Selection of highly informative and user-friendly microsatellites (SSRs) for genotyping of cultivated potato.

Characterization of nearly 1,000 cultivated potato accessions with simple sequence repeats (SSRs; also referred to as microsatellites) has allowed the identification of a reference set of SSR markers for accurate and efficient genotyping. In addition, 31 SSRs are reported here for a potato genetic map, including new map locations for 24 of them. A first criterion for this proposed reference set was ubiquity of the SSRs in the eight landrace cultivar groups of the potato, Solanum tuberosum. All SSRs tested in the present study displayed the same allele phenotypes and allele size range in the diverse germplasm set as in the advanced potato cultivar germplasm in which they were originally discovered. Thirteen of 13 SSR products from all cultivar groups are shown to cross-hybridize with the corresponding SSR product of the source cultivar to ascertain sequence homology. Other important SSR selection criteria are quality of amplification products, locus complexity, polymorphic index content, and well-dispersed location on a potato genetic map. Screening of 156 SSRs allowed the identification of a highly informative and user-friendly set comprising 18 SSR markers for use in characterization of potato genetic resources. In addition, we have identified true- and pseudo-multiplexing SSRs for even greater efficiency.

Construction of a genetic linkage map for Camellia sinensis (tea).

Genetic maps are a vital tool in cultivar improvement programmes for woody perennial tree crops such as tea (Camellia sinensis). A population thought to be derived from two known, noninbred parents was scored for RAPD and AFLP markers, in order to develop a linkage map. However, a very high proportion of the markers exhibited unexpected segregation ratios in the light of their configurations in the parents, and an exploratory statistical analysis revealed patterns in the marker scores which can most easily be explained by the hypothesis of three male parents contributing pollen to this cross. We discuss the evidence for this and the subsequent analysis required to assemble the markers from the female parent into the first linkage map for tea. The map has 15 linkage groups of three or more markers, agreeing with the haploid chromosome number of tea. The statistical methods that revealed the subpopulations are easy to apply routinely, and may prove a useful diagnostic tool for the analysis of noninbred mapping populations.

Isolation, characterisation and mapping of simple sequence repeat loci in potato.

Solanum tuberosum L. DNA sequences containing simple sequence repeat (SSR) motifs were extracted from the EMBL database, cDNA and selectively enriched small-insert DNA libraries. Enrichment was achieved using either triplex affinity capture or single-strand hybridisation selection. One hundred and twelve primer pairs which successfully amplified products of the correct size from potato DNA were ultimately designed and synthesised. Ninety-eight of these revealed length polymorphisms in a panel of four diploid and two tetraploid clones, in agreement with the high information content of this class of markers which has been found in other species. All of the markers were assigned a quality score of 1-5 based on their potential usefulness. Eighty-nine loci from 65 of the primer pairs were located on two genetic linkage maps of potato by segregation analysis of the amplified alleles. Fifty-two of the SSRs were clearly single locus. The maps were aligned using 23 SSR primer pairs and 13 RFLP loci mapped in both populations. The markers described constitute a class which should replace Restriction Fragment Length Polymorphisms (RFLP) as the markers of choice for future genetic studies in potato. The sequences of the primers, together with other information on these markers are provided.

Linkage analysis in tetraploid potato and association of markers with quantitative resistance to late blight (Phytophthora infestans).

We have constructed a partial linkage map in tetraploid potato which integrates simplex, duplex and double-simplex AFLP markers. The map consists of 231 maternal and 106 paternal markers with total map lengths of 990.9 cM and 484.6 cM. The longer of the two cumulative map lengths represents approximately 25% coverage of the genome. In tetraploids, much of the polymorphism between parental clones is masked by 'dosage' which significantly reduces the number of individual markers that can be scored in a population. Consequently, the major advantage of using AFLPs--their high multiplex ratio--is reduced to the point where the use of alternative multi-allelic marker types would be significantly more efficient. The segregation data and map information have been used in a QTL analysis of late blight resistance, and a multi-allelic locus at the proximal end of chromosome VIII has been identified which contributes significantly to the expression of resistance. No late blight resistance genes or QTLs have previously been mapped to this location.

Exon skipping induced by cold stress in a potato invertase gene transcript.

We show that two invertase genes in potato, like most other plant invertase genes, include a very short second exon of 9 bp which encodes the central three amino acids of a motif highly conserved in invertases of diverse origin. This mini-exon is one of the smallest known in plants and pre-mRNA from these genes may be susceptible to alternative splicing, because of a potential requirement for specialized interaction with the splicing machinery to ensure correct processing for the production of a mature mRNA. No evidence of aberrant post-transcriptional processing was observed during normal invertase gene expression in potato. The fidelity of post-transcriptional processing of the pre-mRNA from one of the genes was perturbed by cold stress, resulting in the deletion of the mini-exon from some transcripts. This alternative splicing event occurred under cold stress in both leaf and stem, but was not induced by wounding. This adds an example of exon skipping and the induction of alternative processing by cold stress to the small number of transcripts which have been shown to exhibit alternative splicing in plants. The differential sensitivity of post-transcriptional processing to cold stress observed for the two transcripts examined will permit further dissection of the nucleotide sequence requirements for their accurate splicing.

Detection and pattern of interspecific hybridization between Gliricida sepium and G. maculata in Meso-America revealed by PCR-based assays.

Gliricidia sepium provides a variety of products important for rural communities in tropical countries. Native populations in Meso-America currently form an important source of seed for distribution to farmers, but concerns centre on mechanisms which may lead to their genetic erosion, including anthropogenic dispersal and subsequent introgression from the related species, G. maculata. Populations of Gliricidia were examined genetically using approaches based on the polymerase chain reaction to test for interspecific hybridization and introgression between G. sepium and G. maculata. Analysis involved 13 RAPD and two RFLP-PCR markers which were identified to have species-diagnostic distributions. Data from both approaches corresponded and indicated three locations where multilocus genotypes were consistent with an hybrid origin. Data at one of these sites was consistent with introgression following hybridization. The hybrid origin of populations was supported by the intermediate geographical location of these sites to 'pure' populations of each species. Analysis of maternally inherited organellar DNA, which involved the detection of SSCPs in mitochondrial DNA amplification products, allowed further delineation of genetic structure among Gliricidia populations. Mitochondrial data indicated a high degree of organelle differentiation between sampled locations and identified G. sepium- and G. maculata-diagnostic haplotypes. This data supported the interpretation of genetic structure based on RAPDs and RFLP-PCR. In addition, cytonuclear analysis allowed the directionality of gene transfer during the formation of hybrid populations to be described. Despite evidence for the occurrence of interspecific hybridization and introgression in Gliricidia, important resource populations of G. sepium on the Pacific coast appear to have retained their genetic integrity. Implications in terms of the conservation and utilization of genetic resources within the genus are discussed.

Detection of genetic diversity in tea (Camellia sinensis) using RAPD markers.

Camellia sinensis is a beverage tree crop native to Southeast Asia and introductions have been made into several nonindigenous countries. No systematic assessment of genetic variability in tea has been done anywhere. In this study, random amplified polymorphic DNA (RAPD) analysis was used to estimate genetic diversity and taxonomic relationships in 38 clones belonging to the three tea varieties, assamica, sinensis, and assamica ssp. lasiocalyx. Extensive genetic variability was detected between species, which was partitioned into between and within population components. Seventy percent of the variation was detected within populations. Analyses based on band sharing separated the three populations in a manner consistent with both the present taxonomy of tea and with the known pedigrees of some clones. RAPD analysis also discriminated all of the 38 commercial clones, even those which cannot be distinguished on the basis of morphological and phenotypic traits.

Molecular characterisation of plant U14 small nucleolar RNA genes: closely linked genes are transcribed as polycistronic U14 transcripts.

U14snoRNAs are highly conserved eukaryotic nucleolar small RNAs involved in precursor ribosomal RNA processing. In vertebrates, U14snoRNAs and a number of other snoRNAs are transcribed within introns of protein coding genes and are released by processing. We have isolated potato and maize genomic U14 clones using PCR-amplified plant U14 probes. Plant U14s show extensive homology to those from yeast and animals but contain plant-specific sequences. One of the isolated maize clones contains a cluster of four U14 genes in a region of only 761 bp, confirming the close linkage of U14 genes in maize, potato and barley as established by PCR. The absence of known plant promoter elements, the proximity of the genes and the detection of transcripts containing linked U14s by RT-PCR indicates that some plant U14snoRNAs are transcribed as precursor RNAs which are then processed to release individual U14s. Whether plant U14snoRNAs are intron-encoded or transcribed from novel promoter sequences, remains to be established.

Complementary deletions in expressed potato U2snRNA gene variants support the hypothesis that stem-loop IIb is dispensable for splicing.

A polymerase chain reaction (PCR) strategy designed to amplify DNA sequences between closely linked U2snRNA genes has generated extensive coding and 5' regulatory sequence information on the potato U2snRNA multigene family. Two of the U2snRNA coding sequences isolated differed substantially from normal U2snRNAs by containing both complementary deletions and regions of novel sequence. However, sequences such as Sm-binding sites and loops of stem-loops III and IV, which are some of the most highly conserved regions in U2snRNA, remain highly conserved in these genes. The complementary deletions would effectively remove stem-loop IIb which has been shown in yeast to be unnecessary for pre-mRNA splicing. Transcripts from one of the genes have been detected by reverse transcriptase-PCR (RT-PCR) in total RNA. These novel U2snRNA genes represent the first reported example of naturally occurring structural variants and provide support for the proposed non-essential role of U2snRNA stem-loop IIb.