Isolation of a set of ripening-related genes from strawberry: their identification and possible relationship to fruit quality traits

The ripening of strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, is a complex developmental process that involves many changes in gene expression. To understand how these changes relate to the biochemistry and composition of the fruit the specific genes involved have been examined. A high-quality cDNA library prepared from ripe strawberry fruit was differentially screened for ripening-related clones using cDNA from ripe and white fruits. From 112 up-regulated clones obtained in the primary screen, 66 differentially expressed clones were isolated from the secondary screen. The partial sequences of these cDNAs were compared with database sequences and 26 families of non-redundant clones were identified. Northern analysis confirmed that all of these cDNAs were ripening-enhanced. The expression of many of their corresponding genes was negatively regulated in auxin-treated fruit. These sequences, several of which are novel to fruits, encode proteins involved in key metabolic events including anthocyanin biosynthesis, cell wall degradation, sucrose and lipid metabolism, protein synthesis and degradation, and respiration. These findings are discussed in relation to the role of these genes in determining fruit quality characteristics. Received: 19 January 1998 / Accepted: 5 February 1998     

Construction of a bacterial artificial chromosome (BAC) library for citrus and identification of BAC contigs containing resistance gene candidates

A BAC library was constructed from the genomic DNA of an intergeneric Citrus and Poncirus hybrid. The library consists of 24,576 clones with an average insert size of 115 kb, representing approximately seven haploid genome equivalents and is able to give a greater than 99% probability of isolating single-copy citrus DNA sequences from this library. High-density colony hybridization-based library screening was performed using DNA markers linked to the citrus tristeza virus (CTV) resistance gene and citrus disease resistance gene candidate (RGC) sequences. Between four and eight clones were isolated with each of the CTV resistance gene-linked markers, which agrees with the library’s predicted genome coverage. Three hundred and twenty-two clones were identified using 13 previously cloned citrus RGC sequences as probes in library screening. One to four fragments in each BAC were shown to hybridize with RGC sequences. One hundred and nine of the RGC BAC clones were fingerprinted using a sequencing gel-based procedure. From the fingerprints, 25 contigs were assembled, each having a size of 120–250 kb and consisting of 2–11 clones. These results indicate that the library is a useful resource for BAC contig construction and molecular isolation of disease resistance genes. Received: 22 May 2000 / Accepted: 25 September 2000     

Molecular characterization and genomic organization of low molecular weight glutenin subunit genes at the Glu-3 loci in hexaploid wheat (Triticum aestivum L.)

In this study, we report on the molecular characterization and genomic organization of the low molecular weight glutenin subunit (LMW-GS) gene family in hexaploid wheat (Triticum aestivum L.). Eighty-two positive BAC clones were identified to contain LMW-GS genes from the hexaploid wheat ‘Glenlea’ BAC library via filter hybridization and PCR validation. Twelve unique LMW glutenin genes and seven pseudogenes were isolated from these positive BAC clones by primer-template mismatch PCR and subsequent primer walking using hemi-nested touchdown PCR. These genes were sequenced and each consisted of a single-open reading frame (ORF) and untranslated 5′ and 3′ flanking regions. All 12 LMW glutenin subunits contained eight cysteine residues. The LMW-m-type subunits are the most abundant in hexaploid wheat. Of the 12 LMW-GS, 1, 2 and 9 are i-type, s-type and m-type, respectively. The phylogenetic analysis suggested that the LMW-i type gene showed greater differences to LMW-s and LMW-m-type genes, which, in turn, were more closely related to one another. On the basis of their N-terminal sequences, they were classified into nine groups. Fingerprinting of the 82 BAC clones indicated 30 BAC clones assembled into eight contigs, while the remaining clones were singletons. BAC end sequencing of the 82 clones revealed that long terminal repeat (LTR) retrotransposons were abundant in the Glu-3 regions. The average physical distance between two adjacent LMW-GS genes was estimated to be 81 kb. Most of LMW-GS genes are located in the d-genome, suggesting that the Glu-D3 locus is much larger than the Glu-B3 locus and Glu-A3 locus. Alignments of sequences indicated that the same type (starting with the same N-terminal sequence) LMW-GS genes were highly conserved in the homologous genomes between hexaploid wheat and its donors such as durum wheat and T. tauschii. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fosmid Library Construction and Initial Analysis of End Sequences in Female Half-smooth Tongue Sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis: Pleuronectiformes) is a commercially important cultured marine flatfish in China and forms an important fishery resource, but the research of its genome is underdeveloped. In this study, we constructed a female C. semilaevis fosmid library and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of 49,920 clones with an average insert size of about 39 kb, amounting to 3.23 genome equivalents. Fosmid stability assays indicate that female C. semilaevis DNA was stable during propagation in the fosmid system. Library screening with eight microsatellite markers yielded between two and five positive clones, and none of those tested was absent from the library. End-sequencing of both 5′ and 3′ ends of 1,152 individual clones generated 2,247 sequences after trimming, with an average sequence length of 855 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 259 (11.53%) and 287 (12.77%) significant hits (E < e ⁻⁵), respectively. Repetitive sequences analysis resulted in 5.23% of base pairs masked using both the Fugu and Danio databases, repetitive elements were composed of retroelements, DNA transposons, satellites, simple repeats, and low-complexity sequences. The fosmid library, in conjunction with the fosmid end sequences, will serve as a useful resource for large-scale genome sequencing, physical mapping, and positional cloning, and provide a better understanding of female C. semilaevis genome.     

Construction of a bacterial artificial chromosome library of Medicago truncatula and identification of clones containing ethylene-response genes

 To facilitate genome analysis and map-based cloning of symbiotic genes in the model legume Medicago truncatula, a bacterial artificial chromosome (BAC) library was constructed. The library consists of 30 720 clones with an average insert size of approximately 100 kb, representing approximately five haploid-genome equivalents. The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be 1.4%. Screening of the library with single- or low-copy genes as hybridization probes resulted in the detection of 1–12 clones per gene. Hybridization of the library with repeated sequences such as rDNA genes and transposon-like elements of M. truncatula revealed the presence of 60 and 374 BAC clones containing the two sequences, respectively. The BAC library was pooled for screening by polymerase chain reaction (PCR)-amplification. To demonstrate the utility of this system, we used primers designed from a conserved region of the ein3-like loci of Arabidopsis thaliana and isolated six unique BAC clones from the library. DNA gel-blot and sequence analyses showed that these ein3-like clones could be grouped into three classes, an observation consistent with the presence of multiple ein3-like loci in M. truncatula. These results indicate that the BAC library represents a central resource for the map-based cloning and physical mapping in M. truncatula and other legumes. Received: 27 July 1998 / Accepted: 5 August 1998     

Construction of a Full-Length cDNA Library and Analysis of Expressed Sequence Tags from Inflorescence of Apomictic Sabaigrass (Eulaliopsis binata)

Eulaliopsis binata, which is a close relative of cereal crops, was recognized as an important research material owing to its high frequency of apospory and autonomous endosperm formation. However, little information is known about its genomics and regulatory pathway participating in reproductive development. For the first step to understand molecular basis in sabaigrass (E. binata), a SMART complementary DNA library from the inflorescence tissue was constructed and characterized. The titers of original and amplified libraries were 5.53 × 10⁶ and 1.49 × 10¹⁰ pfu/ml, respectively. The percentage of recombinants was 96% in the original library. Analysis of sequencing results of 398 out of 437 randomly picked clones showed that 271 (68.1%) expressed sequence tags (ESTs) exhibited significant similarity with known putative functional nucleotide sequences in the GenBank databases, 25 (6.3%) ESTs have significant matches with hypothetical proteins, putative proteins, and unknown proteins, and the other 25.6% ESTs had no significant similarity to sequences in the public databases. Based on molecular function of GO annotation, the four most abundant terms are nucleotide binding, hydrolase activity, ion binding, and protein binding, and these genes were involved in 61 different pathways using the Kyoto Encyclopedia of Genes and Genomes pathway analysis. Besides, simple sequence repeats detection in 398 ESTs was carried out, and several genes were chosen to perform expression analysis. This report represents a first step in expanding molecular-genetic analyses in E. binata and can be used to optimally mine useful information from a relatively small data set.     

Expressed sequence tags of young floral buds and characterization of a bud- preferential lectin- like cDNA fromPharbitis nil

To identify the genes involved in flower development, we analyzed 207 expressed sequence tags (ESTs) from a young floral bud cDNA library ofPharbitis nil. Of these, 87 clones (42%) showed significant homology to known protein sequences in the NCBI database. Four of these had not been reported previously in the plant kingdom, indicating that 1.9% of the ESTs were newly identified in plants. Functional categorization revealed that the genes involved in metabolic pathways, such as glycolysis and photosynthesis, were most abundant Reverse-northern and northern analyses showed that one clone,PnFP161, was expressed preferentially in floral buds. DNA sequence analysis indicated that this clone contained 147 bp of 5′-UTR, 264 bp of -UTR, and an open reading frame of 233 amino acids, thereby sharing 33% identity with a lectin fromCalystegia sepium. The C-terminal regionof PnFPI61 had well-conserved residue with that of the lectins. Southern blot analysis demonstrated thatPnFPI61 exists as a multigene family.     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

Generation of a soybean BAC library, and identification of DNA sequences tightly linked to the Rps1-k disease resistance gene

 A soybean bacterial artificial chromosome (BAC) library, comprising approximately 45 000 clones, was constructed from high-molecular-weight nuclear DNA of cultivar Williams 82, which carries the Rps1-k gene for resistance against Phytophthora sojae. The library is stored in 130 pools with about 350 clones per pool. Completeness of the library was evaluated for 21 random sequences including four markers linked to the Rps1 locus and 16 cDNAs. We identified pools containing BACs for all sequences except for one cDNA. Additionally, when screened for possible contaminating BAC clones carrying chloroplast genes, no sequences homologous to two barley chloroplast genes were found. The estimated average insert size of the BAC clones was about 105 kb. The library comprises about four genome equivalents of soybean DNA. Therefore, this gives a probability of 0.98 of finding a specific sequence from this library. This library should be a useful resource for the positional cloning of Rps1-k, and other soybean genes. We have also evaluated the feasibility of an RFLP-based screening procedure for the isolation of BAC clones specific for markers that are members of repetitive sequence families, and are linked to the Rps1-k gene. We show that BAC clones isolated for two genetically linked marker loci, Tgmr and TC1-2, are physically linked. Application of this method in expediting the map-based cloning of a gene, especially from an organism, such as soybean, maize and wheat, with a complex genome is discussed. Received: 12 May 1998/Accepted: 24 August 1998     

Phylotype diversity in a benthic cyanobacterial mat community on King George Island, maritime Antarctica

Cyanobacterial 16S ribosomal RNA gene diversity was examined in a benthic mat on Fildes Peninsula of King George Island (62o09′54.4′′S, 58o57′20.9′′W), maritime Antarctica. Environmental DNA was isolated from the mat, a clone library of PCR-amplified 16S rRNA gene fragments was prepared, and amplified ribosomal DNA restriction analysis (ARDRA) was done to assign clones to seven groups. Low cyanobacterial diversity in the mat was suggested in that 83% of the clones were represented by one ARDRA group. DNA sequences from this group had high similarity with 16S rRNA genes of Tychonema bourrellyi and T. bornetii isolates, whose geographic origins were southern Norway and Northern Ireland. Cyanobacterial morphotypes corresponding to Tychonema have not been reported in Antarctica, however, this morphotype was previously found at Ward Hunt Lake (83oN), and in western Europe (52oN). DNA sequences of three of the ARDRA groups had highest similarity with 16S rDNA sequences of the Tychonema group accounting for 9.4% of the clones. Sequences of the remaining three groups (7.6%) had highest similarity with 16S rRNA genes of uncultured cyanobacteria clones from benthic mats of Lake Fryxell and fresh meltwater on the McMurdo Ice Shelf.