Recent insights into the evolution of genetic diversity of maize.

Evolutionary changes that occur within the maize genome can be divided into two classes: In the protein-coding regions, mutations that survive selective pressure primarily consist of single nucleotide polymorphisms which evolve at a relatively slow rate (10-9 mutations/ bp/generation), and functionally detrimental insertions are strongly selected against. In intergenic regions rapidly evolving (10-4 10-8 mutations/bp/generation) transposon insertions and deletions predominate. While genic single nucleotide changes are expected in part to result in amino acid sequence variants leading to the modification of protein properties (catalytic properties of enzymes, binding constants, etc.), transposable elements and other large insertions and deletions, when functionally relevant, are predicted to be regulatory in nature and may affect gene expression at distances of up to 100 kb away. Here, we discuss recent experimental evidence for massive dynamic changes of maize intergenic regions and the predicted functional consequences of genome diversity within this species.

Development of a high-throughput yeast two-hybrid screening system to study protein-protein interactions in plants.

We have developed a high-throughput yeast two-hybrid screening system (HTP-YTH) that incorporates yeast gap-repair cloning, multiple positive ( ADE2, HIS3, lacZ) and negative ( URA3-based) selection schemes to reduce the incidence of negative and false positive clones, and automation of laboratory procedures to increase throughput. This HTP-YTH system has been applied to the study of protein-protein interactions that are involved in rice defense signal transduction pathways. More than 100 genes involved in plant defense responses were selected from DuPont's rice expressed sequence tag (EST) databases as baits for HTP-YTH screening. Results from YTH screening of eight of these rice genes are presented in this paper. Not only have we identified known protein-protein interactions, but we have also discovered novel interactions, which may ultimately reveal the regulatory network of host defense signal transduction pathways. We have demonstrated that our HTP-YTH method can be used to map protein-protein interaction networks and signal transduction pathways in any system. In combination with other approaches, such efficient YTH screens can help us systemically to study the functions of known and unknown genes in the genomics era.

Comparative analysis of the complete genome sequences of Helicoverpa zea and Helicoverpa armigera single-nucleocapsid nucleopolyhedroviruses.

The complete nucleotide sequence of Helicoverpa zea single-nucleocapsid nucleopolyhedrovirus (HzSNPV) has been determined (130869 bp) and compared to the nucleotide sequence of Helicoverpa armigera (Ha) SNPV. These two genomes are very similar in their nucleotide (97% identity) and amino acid (99% identity) sequences. The coding regions are much more conserved than the non-coding regions. In HzSNPV/HaSNPV, the 63 open reading frames (ORFs) present in all baculoviruses sequenced so far are much more conserved than other ORFs. HzSNPV has four additional small ORFs compared with HaSNPV, one of these (Hz42) being in a correct transcriptional context. The major differences between HzSNPV and HaSNPV are found in the sequence and organization of the homologous regions (hrs) and the baculovirus repeat ORFs (bro genes). The sequence identity between the HzSNPV and HaSNPV hrs ranges from 90% (hr1) to almost 100% (hr5) and the hrs differ in the presence/absence of one or more type A and/or B repeats. The three HzSNPV bro genes differ significantly from those in HaSNPV and may have been acquired independently in the ancestral past. The sequence data suggest strongly that HzSNPV and HaSNPV are variants of the same virus species, a conclusion that is supported by the physical and biological data.

The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Abundance, distribution, and transcriptional activity of repetitive elements in the maize genome.

Long terminal repeat (LTR) retrotransposons have been shown to make up much of the maize genome. Although these elements are known to be prevalent in plant genomes of a middle-to-large size, little information is available on the relative proportions composed by specific families of elements in a single genome. We sequenced a library of randomly sheared genomic DNA from maize to characterize this genome. BLAST analysis of these sequences demonstrated that the maize genome is composed of diverse sequences that represent numerous families of retrotransposons. The largest families contain the previously described elements Huck, Ji, and Opie. Approximately 5% of the sequences are predicted to encode proteins. The genomic abundance of 16 families of elements was estimated by hybridization to an array of 10,752 maize bacterial artificial chromosome (BAC) clones. Comparisons of the number of elements present on individual BACs indicated that retrotransposons are in general randomly distributed across the maize genome. A second library was constructed that was selected to contain sequences hypomethylated in the maize genome. Sequence analysis of this library indicated that retroelements abundant in the genome are poorly represented in hypomethylated regions. Fifty-six retroelement sequences corresponding to the integrase and reverse transcriptase domains were isolated from approximately 407,000 maize expressed sequence tags (ESTs). Phylogenetic analysis of these and the genomic retroelement sequences indicated that elements most abundant in the genome are less abundant at the transcript level than are more rare retrotransposons. Additional phylogenies also demonstrated that rice and maize retrotransposon families are frequently more closely related to each other than to families within the same species. An analysis of the GC content of the maize genomic library and that of maize ESTs did not support recently published data that the gene space in maize is found within a narrow GC range, but does indicate that genic sequences have a higher GC content than intergenic sequences (52% vs. 47% GC).

Conservation of microstructure between a sequenced region of the genome of rice and multiple segments of the genome of Arabidopsis thaliana.

The nucleotide sequence was determined for a 340-kb segment of rice chromosome 2, revealing 56 putative protein-coding genes. This represents a density of one gene per 6.1 kb, which is higher than was reported for a previously sequenced segment of the rice genome. Sixteen of the putative genes were supported by matches to ESTs. The predicted products of 29 of the putative genes showed similarity to known proteins, and a further 17 genes showed similarity only to predicted or hypothetical proteins identified in genome sequence data. The region contains a few transposable elements: one retrotransposon, and one transposon. The segment of the rice genome studied had previously been identified as representing a part of rice chromosome 2 that may be homologous to a segment of Arabidopsis chromosome 4. We confirmed the conservation of gene content and order between the two genome segments. In addition, we identified a further four segments of the Arabidopsis genome that contain conserved gene content and order. In total, 22 of the 56 genes identified in the rice genome segment were represented in this set of Arabidopsis genome segments, with at least five genes present, in conserved order, in each segment. These data are consistent with the hypothesis that the Arabidopsis genome has undergone multiple duplication events. Our results demonstrate that conservation of the genome microstructure can be identified even between monocot and dicot species. However, the frequent occurrence of duplication, and subsequent microstructure divergence, within plant genomes may necessitate the integration of subsets of genes present in multiple redundant segments to deduce evolutionary relationships and identify orthologous genes.

Rice ESTs with disease-resistance gene- or defense-response gene-like sequences mapped to regions containing major resistance genes or QTLs.

The chromosomal locations of 109 rice expressed sequence tags (ESTs) in the rice genome were determined using a doubled haploid mapping population. These ESTs show high similarity to disease resistance genes or to defense response genes. Nine of the ESTs were mapped to three regions that contain genetically defined resistance genes on chromosomes 6 and 11. Clustering of the ESTs in the rice genome was observed at several chromosomal regions. Some of the clusters were located in regions where quantitative trait loci (QTL) associated with partial resistance to rice blast, bacterial blight and sheath blight are known to lie. Three ESTs that were mapped to the regions containing blast resistance genes Pi2 and Pia were chosen for Northern analysis after inoculation of plants with the blast fungus. Two of them, which code for a receptor-like kinase and a putative membrane channel protein, respectively, and were mapped to the Pi2 locus, were induced by rice blast infection as early as 4 h after inoculation. Transcription of another EST, which codes for a homolog of a putative human tumor suppressor and was mapped to the region containing Pia, was repressed after blast infection. These findings demonstrate that the candidate-gene approach is an efficient way of mapping resistance genes or resistance QTLs in rice.

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.

Gene discovery and product development for grain quality traits.

The composition of oils, proteins, and carbohydrates in seeds of corn, soybean, and other crops has been modified to produce grains with enhanced value. Both plant breeding and molecular technologies have been used to produce plants carrying the desired traits. Genomics-based strategies for gene discovery, coupled with high-throughput transformation processes and miniaturized, automated analytical and functionality assays, have accelerated the identification of product candidates. Molecular marker-based breeding strategies have been used to accelerate the process of moving trait genes into high-yielding germplasm for commercialization. These products are being tested for applications in food, feed, and industrial markets.

New experimental and computational approaches to the analysis of gene expression.

Public and private EST (Expressed Sequence Tag) programs provide access to a large number of ESTs from a number of plant species, including Arabidopsis, corn, soybean, rice, wheat. In addition to the homology of each EST to genes in GenBank, information about homology to all other ESTs in the data base can be obtained. To estimate expression levels of genes represented in the DuPont EST data base we count the number of times each gene has been seen in different cDNA libraries, from different tissues, developmental stages or induction conditions. This quantitation of message levels is quite accurate for highly expressed messages and, unlike conventional Northern blots, allows comparison of expression levels between different genes. Lists of most highly expresses genes in different libraries can be compiled. Also, if EST data is available for cDNA libraries derived from different developmental stages, gene expression profiles across development can be assembled. We present an example of such a profile for soybean seed development. Gene expression data obtained from Electronic Northern analysis can be confirmed and extended beyond the realm of highly expressed genes by using high density DNA arrays. The ESTs identified as interesting can be arrayed on nylon or glass and probed with total labeled cDNA first strand from the tissue of interest. Two-color fluorescent labeling allows accurate mRNA ratio measurements. We are currently using the DNA array technology to study chemical induction of gene expression and the biosynthesis of oil, carbohydrate and protein in developing seeds.

Genepool variation in genus Glycine subgenus Soja revealed by polymorphic nuclear and chloroplast microsatellites.

A combination of nuclear and chloroplast simple sequence repeats (SSRs) have been used to investigate the levels and pattern of variability detected in Glycine max and G. soja genotypes. Based on the analysis of 700 soybean genotypes with 115 restriction fragment length polymorphism (RFLP) probes, 12 accessions were identified that represent 92% of the allelic variability detected in this genepool. These 12 core genotypes together with a sample of G. max and G. soja accessions were evaluated with 11 nuclear SSRs that detected 129 alleles. Compared with the other G. max and G. soja genotypes sampled, the core genotypes represent 40% of the allelic variability detected with SSRs. Despite the multi-allelic nature of soybean SSRs, dendrograms representing phenetic relationships between accessions clustered according to their subspecies origin. In addition to biparentally inherited nuclear SSRs, two uniparentally (maternally) transmitted chloroplast SSRs were also studied. A total of seven haplotypes were identified, and diversity indices of 0.405 +/- 0.088 and 0.159 +/- 0.071 were obtained for the two chloroplast SSRs. The availability of polymorphic SSR loci in the chloroplast genome provides new opportunities to investigate cytonuclear interactions in plants.

Simple sequence repeats for germplasm analysis and mapping in maize.

Simple sequence repeats (SSRs) are a relatively new class of DNA markers consisting of short runs of tandemly repeated sequence motifs evenly distributed throughout eukaryotic genomes. Owing to the high rate of variation in the number of repeat units, the polymorphism level shown by SSRs is high. Furthermore, they are easy to analyze by means of the polymerase chain reaction, using flanking unique sequence primers. In order to establish the utility of SSR markers for genetic mapping and for the analysis of corn germplasm, corn genomic libraries were constructed and screened for clones containing dinucleotide and trinucleotide repeats. One hundred and fifty clones were isolated and 34 of them were used in this study to analyze 15 (AG)n repeats, 15 (AC)n repeats, and 4 trinucleotide repeats. Twelve corn inbred lines, representing 87% of the RFLP alleles present in a collection of public corn cultivars, were used to assess the information content of the SSR markers. The expected heterozygosity of each SSR marker was compared with the expected heterozygosity of 100 different RFLP markers. The stability of SSRs was also tested through segregation analysis on an existing mapping population.

Hypervariable microsatellites provide a general source of polymorphic DNA markers for the chloroplast genome.

BACKGROUND: The study of plant populations is greatly facilitated by the deployment of chloroplast DNA markers. Asymmetric inheritance, lower effective population sizes and perceived lower mutation rates indicate that the chloroplast genome may have different patterns of genetic diversity compared to nuclear genomes. Convenient assays that would allow intraspecific chloroplast variability to be detected are required. RESULTS: Eukaryote nuclear genomes contain ubiquitous simple sequence repeat (microsatellite) loci that are highly polymorphic in length; these polymorphisms can be rapidly typed by the polymerase chain reaction (PCR). Using primers flanking simple mononucleotide repeat motifs in the chloroplast DNA of annual and perennial soybean species, we demonstrate that microsatellites in the chloroplast genome also exhibit length variation, and that this polymorphism is due to changes in the repeat region. Furthermore, we have observed a nonrandom geographic distribution of variations at these loci, and have examined the number and location of such repeats within the chloroplast genomes of other species. CONCLUSIONS: PCR-based analysis of mononucleotide repeats may be used to detect both intraspecific and interspecific variability in the chloroplast genomes of seed plants. The analysis of polymorphic microsatellites thus provides an important experimental tool to examine a range of issues in plant genetics.

Genetic mapping and variability of seven soybean simple sequence repeat loci.

Microsatellites or simple sequence repeats are stretches of short tandemly repeated DNA sequence motifs, dispersed throughout the genomes of most eukaryotes. Simple sequence repeat polymorphisms (SSRPs) have recently been reported in plants. Here we present the genetic map position of seven different soybean (Glycine max (L.) Merr. and Glycine soja Sieb. and Zucc.) SSRPs contained in sequenced genes, four of which represent newly mapped positions for these genes. The other three SSRPs coincided with independently established RFLP map positions for the corresponding genes. When a set of 61 soybean accessions was screened at four of these loci by using agarose gels, the average number of alleles per locus was 7.75, the effective number of alleles (ne) was 2.57, and the level of allele differentiation (delta(t)) was 0.62. Allelic variation decreased sharply with increasing levels of domestication, with the level of differentiation going from 84% in the wild soybean to 43% in the elite germplasm. Variation levels observed on a subset made of 19 of the 61 lines were always higher for SSRPs than for RFLP markers, with the average number of alleles per locus going from 4.25 to 2.15. In comparison with RFLP markers, SSRPs are more informative and are easier to analyse but require more effort to develop.

Generation-means analysis and quantitative trait locus mapping of anthracnose stalk rot genes in maize.

A generation-means analysis was performed on two maize populations, each segregating for genes conferring resistance to anthracnose stalk rot (ASR). The populations were derived from a cross of DE811ASR x DE811 and of DE811ASR x LH132. The resistant parent, DE811ASR, was obtained through introgression with MP305 as the donor and DE811 as the recurrent parent. The analysis revealed significant additive effects in both populations and a significant additive x dominant effect in the DES11ASR x DES11 population. Quantitative trait locus (QTL) mapping, using restriction fragment length polymorphism (RFLP)-based molecular markers, indicated a significant QTL on linkage group 4 in both populations. The QTL analysis confirmed additive inheritance in both populations. This work demonstrates a close correspondence between generation-means analysis and discrete observations using molecular markers. Linkage of a genetic marker to genes conferring resistance to ASR will be useful for the introgression of resistance into elite germplasm.

Genetic diagnostics in plant breeding: RAPDs, microsatellites and machines.

The science of mapping genetic traits, including those of agronomic interest, is well established and many genetic marker systems are available. However, the application of genetic diagnostics in plant breeding is in its infancy. The sample throughput and cost requirements are very different from those of medical DNA diagnostics. It will be necessary to automate the DNA isolation process, DNA amplification-based allele identification and data handling. Here, we discuss recent progress in the development of molecular technology for plant breeding.