Michele Morgante

The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis

The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.

Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes

Microsatellites are a ubiquitous class of simple repetitive DNA sequence. An excess of such repetitive tracts has been described in all eukaryotes analyzed and is thought to result from the mutational effects of replication slippage. Large-scale genomic and EST sequencing provides the opportunity to evaluate the abundance and relative distribution of microsatellites between transcribed and nontranscribed regions and the relationship of these features to haploid genome size. Although this has been studied in microbial and animal genomes, information in plants is limited. We assessed microsatellite frequency in plant species with a 50-fold range in genome size that is mostly attributable to the recent amplification of repetitive DNA. Among species, the overall frequency of microsatellites was inversely related to genome size and to the proportion of repetitive DNA but remained constant in the transcribed portion of the genome. This indicates that most microsatellites reside in regions pre-dating the recent genome expansion in many plants. The microsatellite frequency was higher in transcribed regions, especially in the untranslated portions, than in genomic DNA. Contrary to previous reports suggesting a preferential mechanism for the origin of microsatellites from repetitive DNA in both animals and plants, our findings show a significant association with the low-copy fraction of plant genomes.

SNP frequency, haplotype structure and linkage disequilibrium in elite maize inbred lines

BackgroundRecent studies of ancestral maize populations indicate that linkage disequilibrium tends to dissipate rapidly, sometimes within 100 bp. We set out to examine the linkage disequilibrium and diversity in maize elite inbred lines, which have been subject to population bottlenecks and intense selection by breeders. Such population events are expected to increase the amount of linkage disequilibrium, but reduce diversity. The results of this study will inform the design of genetic association studies.ResultsWe examined the frequency and distribution of DNA polymorphisms at 18 maize genes in 36 maize inbreds, chosen to represent most of the genetic diversity in U.S. elite maize breeding pool. The frequency of nucleotide changes is high, on average one polymorphism per 31 bp in non-coding regions and 1 polymorphism per 124 bp in coding regions. Insertions and deletions are frequent in non-coding regions (1 per 85 bp), but rare in coding regions. A small number (2–8) of distinct and highly diverse haplotypes can be distinguished at all loci examined. Within genes, SNP loci comprising the haplotypes are in linkage disequilibrium with each other.ConclusionsNo decline of linkage disequilibrium within a few hundred base pairs was found in the elite maize germplasm. This finding, as well as the small number of haplotypes, relative to neutral expectation, is consistent with the effects of breeding-induced bottlenecks and selection on the elite germplasm pool. The genetic distance between haplotypes is large, indicative of an ancient gene pool and of possible interspecific hybridization events in maize ancestry.

4 – Generating and Using DNA Markers in Plants

This chapter presents description and comparison of different DNA marker systems. DNA markers are being applied to a wide variety of problems central to plant genome analysis. Each marker system is characterized by a unique combination of advantages and disadvantages and the choice of a marker system is dictated to a significant extent by the application. Factors to consider in choosing a marker system include the amount of available plant material, the quality of the DNA, and the availability of public collections of DNA markers for the species being examined. Restriction fragment length polymorphism (RFLP) markers are generally codominant, allowing detection and characterization of multiple alleles at a given RELP locus among individuals in a population. Several types of polymorphism can be detected, including single base substitutions, insertions, and deletions. One clear disadvantage to using RFLP markers is the large amount of high-quality genomic DNA required from each individual. It is suggested that once the mapping parents have been chosen and low-copy RFLP probes obtained, those probes that detect useful polymorphisms in the segregating population must be identified. The polymerase chain reaction analysis and identification of polymorphisms are also elaborated.

Contrasting Effects of Selection on Sequence Diversity and Linkage Disequilibrium at Two Phytoene Synthase Loci

We investigated the effects of human selection for yellow endosperm color, representing increased carotenoid content, on two maize genes, the Y1 phytoene synthase and PSY2, a putative second phytoene synthase. Multiple polymorphic sites were identified at Y1 and PSY2 in 75 white and yellow maize inbred lines. Many polymorphic sites showed strong association with the endosperm color phenotype at Y1, but no detectable association was found at PSY2. Nucleotide diversity was equivalent for whites and yellows at PSY2 but was 19-fold less in yellows than in whites at Y1, consistent with the white ancestral state of the gene. The strong sequence haplotype conservation within yellows at Y1 and a significant, negative Tajimas D both verified positive selection for yellow endosperm. We propose that two independent gain-of-function events associated with insertions into the promoter of the Y1 gene and upregulation of expression in endosperm have been incorporated into yellow maize.

Monitoring genome‐wide changes in gene expression in response to endogenous cytokinin reveals targets in Arabidopsis thaliana

Cytokinins have been implicated in developmental and growth processes in plants including cell division, chloroplast biogenesis, shoot meristem initiation and senescence. The regulation of these processes requires changes in cytokinin‐responsive gene expression. Here, we induced the expression of a bacterial isopentenyl transferase gene, IPT, in transgenic Arabidopsis thaliana seedlings to study the regulation of genome‐wide gene expression in response to endogenous cytokinin. Using MPSS (massively parallel signature sequencing) we identified 823 and 917 genes that were up‐ and downregulated, respectively, following 24 h of IPT induction. When comparing the response to cytokinin after 6 and 24 h, we identified different clusters of genes showing a similar course of regulation. Our study provides researchers with the opportunity to rapidly assess whether genes of interest are regulated by cytokinins.