AK karyotype prior to the divergence of the Brassiceae from other crucifers. The presented data also refute previous phylogenetic reports that suggest S. alba was more closely related to Brassica nigra (B genome) than to B. rapa (A genome) and B. oleracea (C genome). A comparison of the S. alba and Arabidopsis thaliana genomes revealed many regions of conserved gene order, which will facilitate access to the rich genomic resources available in the model species A. thaliana for genetic research in the less well-resourced crop species S. alba.     

Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives

The cultivated Brassica species are the group of crops most closely related to Arabidopsis thaliana (Arabidopsis). They represent models for the application in crops of genomic information gained in Arabidopsis and provide an opportunity for the investigation of polyploid genome formation and evolution. The scientific literature contains contradictory evidence for the dynamics of the evolution of polyploid genomes. We aimed at overcoming the inherent complexity of Brassica genomes and clarify the effects of polyploidy on the evolution of genome microstructure in specific segments of the genome. To do this, we have constructed bacterial artificial chromosome (BAC) libraries from genomic DNA of B. rapa subspecies trilocularis (JBr) and B. napus var Tapidor (JBnB) to supplement an existing BAC library from B. oleracea. These allowed us to analyse both recent polyploidization (under 10,000 years in B. napus) and more ancient polyploidization events (ca. 20 Myr for B. rapa and B. oleracea relative to Arabidopsis), with an analysis of the events occurring on an intermediate time scale (over the ca. 4 Myr since the divergence of the B. rapa and B. oleracea lineages). Using the Arabidopsis genome sequence and clones from the JBr library, we have analysed aspects of gene conservation and microsynteny between six regions of the genome of B. rapa with the homoeologous regions of the genomes of B. oleracea and Arabidopsis. Extensive divergence of gene content was observed between the B. rapa paralogous segments and their homoeologous segments within the genome of Arabidopsis. A pattern of interspersed gene loss was identified that is similar, but not identical, to that observed in B. oleracea. The conserved genes show highly conserved collinearity with their orthologues across genomes, but a small number of species-specific rearrangements were identified. Thus the evolution of genome microstructure is an ongoing process. Brassica napus is a recently formed polyploid resulting from the hybridization of B. rapa (containing the Brassica A genome) and B. oleracea (containing the Brassica C genome). Using clones from the JBnB library, we have analysed the microstructure of the corresponding segments of the B. napus genome. The results show that there has been little or no change to the microstructure of the analysed segments of the Brassica A and C genomes as a consequence of the hybridization event forming natural B. napus. The observations indicate that, upon polyploid formation, these segments of the genome did not undergo a burst of evolution discernible at the scale of microstructure.     

Chromosomal localization and characterization of rDNA loci in the Brassica A and C genomes.

Using fluorescence in situ hybridization, we located ribosomal DNA loci on prometaphase chromosomes of the diploid species Brassica rapa and Brassica oleracea and their amphidiploid Brassica napus. Based on comparisons of chromosome morphology and hybridization patterns, we characterized the individual B. napus rDNA loci according to their presumed origins in the Brassica A and C genomes. As reported in other studies, the sum of rDNA loci observed on B. rapa (AA genome) and B. oleracea (CC genome) chromosomes was one greater than the total number of loci seen in their amphidiploid B. napus (AACC). Evidence is presented that this reduction in B. napus rDNA locus number results from the loss of the smallest A genome rDNA site in the amphidiploid.     

Characterization of seedling proteomes and development of markers to distinguish the Brassica A and C genomes

The diploid species Brassica rapa(genome AA)and B.oleracea(genome CC)were compared by full-scale proteome analyses of seedling.A total of 28.2% of the proteins was common to both species,indicating the existence of a basal or ubiquitous proteome.However,a number of discriminating proteins(32.0%)and specific proteins(39.8%)of the Brassica A and C genomes,respectively,were identified,which could represent potentially species-specific functions.Based on these A or C genome-specific proteins,a number of PCR-bas...     

Structural and functional comparative mapping between the Brassica A genomes in allotetraploid Brassica napus and diploid Brassica rapa

Brassica napus (AACC genome) is an important oilseed crop that was formed by the fusion of the diploids B. rapa (AA) and B. oleracea (CC). The complete genomic sequence of the Brassica A genome will be available soon from the B. rapa genome sequencing project, but it is not clear how informative the A genome sequence in B. rapa (A(r)) will be for predicting the structure and function of the A subgenome in the allotetraploid Brassica species B. napus (A(n)). In this paper, we report the results of structural and functional comparative mapping between the A subgenomes of B. napus and B. rapa based on genetic maps that were anchored with bacterial artificial chromosomes (BACs)-sequence of B. rapa. We identified segmental conservation that represented by syntenic blocks in over one third of the A genome; meanwhile, comparative mapping of quantitative trait loci for seed quality traits identified a dozen homologous regions with conserved function in the A genome of the two species. However, several genomic rearrangement events, such as inversions, intra- and inter-chromosomal translocations, were also observed, covering totally at least 5% of the A genome, between allotetraploid B. napus and diploid B. rapa. Based on these results, the A genomes of B. rapa and B. napus are mostly functionally conserved, but caution will be necessary in applying the full sequence data from B. rapa to the B. napus as a result of genomic rearrangements in the A genome between the two species.     

Studies on the Relationships Between Moricandia and Brassica Species

Moricandia is the only genus with C3-C4 species within the family of Cruciferae. To provide the basic information of transferring C3-C4 and other important characteristics from Moricandia to Brassica crops, the relationships between Moricandia and Brassica species were studied based on crossability and RFLP fingerprinting. The crossability was very low between the two genera in the experiment. There was no hybrid seed obtained between M. arvensis and B. rapa though 8 000 flowers were crossed. 2 989 cross-pollinated ovaries were cultured and also no hybrid embryo was developed. However, four intergeneric hybrid shoots were generated from 105 cultured ovaries in the combination of M. arvensis x B. napus. The nucleus DNA polymorphism of restriction loci was detected with 23 genic DNA clones of B. napus for the samples of B. napus, B. rapa and B. oleracea, M. arvensis and M. nit, ns. A high homology was found between Moricandia and Brassica species. The similarity between M. nitens and B. rapa was even greater than that between B. rapa and B. napus. The close relationships between Moricandia species and Brassica crops, especially European B. rapa, were also detected with 4 beta mitochondria probes. The intensive homology between Moricandia C3-C4 species and Brassica crops evaluated with the RFLP markers revealed the possibility of transferring some important genes from the C3-C4 species to the domesticated species by sexual hybridization or protoplast fusion followed by recombination of homoeologous chromosomes.