Antoni Rafalski

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.

Applications of single nucleotide polymorphisms in crop genetics.

The discovery of single nucleotide polymorphisms (SNPs) and insertions/deletions, which are the basis of most differences between alleles, has been simplified by recent developments in sequencing technology. SNP discovery in many crop species, such as corn and soybean, is relatively straightforward because of their high level of intraspecific nucleotide diversity, and the availability of many gene and expressed sequence tag (EST) sequences. For these species, direct readout of SNP haplotypes is possible. Haplotype-based analysis is more informative than analysis based on individual SNPs, and has more power in analyzing association with phenotypes. The elite germplasm of some crops may have been subjected to bottlenecks relatively recently, increasing the amount of linkage disequilibrium (LD) present and facilitating the association of SNP haplotypes at candidate gene loci with phenotypes. Whole-genome scans may help identify genome regions that are associated with interesting phenotypes if sufficient LD is present. Technological improvements make the use of SNP and indel markers attractive for high-throughput use in marker-assisted breeding, EST mapping and the integration of genetic and physical maps.

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.

Gene duplication and exon shuffling by helitron-like transposons generate intraspecies diversity in maize

We report a whole-genome comparison of gene content in allelic BAC contigs from two maize inbred lines. Genic content polymorphisms involve as many as 10,000 sequences and are mainly generated by DNA insertions. The termini of eight of the nine genic insertions that we analyzed shared the structural hallmarks of helitron rolling-circle transposons. DNA segments defined by helitron termini contained multiple gene-derived fragments and had a structure typical of nonautonomous helitron-like transposons. Closely related insertions were found in multiple genomic locations. Some of these produced transcripts containing segments of different genes, supporting the idea that these transposition events have a role in exon shuffling and the evolution of new proteins. We identified putative autonomous helitron elements and found evidence for their transcription. Helitrons in maize seem to continually produce new nonautonomous elements responsible for the duplicative insertion of gene segments into new locations and for the unprecedented genic diversity. The maize genome is in constant flux, as transposable elements continue to change both the genic and nongenic fractions of the genome, profoundly affecting genetic diversity.

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.

The Complete Sequence of 340 kb of DNA around the Rice Adh1–Adh2 Region Reveals Interrupted Colinearity with Maize Chromosome 4

A 2.3-centimorgan (cM) segment of rice chromosome 11 consisting of 340 kb of DNA sequence around the alcohol dehydrogenase Adh1 and Adh2 loci was completely sequenced, revealing the presence of 33 putative genes, including several apparently involved in disease resistance. Fourteen of the genes were confirmed by identifying the corresponding transcripts. Five genes, spanning 1.9 cM of the region, cross-hybridized with maize genomic DNA and were genetically mapped in maize, revealing a stretch of colinearity with maize chromosome 4. The Adh1 gene marked one significant interruption. This gene mapped to maize chromosome 1, indicating a possible translocation of Adh1 after the evolutionary divergence leading to maize and sorghum. Several other genes, most notably genes similar to known disease resistance genes, showed no cross-hybridization with maize genomic DNA, suggesting sequence divergence or absence of these sequences in maize, which is in contrast to several other well-conserved genes, including Adh1 and Adh2. These findings indicate that the use of rice as the model system for other cereals may sometimes be complicated by the presence of rapidly evolving gene families and microtranslocations. Seven retrotransposons and eight transposons were identified in this rice segment, including a Tc1/Mariner–like element, which is new to rice. In contrast to maize, retroelements are less frequent in rice. Only 14.4% of this genome segment consist of retroelements. Miniature inverted repeat transposable elements were found to be the most frequently occurring class of repetitive elements, accounting for 18.8% of the total repetitive DNA.

Insertion-deletion polymorphisms in 3′ regions of maize genes occur frequently and can be used as highly informative genetic markers

Single-nucleotide polymorphisms (SNPs) are the most frequent variations in the genome of any organism. SNP discovery approaches such as resequencing or data mining enable the identification of insertion deletion (indel) polymorphisms. These indels can be treated as biallelic markers and can be utilized for genetic mapping and diagnostics. In this study 655 indels have been identified by resequencing 502 maize (Zea mays) loci across 8 maize inbreds (selected for their high allelic variation). Of these 502 loci, 433 were polymorphic, with indels identified in 215 loci. Of the 655 indels identified, single-nucleotide indels accounted for more than half (54.8%) followed by two- and three-nucleotide indels. A high frequency of 6-base (3.4%) and 8-base (2.3%) indels were also observed. When analysis is restricted to the B73 and Mo17 genotypes, 53% of the loci analyzed contained indels, with 42% having an amplicon size difference. Three novel miniature inverted-repeat transposable element (MITE)-like sequences were identified as insertions near genes. The utility of indels as genetic markers was demonstrated by using indel polymorphisms to map 22 loci in a B73 × Mo17 recombinant inbred population. This paper clearly demonstrates that the resequencing of 3′ EST sequence and the discovery and mapping of indel markers will position corresponding expressed genes on the genetic map.

DNA array profiling of gene expression changes during maize embryo development

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)+RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)+ RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6.

Random amplified polymorphic DNA (RAPD) markers

Williams [14] and Welsh and McClelland [13] demonstrated the utility of single short oligonucleotide primers of arbitrary sequence for the amplification of DNA segments distributed randomly throughout the genome. Welsh and McClelland showed that the pattern of amplified bands could be used for genome fingerprinting [13] and Williams et al. [ 14] showed that the differences (polymorphisms) in the pattern of bands amplified from genetically distinct individuals behaved as mendelian genetic markers (named RAPDs, for Random Amplified Polymorphic DNA). Well-saturated maps of the Arabidopsis [11] and pine [4] genomes have been constructed using RAPD technology. A single set of arbitrary-sequence 10-mers may be used for mapping or fingerprinting any species. The many advantages of RAPD markers over RFLPs or isozyme markers accelerated the adoption of RAPD technology for the construction of genetic maps, fingerprinting, and population genetic studies [6]. Current reviews of the applications of RAPD technology are available [ 10, 12].

Genetic Diversity, Linkage Disequilibrium and Association Mapping

Maize, at all levels of resolution, is one of the most diverse crop species. Large insertions and deletions are common between maize inbreds, and include tandem repeat clusters, abundant retroelement and transposons. At the gene level, single nucleotide polymorphisms are common, especially in introns and untranslated regions of genes. Depending on choice of experimental population and region in the genome, linkage disequilibrium between polymorphic sites could decay very rapidly, or persist for hundreds of Kb. Appropriately chosen germplasm collections may be used for genetic association mapping (also called linkage disequilibrium mapping), either with candidate genes, or by scanning the whole genome with thousands of markers at high density. This approach, in favorite circumstances, could provide high resolution. The power of association mapping is variable, and has not been thoroughly investigated. Rapid advances in genome sequencing and high density genotyping are making this approach to relating genotype with phenotype increasingly attractive.