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Randy C Shoemaker

Publications and source records attributed to Randy C Shoemaker.

18 recordsLinked to original sources

Impacts of genetic bottlenecks on soybean genome diversity.

Soybean has undergone several genetic bottlenecks. These include domestication in Asia to produce numerous Asian landraces, introduction of relatively few landraces to North America, and then selective breeding over the past 75 years. It is presumed that these three human-mediated events have reduced genetic diversity. We sequenced 111 fragments from 102 genes in four soybean populations representing the populations before and after genetic bottlenecks. We show that soybean has lost many rare sequence variants and has undergone numerous allele frequency changes throughout its history. Although soybean genetic diversity has been eroded by human selection after domestication, it is notable that modern cultivars have retained 72% of the sequence diversity present in the Asian landraces but lost 79% of rare alleles (frequency </=0.10) found in the Asian landraces. Simulations indicated that the diversity lost through the genetic bottlenecks of introduction and plant breeding was mostly due to the small number of Asian introductions and not the artificial selection subsequently imposed by selective breeding. The bottleneck with the most impact was domestication; when the low sequence diversity present in the wild species was halved, 81% of the rare alleles were lost, and 60% of the genes exhibited evidence of significant allele frequency changes.

Alleles↗

Sequence conservation of homeologous bacterial artificial chromosomes and transcription of homeologous genes in soybean (Glycine max L. Merr.).

The paleopolyploid soybean genome was investigated by sequencing homeologous BAC clones anchored by duplicate N-hydroxycinnamoyl/benzoyltransferase (HCBT) genes. The homeologous BACs were genetically mapped to linkage groups C1 and C2. Annotation of the 173,747- and 98,760-bp BACs showed that gene conservation in both order and orientation is high between homeologous regions with only a single gene insertion/deletion and local tandem duplications differing between the regions. The nucleotide sequence conservation extends into intergenic regions as well, probably due to conserved regulatory sequences. Most of the homeologs appear to have a role in either transcription/DNA binding or cellular signaling, suggesting a potential preference for retention of duplicate genes with these functions. Reverse transcriptase-PCR analysis of homeologs showed that in the tissues sampled, most homeologs have not diverged greatly in their transcription profiles. However, four cases of changes in transcription were identified, primarily in the HCBT gene cluster. Because a mapped locus corresponds to a soybean cyst nematode (SCN) QTL, the potential role of HCBT genes in response to SCN is discussed. These results are the first sequenced-based analysis of homeologous BACs in soybean, a diploidized paleopolyploid.

Base Sequence↗

Paleopolyploidy and gene duplication in soybean and other legumes.

Two of the most important observations from whole-genome sequences have been the high rate of gene birth and death and the prevalence of large-scale duplication events, including polyploidy. There is also a growing appreciation that polyploidy is more than the sum of the gene duplications it creates, in part because polyploidy duplicates the members of entire regulatory networks. Thus, it may be important to distinguish paralogs that are produced by individual gene duplications from the homoeologous sequences produced by (allo)polyploidy. This is not a simple task, for several reasons, including the chromosomally cryptic nature of many duplications and the variable rates of gene evolution. Recent progress has been made in understanding patterns of gene and genome duplication in the legume family, specifically in soybean.

Biological Evolution↗

Pericentromeric regions of soybean (Glycine max L. Merr.) chromosomes consist of retroelements and tandemly repeated DNA and are structurally and evolutionarily labile.

Little is known about the physical makeup of heterochromatin in the soybean (Glycine max L. Merr.) genome. Using DNA sequencing and molecular cytogenetics, an initial analysis of the repetitive fraction of the soybean genome is presented. BAC 076J21, derived from linkage group L, has sequences conserved in the pericentromeric heterochromatin of all 20 chromosomes. FISH analysis of this BAC and three subclones on pachytene chromosomes revealed relatively strict partitioning of the heterochromatic and euchromatic regions. Sequence analysis showed that this BAC consists primarily of repetitive sequences such as a 102-bp tandem repeat with sequence identity to a previously characterized approximately 120-bp repeat (STR120). Fragments of Calypso-like retroelements, a recently inserted SIRE1 element, and a SIRE1 solo LTR were present within this BAC. Some of these sequences are methylated and are not conserved outside of G. max and G. soja, a close relative of soybean, except for STR102, which hybridized to a restriction fragment from G. latifolia. These data present a picture of the repetitive fraction of the soybean genome that is highly concentrated in the pericentromeric regions, consisting of rapidly evolving tandem repeats with interspersed retroelements.

Base Sequence↗

ESTminer: a suite of programs for gene and allele identification.

UNLABELLED: ESTminer is a collection of programs that use expressed sequence tag (EST) data from inbred genomes to identify unique genes within gene families. The algorithm utilizes Cap3 to perform an initial clustering of related EST sequences to produce a consensus sequence of a gene family. These consensus sequences are then used to collect all ESTs in the original EST library that are related using BLAST. A redundancy based criterion is applied to each EST to identify reliable unique gene-sequences. Using a highly inbred genome as a source of ESTs eliminates the necessity of computing covariance on each polymorphism to identify alleles of the same gene, thus making this algorithm more streamlined than other alternatives which must computationally attempt to distinguish genes from alleles. AVAILABILITY: The programs were written in PERL and are freely available at http://www.soybase.org/publication_data/Nelson/ESTminer/ESTminer.html CONTACT: nelsonrt@iastate.edu SUPPLEMENTARY INFORMATION: Figures and dataset can be obtained from: http://www.soybase.org/publication_data/Nelson/ESTminer/ESTminer.html.

Algorithms↗

Microarrays for global expression constructed with a low redundancy set of 27,500 sequenced cDNAs representing an array of developmental stages and physiological conditions of the soybean plant.

BACKGROUND: Microarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays. RESULTS: We report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants. The set was assembled from the 5' sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3' end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines. CONCLUSIONS: Global expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype.

Cluster Analysis↗

National Science Foundation-sponsored workshop report. Draft plan for soybean genomics.

Recent efforts to coordinate and define a research strategy for soybean (Glycine max) genomics began with the establishment of a Soybean Genetics Executive Committee, which will serve as a communication focal point between the soybean research community and granting agencies. Secondly, a workshop was held to define a strategy to incorporate existing tools into a framework for advancing soybean genomics research. This workshop identified and ranked research priorities essential to making more informed decisions as to how to proceed with large scale sequencing and other genomics efforts. Most critical among these was the need to finalize a physical map and to obtain a better understanding of genome microstructure. Addressing these research needs will require pilot work on new technologies to demonstrate an ability to discriminate between recently duplicated regions in the soybean genome and pilot projects to analyze an adequate amount of random genome sequence to identify and catalog common repeats. The development of additional markers, reverse genetics tools, and bioinformatics is also necessary. Successful implementation of these goals will require close coordination among various working groups.

Computational Biology↗

Soybean FGAM synthase promoters direct ectopic nematode feeding site activity.

Soybean cyst nematode (SCN) resistance in soybean is a complex oligogenic trait. One of the most important nematode resistance genes, rhg1, has been mapped to a distal region of molecular linkage group G in soybean. A simplified genetic system to identify soybean genes with modified expression in response to SCN led to the identification of several genes within the nematode feeding sites. The genes were mapped to reveal their linkage relationship to known QTLs associated with soybean cyst nematode (SCN) resistance. One candidate, a phosphoribosyl formyl glycinamidine (FGAM) synthase (EC 6.3.5.3) gene, mapped to the same genomic interval as the major SCN resistance gene rhg1 within linkage group G. Isolation of FGAM synthase from a soybean bacterial artificial chromosome (BAC) library revealed two highly homologous paralogs. The genes appeared to be well conserved between bacteria and humans. Promoter analysis of the two soybean homologs was carried out with the Arabidopsis thaliana - Heterodera schachtii system to investigate gene response to nematode feeding. The two promoters and their derived deletion constructions effected green fluorescent protein (GFP) expression within nematode feeding sites. The 1.0-kb promoter sequence immediately adjacent to the translation start site was sufficient to direct expression of GFP within syncytia. A wound-inducible element and a floral organ expression sequence were also identified within these promoters. Although a nematode-responsive element could not be identified, the observed expression of GFP within feeding sites supports the hypothesis that plant gene expression is redirected within feeding sites to benefit the parasite.

Animals↗

Soybean bacterial artificial chromosome contigs anchored with RFLPs: insights into genome duplication and gene clustering.

Surveying the soybean genome with 683 bacterial artificial chromosome (BAC) contiguous groups (contigs) anchored by restriction fragment length polymorphisms (RFLPs) enabled us to explore microsyntenic relationships among duplicated regions and also to examine the physical organization of hypomethylated (and presumably gene-rich) genomic regions. Numerous cases where nonhomologous RFLPs hybridized to common BAC clones indicated that RFLPs were physically clustered in soybean, apparently in less than 25% of the genome. By extension, we speculate that most of the genes are clustered in less than 275 M of the soybean genome. Approximately 40%-45% of this gene-rich portion is associated with the RFLP-anchored contigs described in this study. Similarities in genome organization among BAC contigs from duplicate genomic regions were also examined. Homoeologous BAC contigs often exhibited extensive microsynteny. Furthermore, paralogs recovered from duplicate contigs shared 86%-100% sequence identity.

Chromosomes, Artificial, Bacterial↗

Segmental duplications within the Glycine max genome revealed by fluorescence in situ hybridization of bacterial artificial chromosomes.

Soybean (Glycine max L. Merr.) is presumed to be an ancient polyploid based on chromosome number and multiple RFLP fragments in genetic mapping. Direct cytogenetic observation of duplicated regions within the soybean genome has not heretofore been reported. Employing fluorescence in situ hybridization (FISH) of genetically anchored bacterial artificial chromosomes (BACs) in soybean, we were able to observe that the distal ends of molecular linkage group E had duplicated regions on linkage groups A2 and B2. Further, using fiber-FISH, it was possible to measure the molecular size and organization of one of the duplicated regions. As FISH did not require repetitive DNA for blocking fluorescence signals, we assume that the 200-kb genome region is relatively low in repetitive sequences. This observation, along with the observation that the BACs are located in distal euchromatin regions, has implications for genome structure/evolution and the approach used to sequence the soybean genome.

Biological Evolution↗

Mining EST databases to resolve evolutionary events in major crop species.

Using plant EST collections, we obtained 1392 potential gene duplicates across 8 plant species: Zea mays, Oryza sativa, Sorghum bicolor, Hordeum vulgare, Solanum tuberosum, Lycopersicon esculentum, Medicago truncatula, and Glycine max. We estimated the synonymous and nonsynonymous distances between each gene pair and identified two to three mixtures of normal distributions corresponding to one to three rounds of genome duplication in each species. Within the Poaceae, we found a conserved duplication event among all four species that occurred approximately 50-60 million years ago (Mya); an event that probably occurred before the major radiation of the grasses. In the Solanaceae, we found evidence for a conserved duplication event approximately 50-52 Mya. A duplication in soybean occurred approximately 44 Mya and a duplication in Medicago about 58 Mya. Comparing synonymous and nonsynonymous distances allowed us to determine that most duplicate gene pairs are under purifying, negative selection. We calculated Pearson's correlation coefficients to provide us with a measure of how gene expression patterns have changed between duplicate pairs, and compared this across evolutionary distances. This analysis showed that some duplicates seemed to retain expression patterns between pairs, whereas others showed uncorrelated expression.

Computational Biology↗

Features of a 103-kb gene-rich region in soybean include an inverted perfect repeat cluster of CHS genes comprising the I locus.

The I locus in soybean (Glycine max) corresponds to a region of chalcone synthase (CHS) gene duplications affecting seed pigmentation. We sequenced and annotated BAC clone 104J7, which harbors a dominant i(i) allele from Glycine max 'Williams 82', to gain insight into the genetic structure of this multigenic region in addition to examining its flanking regions. The 103-kb BAC encompasses a gene-rich region with 11 putatively expressed genes. In addition to six copies of CHS, these genes include: a geranylgeranyltransferase type II beta subunit (E.C.2.5.1.60), a beta-galactosidase, a putative spermine and (or) spermidine synthase (E.C.2.5.1.16), and an unknown expressed gene. Strikingly, sequencing data revealed that the 10.91-kb CHS1, CHS3, CHS4 cluster is present as a perfect inverted repeat separated by 5.87 kb. Contiguous arrangement of CHS paralogs could lead to folding into multiple secondary structures, hypothesized to induce deletions that have previously been shown to effect CHS expression. BAC104J7 also contains several gene fragments representing a cation/hydrogen exchanger, a 40S ribosomal protein, a CBL-interacting protein kinase, and the amino terminus of a subtilisin. Chimeric ESTs were identified that may represent read-through transcription from a flanking truncated gene into a CHS cluster, generating aberrant CHS RNA molecules that could play a role in CHS gene silencing.

Acyltransferases↗

Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes.

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine. Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency. At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.

Amino Acid Sequence↗

Organization, expression and evolution of a disease resistance gene cluster in soybean.

PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar "Williams 82" [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca(2+)-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.

Amino Acid Sequence↗

Identification, structure, and differential expression of members of a BURP domain containing protein family in soybean.

Expressed sequence tags (ESTs) exhibiting homology to a BURP domain containing gene family were identified from the Glycine max (L.) Merr. EST database. These ESTs were assembled into 16 contigs of variable sizes and lengths. Consistent with the structure of known BURP domain containing proteins, the translation products exhibit a modular structure consisting of a C-terminal BURP domain, an N-terminal signal sequence, and a variable internal region. The soybean family members exhibit 35-98% similarity in a -100-amino-acid C-terminal region, and a phylogenetic tree constructed using this region shows that some soybean family members group together in closely related pairs, triplets, and quartets, whereas others remain as singletons. The structure of these groups suggests that multiple gene duplication events occurred during the evolutionary history of this family. The depth and diversity of G. max EST libraries allowed tissue-specific expression patterns of the putative soybean BURPs to be examined. Consistent with known BURP proteins, the newly identified soybean BURPs have diverse expression patterns. Furthermore, putative paralogs can have both spatially and quantitatively distinct expression patterns. We discuss the functional and evolutionary implications of these findings, as well as the utility of EST-based analyses for identifying and characterizing gene families.

Amino Acid Sequence↗