Localization of jointless-2 gene in the centromeric region of tomato chromosome 12 based on high resolution genetic and physical mapping.

Abscission is a universal process whereby plants shed their organs, such as flowers, fruit and leaves. In tomato, the non-allelic mutations jointless and jointless-2 have been discovered as recessive mutations that completely suppress the formation of pedicel abscission zones. A high resolution genetic map of jointless-2 was constructed using 1,122 jointless F2 plants. Restriction fragment length polymorphism (RFLP) marker RPD140 completely co-segregated with the jointless-2 locus and mapped in a 2.4 cM interval between RFLP markers CD22 and TG618. To chromosome walk to jointless-2, all three markers were used to screen a bacterial artificial chromosome (BAC) library and contigs were developed. Intensive efforts to expand and merge the BAC contigs were unsuccessful because of the highly repetitive sequence content on the distal ends of each contig. To determine the physical distance between and the orientation of the three contigs, we used high resolution pachytene fluorescence in situ hybridization (FISH) mapping. The RPD140 contig was positioned in the centromeric region of chromosome 12 between two large pericentric heterochromatin blocks, about 50 Mb from the TG618 contig on the short arm and 10 Mb from the CD22 contig on the long arm, respectively. Based on high resolution genetic and physical mapping, we conclude that the jointless-2 gene is located within or near the chromosome 12 centromere where 1 cM is approximately 25 Mb in length.

Enrichment of gene-coding sequences in maize by genome filtration.

Approximately 80% of the maize genome comprises highly repetitive sequences interspersed with single-copy, gene-rich sequences, and standard genome sequencing strategies are not readily adaptable to this type of genome. Methodologies that enrich for genic sequences might more rapidly generate useful results from complex genomes. Equivalent numbers of clones from maize selected by techniques called methylation filtering and High C0t selection were sequenced to generate approximately 200,000 reads (approximately 132 megabases), which were assembled into contigs. Combination of the two techniques resulted in a sixfold reduction in the effective genome size and a fourfold increase in the gene identification rate in comparison to a nonenriched library.

A framework for sequencing the rice genome.

Rice is an important food crop and a model plant for other cereal genomes. The Clemson University Genomics Institute framework project, begun two years ago in anticipation of the now ongoing international effort to sequence the rice genome, is nearing completion. Two bacterial artificial chromosome (BAC) libraries have been constructed from the Oryza sativa cultivar Nipponbare. Over 100,000 BAC end sequences have been generated from these libraries and, at a current total of 28 Mbp, represent 6.5% of the total rice genome sequence. This sequence information has allowed us to draw first conclusions about unique and redundant rice genomic sequences. In addition, more than 60,000 clones (19 genome equivalents) have been successfully fingerprinted and assembled into contigs using FPC software. Many of these contigs have been anchored to the rice chromosomes using a variety of techniques. Hybridization experiments have shown these contigs to be very robust. Contig assembly and hybridization experiments have revealed some surprising insights into the organization of the rice genome, which will have significant repercussions for the sequencing effort. Integration of BAC end sequence data with anchored contig information has provided unexpected revelations on sequence organization at the chromosomal level.

JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development.

Abscission is a universal and dynamic process in plants whereby organs such as leaves, flowers and fruit are shed, both during normal development, and in response to tissue damage and stress. Shedding occurs by separation of cells in anatomically distinct regions of the plant, called abscission zones (AZs). During abscission, the plant hormone ethylene stimulates cells to produce enzymes that degrade the middle lamella between cells in the AZ. The physiology and regulation of abscission at fully developed AZs is well known, but the molecular biology underlying their development is not. Here we report the first isolation of a gene directly involved in the development of a functional plant AZ. Tomato plants with the jointless mutation fail to develop AZs on their pedicels and so abscission of flowers or fruit does not occur normally. We identify JOINTLESS as a new MADS-box gene in a distinct phylogenetic clade separate from those functioning in floral organs. We propose that a deletion in JOINTLESS accounts for the failure of activation of pedicel AZ development in jointless tomato plants.

Rice transposable elements: a survey of 73,000 sequence-tagged-connectors.

As part of an international effort to sequence the rice genome, the Clemson University Genomics Institute is developing a sequence-tagged-connector (STC) framework. This framework includes the generation of deep-coverage BAC libraries from O. sativa ssp. japonica c.v. Nipponbare and the sequencing of both ends of the genomic DNA insert of the BAC clones. Here, we report a survey of the transposable elements (TE) in >73,000 STCs. A total of 6848 STCs were found homologous to regions of known TE sequences (E<10(-5)) by FASTX search of STCs against a set of 1358 TE protein sequences obtained from GenBank. Of these TE-containing STCs (TE-STCs), 88% (6027) are related to retroelements and the remaining are transposase homologs. Nearly all DNA transposons known previously in plants were present in the STCs, including maize Ac/Ds, En/Spm, Mutator, and mariner-like elements. In addition, 2746 STCs were found to contain regions homologous to known miniature inverted-repeat transposable elements (MITEs). The distribution of these MITEs in regions near genes was confirmed by EST comparisons to MITE-containing STCs, and our results showed that the association of MITEs with known EST transcripts varies by MITE type. Unlike the biased distribution of retroelements in maize, we found no evidence for the presence of gene islands when we correlated TE-STCs with a physical map of the CUGI BAC library. These analyses of TEs in nearly 50 Mb of rice genomic DNA provide an interesting and informative preview of the rice genome.

A deep-coverage tomato BAC library and prospects toward development of an STC framework for genome sequencing.

Recently a new strategy using BAC end sequences as sequence-tagged connectors (STCs) was proposed for whole-genome sequencing projects. In this study, we present the construction and detailed characterization of a 15.0 haploid genome equivalent BAC library for the cultivated tomato, Lycopersicon esculentum cv. Heinz 1706. The library contains 129,024 clones with an average insert size of 117.5 kb and a chloroplast content of 1.11%. BAC end sequences from 1490 ends were generated and analyzed as a preliminary evaluation for using this library to develop an STC framework to sequence the tomato genome. A total of 1205 BAC end sequences (80.9%) were obtained, with an average length of 360 high-quality bases, and were searched against the GenBank database. Using a cutoff expectation value of <10(-6), and combining the results from BLASTN, BLASTX, and TBLASTX searches, 24.3% of the BAC end sequences were similar to known sequences, of which almost half (48.7%) share sequence similarities to retrotransposons and 7% to known genes. Some of the transposable element sequences were the first reported in tomato, such as sequences similar to maize transposon Activator (Ac) ORF and tobacco pararetrovirus-like sequences. Interestingly, there were no BAC end sequences similar to the highly repeated TGRI and TGRII elements. However, the majority (70.3%) of STCs did not share significant sequence similarities to any sequences in GenBank at either the DNA or predicted protein levels, indicating that a large portion of the tomato genome is still unknown. Our data demonstrate that this BAC library is suitable for developing an STC database to sequence the tomato genome. The advantages of developing an STC framework for whole-genome sequencing of tomato are discussed.

Comparative gene expression in sexual and apomictic ovaries of Pennisetum ciliare (L.) Link.

Limited emphasis has been given to the molecular study of apomixis, an asexual method of reproduction where seeds are produced without fertilization. Most buffelgrass (Pennisetum ciliare (L.) Link syn = Cenchrus ciliaris L.) genotypes reproduce by obligate apomixis (apospory); however, rare sexual plants have been recovered. A modified differential display procedure was used to compare gene expression in unpollinated ovaries containing ovules with either sexual or apomictic female gametophytes. The modification incorporated end-labeled poly(A)+ anchored primers as the only isotopic source, and was a reliable and consistent approach for detecting differentially displayed transcripts. Using 20 different decamers and two anchor primers, 2268 cDNA fragments between 200 and 600 bp were displayed. From these, eight reproducible differentially displayed cDNAs were identified and cloned. Based on northern analysis, one cDNA was detected in only the sexual ovaries, two cDNAs in only apomictic ovaries and one cDNA was present in both types of ovaries. Three fragments could not be detected and one fragment was detected in ovaries, stems, and leaves. Comparison of gene expression during sexual and apomictic development in buffelgrass represents a new model system and a strategy for investigating female reproductive development in the angiosperms.