Abstract: Three putative exo-1,3-&b-glucanase genes (Piexo1, Piexo2, Piexo3), one endo-1,3-b-glucanase (Piendo1) and one endo-1,3;1,4-b-glucanase (Piendo2) gene were cloned and characterized from the oomycete Phytophthora infestans. Southern hybridization revealed that Piexo1, Piexo2, Piexo3, and Piendo2 are single copy, and that Piendo1 is encoded by two copies. Furthermore, the analyses showed that for each gene, one or two closely related gene family members were present. The genes contain no introns. Nucleotide sequence analysis of the promoter regions (200 nt upstream of ATG start codon) showed that the regions have 56–81% similarity to a 16-nt core sequence hypothesized to be the initiation of transcription point in oomycetes. The predicted molecular weights (32–83 kDa), iso-electric points (4.2–6.7) and amino acid sequences of the five proteins are diverse. All the genes are expressed in in vitro grown mycelia and sporangia, as well as during infection of potatoes. Further, Piendo1 and Piendo2 are also expressed in germinating cysts, and Piendo2 in zoospores.
Abstract: Over three million sequences from approximately 200 plant species have been deposited in the publicly available plant expressed sequence tag (EST) sequence databases. Many of the ESTs have been sequenced as an alternative to complete genome sequencing or as a substrate for cDNA array-based expression analyses. This creates a formidable resource from both biodiversity and gene-discovery standpoints. Bioinformatics-based sequence analysis tools have extended the scope of EST analysis into the fields of proteomics, marker development and genome annotation. Although EST collections are certainly no substitute for a whole genome scaffold, this poor mans genome resource forms the core foundations for various genome-scale experiments within the as yet unsequenceable plant genomes.
Abstract: The oomycete plant pathogen Phytophthora nicotianae causes diseases on a wide range of plant species. To facilitate isolation and functional characterization of pathogenicity genes, we have constructed a large-insert bacterial artificial chromosome (BAC) library using nuclear DNA from P. nicotianae H1111. The library contains 10,752 clones with an average insert size of 90 kb and is free of mitochondrial DNA. The quality of the library was verified by hybridization with 37 genes, all of which resulted in the identification of multiple positive clones. The library is estimated to be 10.6 haploid genome equivalents based on hybridization of 23 single-copy genes and the genome size of P. nicotianae was estimated to be 95.5 Mb. Hybridization with a nuclear repetitive DNA probe revealed that 4.4% of clones in the library contained 28S rDNA. Hybridization of total genomic DNA to the library indicated that at least 39% of the BAC library contains repetitive DNA sequences. A BAC pooling strategy was developed for efficient library screening. The library was used to identify and characterize BAC clones containing an Hsp70 gene family whose four members were identified to be clustered within ~ 18 kb in the P. nicotianae genome based on the physical mapping of eight BACs spanning a genomic region of ~ 186 kb. The BAC library created provides an invaluable resource for the isolation of P. nicotianae genes and for comparative genomics studies.
Abstract: The sequences of the mitochondrial genomes of the oomycetes Phytophthora ramorum and P. sojae were determined during the course of complete nuclear genome sequencing (Tyler et al., Science, 313:1261,2006). Both mitochondrial genomes are circular mapping, with sizes of 39,314 bp for P. ramorum and 42,977 bp for P. sojae. Each contains a total of 37 recognizable protein-encoding genes, 26 or 25 tRNAs (P. ramorum and P. sojae, respectively) specifying 19 amino acids, six more open reading frames (ORFs) that are conserved, presumably due to functional constraint, across Phytophthora species (P. sojae, P. ramorum, and P. infestans), six ORFs that are unique for P. sojae and one that is unique for P. ramorum. Non-coding regions comprise about 11.5 and 18.4% of the genomes of P. ramorum and P. sojae, respectively. Relative to P. sojae, there is an inverted repeat of 1,150 bp in P. ramorum that includes an unassigned unique ORF, a tRNA gene, and adjacent non-coding sequences, but otherwise the gene order in both species is identical. Comparisons of these genomes with published sequences of the P. infestans mitochondrial genome reveals a number of similarities, but the gene order in P. infestans differed in two adjacent locations due to inversions and specific regions of the genomes exhibited greater divergence than others. For example, the breakpoints for the inversions observed in P. infestans corresponded to regions of high sequence divergence in comparisons between P. ramorum and P. sojae and the location of a hypervariable microsatellite sequence (eight repeats of 24 bp) in the P. sojae genome corresponds to a site of major length variation in P. infestans. Although the overwhelming majority of each genome is conserved (81–92%), there are a number of genes that evolve more rapidly than others. Some of these rapidly evolving genes appear specific to Phytophthora, arose recently, and future evaluation of their function and the effects of their loss could prove fruitful for understanding the phylogeny of these devastating plant pathogens.
Abstract: Microsatellites or single sequence repeats (SSRs) are a powerful choice of marker in the study of Phytophthora population biology, epidemiology, ecology, genetics and evolution. A strategy was tested in which the publicly available unigene datasets extracted from genome sequences of P. infestans, P. sojae and P. ramorum were mined for candidate SSR markers that could be applied to a wide range of Phytophthora species. A first approach, aimed at the identification of polymorphic SSR loci common to many Phytophthora species, yielded 171 reliable sequences containing 211 SSRs. Microsatellites were identified from 16 target species representing the breadth of diversity across the genus. Repeat number ranged from 3 to 16 with most having seven repeats or less and four being the most commonly found. Trinucleotide repeats such as (AAG)n, (AGG)n and (AGC)n were the most common followed by pentanucleotide, tetranucleotide and dinucleotide repeats. A second approach was aimed at the identification of useful loci common to a restricted number of species more closely related to P. sojae (P. alni, P. cambivora, P. europaea and P. fragariae). This analysis yielded 10 trinucleotide and 2 tetranucleotide SSRs which were repeated 4, 5 or 6 times. Key studies on inter- and intra-specific variation of selected microsatellites remain. Despite the screening of conserved gene coding regions, the sequence diversity between species was high and the identification of useful SSR loci applicable to anything other than the most closely related pairs of Phytophthora species was challenging. That said, many novel SSR loci for species other than the three source species (P. infestans, P. sojae and P. ramorum) are reported, offering great potential for the investigation of Phytophthora populations. In addition to the presence of
microsatellites, many of the amplified regions may represent useful molecular marker regions for other studies as they are highly variable and easily amplifiable from different Phytophthora species.
Abstract: A survey of symptoms of phytophthora root and collar rot of common(
Alnus glutinosa) and grey alder (A.incana) in riparian and forest stands in Bavaria was conducted by the Bavarian State Forestry and river authorities. Symptoms were seen in 1041 out of 3247 forest alder stands. The majority of the affected stands (80·9%) were less than 21 years old; 46% of these young stands were growing on nonflooded sites and 92% had been planted. The riparian survey showed that symptoms were widespread along more than 50% of the river systems. Along some rivers the disease incidence exceeded 50%. The alder Phytophthora was recovered from 166 of 185 riparian and forest alder stands with symptoms. In 58 of the 60 rivers and streams investigated in detail, the source of inoculum was traced back to infested young alder
plantations growing on the river banks or on forest sites that drain into the rivers. Once introduced to a river system, the alder Phytophthora infects alders downstream. Baiting tests showed that the alder Phytophthora was present in rootstocks of alders from three out of four nurseries which regularly bought in alder plants for re-sale, but not in rootstocks from four nurseries that grew their own alders from seed. In addition, the infected nurseries used water from infested water courses for irrigation. The Bavarian State Ministry for Agriculture and Forestry has developed a code of practice for producing healthy alder plants in forest nurseries. This includes a 3-year fallow period between bare-rooted alder crops because of poor survival of the alder Phytophthora in soil.
Abstract: A bacterial artificial chromosome (BAC) library consisting of 235,000 clones with an average insert size of 130 kb was constructed from Capsicum annuum, CM334. Based on a pepper haploid genome size of 2,702 Mbp/C, the BAC library is estimated to contain approximately 12 genome equivalents and represents at least 99% of the pepper genome. Screening of the library with mitochondrial DNA probes (coxII, coxIII, atp6 and atp9) and chloroplast DNA probes (atpB, rbcL) indicated that contamination with cytoplasmic DNA was less than 0.5%. To estimate the possibility of isolating a specific clone, the library was screened with single or low-copy gene-specific probes and RFLP probes. Screening of high density BAC filters with RFLP markers linked to L (TMV resistance), Y (fruit color), C2(fruit color) and C (pungency) loci under high stringency conditions revealed that at least three positive BAC clones were found per each probe. This fact indicates that the library is highly reliable and represents a resource for map-based cloning, physical mapping, and characterization of upstream and downstream regulations of the chili pepper genes.
Abstract: We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F2 population of 107 plants with 150 RFLP and 430 AFLP markers. The resulting linkage map consists of 11 large (206–60.3 cM) and 5 small (32.6–10.3 cM) linkage groups covering 1,320 cM with an average map distance between framework markers of 7.5 cM. Most (80%) of the RFLP markers were pepper-derived clones, and these markers were evenly distributed across the genome. By using 30 primer combinations, we were able to generate 444 AFLP markers in the F2 population. The majority of the AFLP markers clustered in each linkage group, although PstI/MseI markers were more evenly distributed than EcoRI/MseI markers within the linkage groups. Genes for the biosynthesis of carotenoids and capsaicinoids were mapped on our linkage map. This map will provide the basis of studying secondary metabolites in pepper.
Abstract: Black pepper (Piper nigrum L.), member of the family Piperaceae is indigenous to India and is one of the most widely used spices in the world. In this paper we report the results of our attempts to identify a set of genes differentially expressed in the leaves of Piper nigrum, which could facilitate targeted engineering of this valuable crop. A PCR-based Suppression Subtractive Hybridization (SSH) technique was used to generate a leaf-specific subtracted cDNA library of Piper nigrum. A tester population of leaf cDNA was subtracted with a root derived driver cDNA. The efficiency of subtraction was confirmed by PCR analysis using the housekeeping gene actin. On sequence analysis, almost 30% of the clones showed homology to metallothionein type-2 gene. The predominance of metallothionein transcripts in the leaf was further confirmed using Real-Time PCR analyses and Northern blot. The possible role of metallothionein type-2 homologues in the leaf is discussed along with the feasibility of using SSH technique for identification of more number of tissue-specific genes from Piper nigrum.
Abstract: An advanced backcross QTL study was performed in pepper using a cross between the cultivated species Capsicum annuum cv. Maor and the wild C. frutescens BG 2816 accession. A genetic map from this cross was constructed, based on 248 BC2 plants and 92 restriction fragment length polymorphism (RFLP) markers distributed throughout the genome. Ten yield-related traits were analyzed in the BC2 and BC2S1 generations, and a total of 58 quantitative trait loci (QTLs) were detected; the number of QTLs per trait ranged from two to ten. Most of the QTLs were found in 11 clusters, in which similar QTL positions were identified for multiple traits. Unlike the high percentage of favorable QTL alleles discovered in wild species of tomato and rice, only a few such QTL alleles were detected in BG 2816. For six QTLs (10%), alleles with effects opposite to those expected from the phenotype were detected in the wild species. The use of common RFLP markers in the pepper and tomato maps enabled possible orthologous QTLs in the two species to be determined. The degree of putative QTL orthology for the two main fruit morphology traits--weight and shape--varied considerably. While all eight QTLs identified for fruit weight in this study could be orthologous to tomato fruit weight QTLs, only one out of six fruit shape QTLs found in this study could be orthologous to tomato fruit shape QTLs.
Abstract