Jessica A. Schlueter

Genome sequence of the palaeopolyploid soybean

Soybean (Glycine max) is one of the most important crop plants for seed protein and oil content, and for its capacity to fix atmospheric nitrogen through symbioses with soil-borne microorganisms. We sequenced the 1.1-gigabase genome by a whole-genome shotgun approach and integrated it with physical and high-density genetic maps to create a chromosome-scale draft sequence assembly. We predict 46,430 protein-coding genes, 70% more than Arabidopsis and similar to the poplar genome which, like soybean, is an ancient polyploid (palaeopolyploid). About 78% of the predicted genes occur in chromosome ends, which comprise less than one-half of the genome but account for nearly all of the genetic recombination. Genome duplications occurred at approximately 59 and 13 million years ago, resulting in a highly duplicated genome with nearly 75% of the genes present in multiple copies. The two duplication events were followed by gene diversification and loss, and numerous chromosome rearrangements. An accurate soybean genome sequence will facilitate the identification of the genetic basis of many soybean traits, and accelerate the creation of improved soybean varieties.

Placing Paleopolyploidy in Relation to Taxon Divergence: A Phylogenetic Analysis in Legumes Using 39 Gene Families

Young polyploid events are easily diagnosed by various methods, but older polyploid events become increasingly difficult to identify as chromosomal rearrangements, tandem gene or partial chromosome duplications, changes in substitution rates among duplicated genes, pseudogenization or locus loss, and interlocus interactions complicate the means of inferring past genetic events. Genomic data have provided valuable information about the polyploid history of numerous species, but on their own fail to show whether related species, each with a polyploid past, share a particular polyploid event. A phylogenetic approach provides a powerful method to determine this but many processes may mislead investigators. These processes can affect individual gene trees, but most likely will not affect all genes, and almost certainly will not affect all genes in the same way. Thus, a multigene approach, which combines the large-scale aspect of genomics with the resolution of phylogenetics, has the power to overcome these difficulties and allow us to infer genomic events further into the past than would otherwise be possible. Previous work using synonymous distances among gene pairs within species has shown evidence for large-scale duplications in the legumes Glycine max and Medicago truncatula. We present a case study using 39 gene families, each with three or four members in G. max and the putative orthologues in M. truncatula, rooted using Arabidopsis thaliana. We tested whether the gene duplications in these legumes occurred separately in each lineage after their divergence (Hypothesis 1), or whether they share a round of gene duplications (Hypothesis 2). Many more gene family topologies supported Hypothesis 2 over Hypothesis 1 (11 and 2, respectively), even after synonymous distance analysis revealed that some topologies were providing misleading results. Only ca. 33% of genes examined support either hypothesis, which strongly suggests that single gene family approaches may be insufficient when studying ancient events with nuclear DNA. Our results suggest that G. max and M. truncatula, along with approximately 7000 other legume species from the same clade, share an ancient round of gene duplications, either due to polyploidy or to some other process.

Large-scale development of cost-effective SNP marker assays for diversity assessment and genetic mapping in chickpea and comparative mapping in legumes

A set of 2486 single nucleotide polymorphisms (SNPs) were compiled in chickpea using four approaches, namely (i) Solexa/Illumina sequencing (1409), (ii) amplicon sequencing of tentative orthologous genes (TOGs) (604), (iii) mining of expressed sequence tags (ESTs) (286) and (iv) sequencing of candidate genes (187). Conversion of these SNPs to the cost-effective and flexible throughput Competitive Allele Specific PCR (KASPar) assays generated successful assays for 2005 SNPs. These marker assays have been designated as Chickpea KASPar Assay Markers (CKAMs). Screening of 70 genotypes including 58 diverse chickpea accessions and 12 BC3F2 lines showed 1341 CKAMs as being polymorphic. Genetic analysis of these data clustered chickpea accessions based on geographical origin. Genotyping data generated for 671 CKAMs on the reference mapping population (Cicer arietinum ICC 4958 × Cicer reticulatum PI 489777) were compiled with 317 unpublished TOG-SNPs and 396 published markers for developing the genetic map. As a result, a second-generation genetic map comprising 1328 marker loci including novel 625 CKAMs, 314 TOG-SNPs and 389 published marker loci with an average inter-marker distance of 0.59 cM was constructed. Detailed analyses of 1064 mapped loci of this second-generation chickpea genetic map showed a higher degree of synteny with genome of Medicago truncatula, followed by Glycine max, Lotus japonicus and least with Vigna unguiculata. Development of these cost-effective CKAMs for SNP genotyping will be useful not only for genetics research and breeding applications in chickpea, but also for utilizing genome information from other sequenced or model legumes.

Defining the Transcriptome Assembly and Its Use for Genome Dynamics and Transcriptome Profiling Studies in Pigeonpea (Cajanus cajan L.)

This study reports generation of large-scale genomic resources for pigeonpea, a so-called ‘orphan crop species’ of the semi-arid tropic regions. FLX/454 sequencing carried out on a normalized cDNA pool prepared from 31 tissues produced 494 353 short transcript reads (STRs). Cluster analysis of these STRs, together with 10 817 Sanger ESTs, resulted in a pigeonpea trancriptome assembly (CcTA) comprising of 127 754 tentative unique sequences (TUSs). Functional analysis of these TUSs highlights several active pathways and processes in the sampled tissues. Comparison of the CcTA with the soybean genome showed similarity to 10 857 and 16 367 soybean gene models (depending on alignment methods). Additionally, Illumina 1G sequencing was performed on Fusarium wilt (FW)- and sterility mosaic disease (SMD)-challenged root tissues of 10 resistant and susceptible genotypes. More than 160 million sequence tags were used to identify FW- and SMD-responsive genes. Sequence analysis of CcTA and the Illumina tags identified a large new set of markers for use in genetics and breeding, including 8137 simple sequence repeats, 12 141 single-nucleotide polymorphisms and 5845 intron-spanning regions. Genomic resources developed in this study should be useful for basic and applied research, not only for pigeonpea improvement but also for other related, agronomically important legumes.

BAC-end Sequence Analysis and a Draft Physical Map of the Common Bean ( Phaseolus vulgaris L.) Genome

Common bean (Phaseolus vulgaris L.) is a legume that is an important source of dietary protein in developing countries throughout the world. Utilizing the G19833 BAC library for P. vulgaris from Clemson University, 89,017 BAC-end sequences were generated giving 62,588,675 base pairs of genomic sequence covering approximately 9.54% of the genome. Analysis of these sequences in combination with 1,404 shotgun sequences from the cultivar Bat7 revealed that approximately 49.2% of the genome contains repetitive sequence and 29.3% is genic. Compared to other legume BAC-end sequencing projects, it appears that P. vulgaris has higher predicted levels of repetitive sequence, but this may be due to a more intense identification strategy combining both similarity-based matches as well as de novo identification of repeats. In addition, fingerprints for 41,717 BACs were obtained and assembled into a draft physical map consisting of 1,183 clone contigs and 6,385 singletons with ~9x coverage of the genome.

Integration of physical and genetic maps of common bean through BAC-derived microsatellite markers

BackgroundCommon bean (Phaseolus vulgaris L.) is the most important legume for direct human consumption and the goal of this study was to integrate a recently constructed physical map for the species with a microsatellite based genetic map using a BAC library from the genotype G19833 and the recombinant inbred line population DOR364 × G19833.ResultsWe searched for simple sequence repeats (SSRs) in the 89,017 BAC-end sequences (BES) from the physical map and genetically mapped any polymorphic BES-SSRs onto the genetic map. Among the BES it was possible to identify 623 contig-linked SSRs, most of which were highly AT-rich. A subgroup of 230 di-nucleotide and tri-nucleotide based SSR primer pairs from these BACs was tested on the mapping parents with 176 single copy loci and 114 found to be polymorphic markers. Of these, 99 were successfully integrated into the genetic map. The 99 linkages between the genetic and physical maps corresponded to an equal number of contigs containing a total of 5,055 BAC clones.ConclusionsClass II microsatellites were more common in the BES than longer class I microsatellites. Both types of markers proved to be valuable for linking BAC clones to the genetic map and were successfully placed across all 11 linkage groups. The integration of common bean physical and genetic maps is an important part of comparative genome analysis and a prelude to positional cloning of agronomically important genes for this crop.

Arabidopsis SCARs Function Interchangeably to Meet Actin-Related Protein 2/3 Activation Thresholds during Morphogenesis

During polarized growth and tissue morphogenesis, cells must reorganize their cytoplasm and change shape in response to growth signals. Dynamic polymerization of actin filaments is one cellular component of polarized growth, and the actin-related protein 2/3 (ARP2/3) complex is an important actin filament nucleator in plants. ARP2/3 alone is inactive, and the Arabidopsis thaliana WAVE complex translates Rho-family small GTPase signals into an ARP2/3 activation response. The SCAR subunit of the WAVE complex is the primary activator of ARP2/3, and plant and vertebrate SCARs are encoded by a small gene family. However, it is unclear if SCAR isoforms function interchangeably or if they have unique properties that customize WAVE complex functions. We used the Arabidopsis distorted group mutants and an integrated analysis of SCAR gene and protein functions to address this question directly. Genetic results indicate that each of the four SCARs functions in the context of the WAVE-ARP2/3 pathway and together they define the lone mechanism for ARP2/3 activation. Genetic interactions among the scar mutants and transgene complementation studies show that the activators function interchangeably to meet the threshold for ARP2/3 activation in the cell. Interestingly, double, triple, and quadruple mutant analyses indicate that individual SCAR genes vary in their relative importance depending on the cell type, tissue, or organ that is analyzed. Differences among SCARs in mRNA levels and the biochemical efficiency of ARP2/3 activation may explain the functional contributions of individual genes.

Large-Scale Development of Cost-Effective Single-Nucleotide Polymorphism Marker Assays for Genetic Mapping in Pigeonpea and Comparative Mapping in Legumes

Single-nucleotide polymorphisms (SNPs, >2000) were discovered by using RNA-seq and allele-specific sequencing approaches in pigeonpea (Cajanus cajan). For making the SNP genotyping cost-effective, successful competitive allele-specific polymerase chain reaction (KASPar) assays were developed for 1616 SNPs and referred to as PKAMs (pigeonpea KASPar assay markers). Screening of PKAMs on 24 genotypes [23 from cultivated species and 1 wild species (Cajanus scarabaeoides)] defined a set of 1154 polymorphic markers (77.4%) with a polymorphism information content (PIC) value from 0.04 to 0.38. One thousand and ninety-four PKAMs showed polymorphisms between parental lines of the reference mapping population (C. cajan ICP 28 × C. scarabaeoides ICPW 94). By using high-quality marker genotyping data on 167 F2 lines from the population, a comprehensive genetic map comprising 875 PKAMs with an average inter-marker distance of 1.11 cM was developed. Previously mapped 35 simple sequence repeat markers were integrated into the PKAM map and an integrated genetic map of 996.21 cM was constructed. Mapped PKAMs showed a higher degree of synteny with the genome of Glycine max followed by Medicago truncatula and Lotus japonicus and least with Vigna unguiculata. These PKAMs will be useful for genetics research and breeding applications in pigeonpea and for utilizing genome information from other legume species.

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.

Comparative Evolution of Photosynthetic Genes in Response to Polyploid and Nonpolyploid Duplication

The likelihood of duplicate gene retention following polyploidy varies by functional properties (e.g. gene ontologies or protein family domains), but little is known about the effects of whole-genome duplication on gene networks related by a common physiological process. Here, we examined the effects of both polyploid and nonpolyploid duplications on genes encoding the major functional groups of photosynthesis (photosystem I, photosystem II, the light-harvesting complex, and the Calvin cycle) in the cultivated soybean (Glycine max), which has experienced two rounds of whole-genome duplication. Photosystem gene families exhibit retention patterns consistent with dosage sensitivity (preferential retention of polyploid duplicates and elimination of nonpolyploid duplicates), whereas Calvin cycle and light-harvesting complex gene families do not. We observed similar patterns in barrel medic (Medicago truncatula), which shared the older genome duplication with soybean but has evolved independently for approximately 50 million years, and in Arabidopsis (Arabidopsis thaliana), which experienced two nested polyploidy events independent from the legume duplications. In both soybean and Arabidopsis, Calvin cycle gene duplicates exhibit a greater capacity for functional differentiation than do duplicates within the photosystems, which likely explains the greater retention of ancient, nonpolyploid duplicates and larger average gene family size for the Calvin cycle relative to the photosystems.