A 2600-locus chromosome bin map of wheat homoeologous group 2 reveals interstitial gene-rich islands and colinearity with rice

The complex hexaploid wheat genome offers many challenges for genomics research. Expressed sequence tags facilitate the analysis of gene-coding regions and provide a rich source of molecular markers for mapping and comparison with model organisms. The objectives of this study were to construct a high-density EST chromosome bin map of wheat homoeologous group 2 chromosomes to determine the distribution of ESTs, construct a consensus map of group 2 ESTs, investigate synteny, examine patterns of duplication, and assess the colinearity with rice of ESTs assigned to the group 2 consensus bin map. A total of 2600 loci generated from 1110 ESTs were mapped to group 2 chromosomes by Southern hybridization onto wheat aneuploid chromosome and deletion stocks. A consensus map was constructed of 552 ESTs mapping to more than one group 2 chromosome. Regions of high gene density in distal bins and low gene density in proximal bins were found. Two interstitial gene-rich islands flanked by relatively gene-poor regions on both the short and long arms and having good synteny with rice were discovered. The map locations of two ESTs indicated the possible presence of a small pericentric inversion on chromosome 2B. Wheat chromosome group 2 was shown to share syntenous blocks with rice chromosomes 4 and 7.     

Deletion mapping of homoeologous group 6-specific wheat expressed sequence tags

To localize wheat (Triticum aestivum L.) ESTs on chromosomes, 882 homoeologous group 6-specific ESTs were identified by physically mapping 7965 singletons from 37 cDNA libraries on 146 chromosome, arm, and sub-arm aneuploid and deletion stocks. The 882 ESTs were physically mapped to 25 regions (bins) flanked by 23 deletion breakpoints. Of the 5154 restriction fragments detected by 882 ESTs, 2043 (loci) were localized to group 6 chromosomes and 806 were mapped on other chromosome groups. The number of loci mapped was greatest on chromosome 6B and least on 6D. The 264 ESTs that detected orthologous loci on all three homoeologs using one restriction enzyme were used to construct a consensus physical map. The physical distribution of ESTs was uneven on chromosomes with a tendency toward higher densities in the distal halves of chromosome arms. About 43% of the wheat group 6 ESTs identified rice homologs upon comparisons of genome sequences. Fifty-eight percent of these ESTs were present on rice chromosome 2 and the remaining were on other rice chromosomes. Even within the group 6 bins, rice chromosomal blocks identified by 1-6 wheat ESTs were homologous to up to 11 rice chromosomes. These rice-block contigs were used to resolve the order of wheat ESTs within each bin.     

Molecular characterization of a set of wheat deletion stocks for use in chromosome bin mapping of ESTs

The objective of this study was molecular characterization of a set of deletion stocks and other aneuploids for use in chromosome bin mapping of ESTs in wheat. Wheat aneuploid stocks including 21 nullisomic-tetrasomic (NT), 24 ditelosomic (Dt), and 101 deletion (del) lines were screened with 526 EST clones. A total of 1,951 loci were detected by 493 informative EST clones and tagged 150 of the 159 deletion intervals or chromosome bins. Previously described deletion lines del1AS-4, del6AL-2, del6BS-6, and del7DS-6 were found to have normal chromosome constitution. The short arm deletion in del3AS-3 may be translocated from an unknown chromosome as this stock is nullisomic for the 3AS arm. Thirty-five new deletions were detected in 26 lines. Most of the new deletions occurred in terminal regions of chromosomes and probably resulted from the loss of very small terminal fragments that were difficult to detect cytologically. Eleven chromosome aberrations were also detected in two NT and five Dt lines. Overall, the chromosome bin map provides a resolution of around 28 Mb for an anchor map of a basic set of seven chromosomes of the Triticeae. Any target gene can be allocated to a specific 28-Mb bin and associated ESTs, anchored to the other Triticeae/grass maps including rice and, therefore, amenable to molecular cloning by comparative and wheat-based positional cloning methods. Electronic Publication     

Utilization of deletion bins to anchor and order sequences along the wheat 7B chromosome

Key message

A total of 3,671 sequence contigs and scaffolds were mapped to deletion bins on wheat chromosome 7B providing a foundation for developing high-resolution integrated physical map for this chromosome.

Abstract

Bread wheat (Triticum aestivum L.) has a large, complex and highly repetitive genome which is challenging to assemble into high quality pseudo-chromosomes. As part of the international effort to sequence the hexaploid bread wheat genome by the international wheat genome sequencing consortium (IWGSC) we are focused on assembling a reference sequence for chromosome 7B. The successful completion of the reference chromosome sequence is highly dependent on the integration of genetic and physical maps. To aid the integration of these two types of maps, we have constructed a high-density deletion bin map of chromosome 7B. Using the 270 K Nimblegen comparative genomic hybridization (CGH) array on a set of cv. Chinese spring deletion lines, a total of 3,671 sequence contigs and scaffolds (~7.8 % of chromosome 7B physical length) were mapped into nine deletion bins. Our method of genotyping deletions on chromosome 7B relied on a model-based clustering algorithm (Mclust) to accurately predict the presence or absence of a given genomic sequence in a deletion line. The bin mapping results were validated using three different approaches, viz. (a) PCR-based amplification of randomly selected bin mapped sequences (b) comparison with previously mapped ESTs and (c) comparison with a 7B genetic map developed in the present study. Validation of the bin mapping results suggested a high accuracy of the assignment of 7B sequence contigs and scaffolds to the 7B deletion bins.     

A chromosome bin map of 16,000 expressed sequence tag loci and distribution of genes among the three genomes of polyploid wheat

Because of the huge size of the common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) genome of 17,300 Mb, sequencing and mapping of the expressed portion is a logical first step for gene discovery. Here we report mapping of 7104 expressed sequence tag (EST) unigenes by Southern hybridization into a chromosome bin map using a set of wheat aneuploids and deletion stocks. Each EST detected a mean of 4.8 restriction fragments and 2.8 loci. More loci were mapped in the B genome (5774) than in the A (5173) or D (5146) genomes. The EST density was significantly higher for the D genome than for the A or B. In general, EST density increased relative to the physical distance from the centromere. The majority of EST-dense regions are in the distal parts of chromosomes. Most of the agronomically important genes are located in EST-dense regions. The chromosome bin map of ESTs is a unique resource for SNP analysis, comparative mapping, structural and functional analysis, and polyploid evolution, as well as providing a framework for constructing a sequence-ready, BAC-contig map of the wheat genome.     

High-density genetic and physical bin mapping of wheat chromosome 1D reveals that the powdery mildew resistance gene <Emphasis Type="Italic">Pm24</Emphasis> is located in a highly recombinogenic region

Genetic maps of wheat chromosome 1D consisting of 57 microsatellite marker loci were constructed using Chinese Spring (CS) × Chiyacao F₂ and the International Triticeae Mapping Initiative (ITMI) recombinant inbred lines (RILs) mapping populations. Marker order was consistent, but genetic distances of neighboring markers were different in two populations. Physical bin map of 57 microsatellite marker loci was generated by means of 10 CS 1D deletion lines. The physical bin mapping indicated that microsatellite marker loci were not randomly distributed on chromosome 1D. Nineteen of the 24 (79.2%) microsatellite markers were mapped in the distal 30% genomic region of 1DS, whereas 25 of the 33 (75.8%) markers were assigned to the distal 59% region of 1DL. The powdery mildew resistance gene Pm24, originating from the Chinese wheat landrace Chiyacao, was previously mapped in the vicinity of the centromere on the short arm of chromosome 1D. A high density genetic map of chromosome 1D was constructed, consisting of 36 markers and Pm24, with a total map length of 292.7 cM. Twelve marker loci were found to be closely linked to Pm24. Pm24 was flanked by Xgwm789 (Xgwm603) and Xbarc229 with genetic distances of 2.4 and 3.6 cM, respectively, whereas a microsatellite marker Xgwm1291 co-segregated with Pm24. The microsatellite marker Xgwm1291 was assigned to the bin 1DS5-0.70-1.00 of the chromosome arm 1DS. It could be concluded that Pm24 is located in the ‘1S0.8 gene-rich region’, a highly recombinogenic region of wheat. The results presented here would provide a start point for the map-based cloning of Pm24.     

Structural and functional analyses of the wheat genomes based on expressed sequence tags (ESTs) related to abiotic stresses.

To gain insights into the structure and function of the wheat (Triticum aestivum L.) genomes, we identified 278 ESTs related to abiotic stress (cold, heat, drought, salinity, and aluminum) from 7671 ESTs previously mapped to wheat chromosomes. Of the 278 abiotic stress related ESTs, 259 (811 loci) were assigned to chromosome deletion bins and analyzed for their distribution pattern among the 7 homoeologous chromosome groups. Distribution of abiotic stress related EST loci were not uniform throughout the different regions of the chromosomes of the 3 wheat genomes. Both the short and long arms of group 4 chromosomes showed a higher number of loci in their distal regions compared with proximal regions. Of the 811 loci, the number of mapped loci on the A, B, and D genomes were 258, 281, and 272, respectively. The highest number of abiotic stress related loci were found in homoeologous chromosome group 2 (142 loci) and the lowest number were found in group 6 (94 loci). When considering the genome-specific ESTs, the B genome showed the highest number of unique ESTs (7 loci), while none were found in the D genome. Similarly, considering homoeologous group-specific ESTs, group 2 showed the highest number with 16 unique ESTs (58 loci), followed by group 4 with 9 unique ESTs (33 loci). Many of the classified proteins fell into the biological process categories associated with metabolism, cell growth, and cell maintenance. Most of the mapped ESTs fell into the category of enzyme activity (28%), followed by binding activity (27%). Enzymes related to abiotic stress such as beta-galactosidase, peroxidase, glutathione reductase, and trehalose-6-phosphate synthase were identified. The comparison of stress-responsive ESTs with genomic sequences of rice (Oryza sativa L.) chromosomes revealed the complexities of colinearity. This bin map provides insight into the structural and functional details of wheat genomic regions in relation to abiotic stress.     

Structural characterization of Brachypodium genome and its syntenic relationship with rice and wheat

Brachypodium distachyon (Brachypodium) has been recently recognized as an emerging model system for both comparative and functional genomics in grass species. In this study, 55,221 repeat masked Brachypodium BAC end sequences (BES) were used for comparative analysis against the 12 rice pseudomolecules. The analysis revealed that ~26.4% of BES have significant matches with the rice genome and 82.4% of the matches were homologous to known genes. Further analysis of paired-end BES and ~1.0 Mb sequences from nine selected BACs proved to be useful in revealing conserved regions and regions that have undergone considerable genomic changes. Differential gene amplification, insertions/deletions and inversions appeared to be the common evolutionary events that caused variations of microcolinearity at different orthologous genomic regions. It was found that ~17% of genes in the two genomes are not colinear in the orthologous regions. Analysis of BAC sequences also revealed higher gene density (~9 kb/gene) and lower repeat DNA content (~13.1%) in Brachypodium when compared to the orthologous rice regions, consistent with the smaller size of the Brachypodium genome. The 119 annotated Brachypodium genes were BLASTN compared against the wheat EST database and deletion bin mapped wheat ESTs. About 77% of the genes retrieved significant matches in the EST database, while 9.2% matched to the bin mapped ESTs. In some cases, genes in single Brachypodium BACs matched to multiple ESTs that were mapped to the same deletion bins, suggesting that the Brachypodium genome will be useful for ordering wheat ESTs within the deletion bins and developing specific markers at targeted regions in the wheat genome.     

A maize map standard with sequenced core markers, grass genome reference points and 932 expressed sequence tagged sites (ESTs) in a 1736-locus map.

We have constructed a 1736-locus maize genome map containing1156 loci probed by cDNAs, 545 probed by random genomic clones, 16 by simple sequence repeats (SSRs), 14 by isozymes, and 5 by anonymous clones. Sequence information is available for 56% of the loci with 66% of the sequenced loci assigned functions. A total of 596 new ESTs were mapped from a B73 library of 5-wk-old shoots. The map contains 237 loci probed by barley, oat, wheat, rice, or tripsacum clones, which serve as grass genome reference points in comparisons between maize and other grass maps. Ninety core markers selected for low copy number, high polymorphism, and even spacing along the chromosome delineate the 100 bins on the map. The average bin size is 17 cM. Use of bin assignments enables comparison among different maize mapping populations and experiments including those involving cytogenetic stocks, mutants, or quantitative trait loci. Integration of nonmaize markers in the map extends the resources available for gene discovery beyond the boundaries of maize mapping information into the expanse of map, sequence, and phenotype information from other grass species. This map provides a foundation for numerous basic and applied investigations including studies of gene organization, gene and genome evolution, targeted cloning, and dissection of complex traits.     

Development and mapping of microsatellite (SSR) markers in wheat

Microsatellite DNA markers are consistently found to be more informative than other classes of markers in hexaploid wheat. The objectives of this research were to develop new primers flanking wheat microsatellites and to position the associated loci on the wheat genome map by genetic linkage mapping in the ITMI W7984 × Opata85 recombinant inbred line (RIL) population and/or by physical mapping with cytogenetic stocks. We observed that the efficiency of marker development could be increased in wheat by creating libraries from sheared rather than enzyme-digested DNA fragments for microsatellite screening, by focusing on microsatellites with the [ATT/TAA]_n motif, and by adding an untemplated G-C clamp to the 5-end of primers. A total of 540 microsatellite-flanking primer pairs were developed, tested, and annotated from random genomic libraries. Primer pairs and associated loci were assigned identifiers prefixed with BARC (the acronym for the USDA-ARS Beltsville Agricultural Research Center) or Xbarc, respectively. A subset of 315 primer sets was used to map 347 loci. One hundred and twenty-five loci were localized by physical mapping alone. Of the 222 loci mapped with the ITMI population, 126 were also physically mapped. Considering all mapped loci, 126, 125, and 96 mapped to the A, B, and D genomes, respectively. Twenty-three of the new loci were positioned in gaps larger than 10 cM in the map based on pre-existing markers, and 14 mapped to the ends of chromosomes. The length of the linkage map was extended by 80.7 cM. Map positions were consistent for 111 of the 126 loci positioned by both genetic and physical mapping. The majority of the 15 discrepancies between genetic and physical mapping involved chromosome group 5.Electronic Supplementary Material Supplementary material is available for this article at     

EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci.

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci.     

Development of chromosome-arm-specific microsatellite markers in Triticum aestivum (Poaceae) using NGS technology

? Premise of the study: The aim of this study was to assess the feasibility of developing chromosome-arm-specific microsatellite markers in wheat on a large scale based on chromosome survey sequences obtained with next-generation sequencing (NGS) technology. ? Methods and Results: The Illumina Hi Seq2000 sequencing platform was used to sequence DNA of isolated wheat chromosome-arm 7DL. The data were assembled and microsatellite loci were identified computationally. In total, 16315 microsatellites were identified from 161061 assembled contigs. Thirty-three markers were randomly selected for validation across 20 diverse wheat cultivars. Two nulli-tetrasomic stocks were also screened to validate the specificity of the newly developed markers. ? Conclusions: This is the first study on identification of chromosome-arm-specific microsatellite markers using NGS technology. These new chromosome-arm-specific markers will facilitate saturation of the 7DL genetic map, and their availability will support genetic mapping and positional cloning in wheat.     

Comparative mapping of wheat chromosome 1AS which contains the tiller inhibition gene (<Emphasis Type="Italic">tin</Emphasis>) with rice chromosome 5S

The capacity to tiller is a key factor that determines plant architecture. Using molecular markers, a single major gene reducing tiller number, formally named the tiller inhibition gene (tin), was mapped to the short arm of chromosome 1A in wheat. We identified a tightly linked microsatellite marker (Xgwm136) that may be useful in future marker-assisted selection. The tin gene was mapped to the distal deletion bin of chromosome 1AS (FLM value 0.86) and wheat ESTs which were previously mapped to the same deletion bin were used to identify 18 closely related sequences in the syntenic region of rice chromosome 5. For a subset of wheat ESTs that detected flanking markers for tin, we identified closely related sequences within the most distal 300 kb of rice chromosome 5S. The synteny between the distal chromosome ends of wheat 1AS and rice 5S appeared to be disrupted at the hairy glume locus and seed storage protein loci. We compared map position of tin with other reduced tillering mutants characterised in other cereals to identify possible orthologous genes.     

Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map

The concept of selective (or bin) mapping is used here for the first time, using as an example the Prunus reference map constructed with an almond x peach F2 population. On the basis of this map, a set of six plants that jointly defined 65 possible different genotypes for the codominant markers mapped on it was selected. Sixty-three of these joint genotypes corresponded to a single chromosomal region (a bin) of the Prunus genome, and the two remaining corresponded to two bins each. The 67 bins defined by these six plants had a 7.8-cM average length and a maximum individual length of 24.7 cM. Using a unit of analysis composed of these six plants, their F1 hybrid parent, and one of the parents of the hybrid, we mapped 264 microsatellite (or simple-sequence repeat, SSR) markers from 401 different microsatellite primer pairs. Bin mapping proved to be a fast and economic strategy that could be used for further map saturation, the addition of valuable markers (such as those based on microsatellites or ESTs), and giving a wider scope to, and a more efficient use of, reference mapping populations.     

A 2500-locus bin map of wheat homoeologous group 5 provides insights on gene distribution and colinearity with rice

We constructed high-density deletion bin maps of wheat chromosomes 5A, 5B, and 5D, including 2338 loci mapped with 1052 EST probes and 217 previously mapped loci (total 2555 loci). This information was combined to construct a consensus chromosome bin map of group 5 including 24 bins. A relatively higher number of loci were mapped on chromosome 5B (38%) compared to 5A (34%) and 5D (28%). Differences in the levels of polymorphism among the three chromosomes were partially responsible for these differences. A higher number of duplicated loci was found on chromosome 5B (42%). Three times more loci were mapped on the long arms than on the short arms, and a significantly higher number of probes, loci, and duplicated loci were mapped on the distal halves than on the proximal halves of the chromosome arms. Good overall colinearity was observed among the three homoeologous group 5 chromosomes, except for the previously known 5AL/4AL translocation and a putative small pericentric inversion in chromosome 5A. Statistically significant colinearity was observed between low-copy-number ESTs from wheat homoeologous group 5 and rice chromosomes 12 (88 ESTs), 9 (72 ESTs), and 3 (84 ESTs).     

Development of a D genome specific marker resource for diploid and hexaploid wheat

Background

Mapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those within the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of polymorphic gene markers and poor genetic recombination in certain genetic regions. Although the abundance of repetitive sequence may pose common problems in genome analysis and sequence assembly of large and complex genomes, they provide repeat junction markers with random and unbiased distribution throughout chromosomes. Hence, development of a high-throughput mapping technology that combine both gene-based and repeat junction-based markers is needed to generate maps that have better coverage of the entire genome.

Results

In this study, the available genomics resource of the diploid Aegilop tauschii, the D genome donor of bread wheat, were used to develop genome specific markers that can be applied for mapping in modern hexaploid wheat. A NimbleGen array containing both gene-based and repeat junction probe sequences derived from Ae. tauschii was developed and used to map the Chinese Spring nullisomic-tetrasomic lines and deletion bin lines of the D genome chromosomes. Based on these mapping data, we have now anchored 5,171 repeat junction probes and 10,892 gene probes, corresponding to 5,070 gene markers, to the delineated deletion bins of the D genome. The order of the gene-based markers within the deletion bins of the Chinese Spring can be inferred based on their positions on the Ae. tauschii genetic map. Analysis of the probe sequences against the Chinese Spring chromosome sequence assembly database facilitated mapping of the NimbleGen probes to the sequence contigs and allowed assignment or ordering of these sequence contigs within the deletion bins. The accumulated length of anchored sequence contigs is about 155 Mb, representing ~ 3.2 % of the D genome. A specific database was developed to allow user to search or BLAST against the probe sequence information and to directly download PCR primers for mapping specific genetic loci.

Conclusions

In bread wheat, aneuploid stocks have been extensively used to assign markers linked with genes/traits to chromosomes, chromosome arms, and their specific bins. Through this study, we added thousands of markers to the existing wheat chromosome bin map, representing a significant step forward in providing a resource to navigate the wheat genome. The database website (http://probes.pw.usda.gov/ATRJM/) provides easy access and efficient utilization of the data. The resources developed herein can aid map-based cloning of traits of interest and the sequencing of the D genome of hexaploid wheat.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1852-2) contains supplementary material, which is available to authorized users.Keyword: Wheat deletion bins, Molecular markers, Repeat junction markers, NimbleGen array, Recombination, Genetic map     

A comparative genetic and cytogenetic mapping of wheat chromosome 5B using introgression lines

The genetic map of chromosome 5B has been constructed by using microsatellite (SSR) analysis of 381 plants from the F2 population produced by cross of the Chinese Spring (CS) and Renan cultivars. Initially, 180 SSR markers for the common wheat 5B chromosome have been used for analysis of these cultivars. The 32 markers able to detect polymorphism between these cultivars have been located on the genetic map of chromosome 5B. Cytogenetic mapping has involved a set of CS 5B chromosome deletion lines. Totally, 51 SSR markers have been located in ten regions (deletion bins) of this chromosome by SSR analysis of these deletion lines. Five genes—TaCBFIIIc-B10, Vrn-B1, Chi-B1, Skr, and Ph1—have been integrated into the cytogenetic map of chromosome 5B using the markers either specific of or tightly linked to the genes in question. Comparison of the genetic and cytogenetic maps suggests that recombination is suppressed in the pericentromeric region of chromosome 5B, especially in the short arm segment. The 18 markers localized to deletion bins 5BL16-0.79-1.00 and 5BL18-0.66-0.79 have been used to analyze common wheat introgression lines L842, L5366-180, L73/00i, and L21-4, carrying fragments of alien genomes in the terminal region of 5B long arm. L5366-180 and L842 lines carry a fragment of the Triticum timopheevii 5GL chromosome, while L73/00i and L21-4 lines, a fragment of the Aegilops speltoides 5SL chromosome. As has been shown, the translocated fragments in these four lines are of different lengths, allowing bin 5BL18-0.66-0.79 to be divided into three shorter regions. The utility of wheat introgression lines carrying alien translocations for increasing the resolution of cytogenetic mapping is discussed.     

Group 3 chromosome bin maps of wheat and their relationship to rice chromosome 1

The focus of this study was to analyze the content, distribution, and comparative genome relationships of 996 chromosome bin-mapped expressed sequence tags (ESTs) accounting for 2266 restriction fragments (loci) on the homoeologous group 3 chromosomes of hexaploid wheat (Triticum aestivum L.). Of these loci, 634, 884, and 748 were mapped on chromosomes 3A, 3B, and 3D, respectively. The individual chromosome bin maps revealed bins with a high density of mapped ESTs in the distal region and bins of low density in the proximal region of the chromosome arms, with the exception of 3DS and 3DL. These distributions were more localized on the higher-resolution group 3 consensus map with intermediate regions of high-mapped-EST density on both chromosome arms. Gene ontology (GO) classification of mapped ESTs was not significantly different for homoeologous group 3 chromosomes compared to the other groups. A combined analysis of the individual bin maps using 537 of the mapped ESTs revealed rearrangements between the group 3 chromosomes. Approximately 232 (44%) of the consensus mapped ESTs matched sequences on rice chromosome 1 and revealed large- and small-scale differences in gene order. Of the group 3 mapped EST unigenes approximately 21 and 32% matched the Arabidopsis coding regions and proteins, respectively, but no chromosome-level gene order conservation was detected.     

Bioinformatic Mining of Type I Microsatellites from Expressed Sequence Tags of Channel Catfish (<Emphasis Type="Italic">Ictalurus punctatus</Emphasis>)

Gene-derived markers are pivotal to the analysis of genome structure, organization, and evolution and necessary for comparative genomics. However, gene-derived markers are relatively difficult to develop. This project utilized the genomic resources of channel catfish expressed sequence tags (ESTs) to identify simple sequence repeats (SSRs), or microsatellites. It took the advantage of ESTs for the establishment of gene identities, and of microsatellites for the acquisition of high polymorphism. When microsatellites are tagged to genes, the microsatellites can then be used as gene markers. A bioinformatic analysis of 43,033 ESTs identified 4855 ESTs containing microsatellites. Cluster analysis indicated that 1312 of these ESTs fell into 569 contigs, and the remaining 3534 ESTs were singletons. A total of 4103 unique microsatellite-containing genes were identified. The dinucleotide CA/TG and GA/TC pairs were the most abundant microsatellites. AT-rich microsatellite types were predominant among trinucleotide and tetranucleotide microsatellites, consistent with our earlier estimation that the catfish genome is highly AT-rich. Our preliminary results indicated that the majority of the identified microsatellites were polymorphic and, therefore, useful for genetic linkage mapping of catfish. Mapping of these gene-derived markers is under way, which will set the foundation for comparative genome analysis in catfish.