Soybean (Glycine max L. Mer.), like many cultivated crops, has a relatively narrow genetic base and lacks diversity for some economically important traits. Glycine latifolia (Benth.) Newell & Hymowitz, one of the 26 perennial wild Glycine species related to soybean in the subgenus Glycine Willd., shows high levels of resistance to multiple soybean pathogens and pests including Alfalfa mosaic virus, Heterodera glycines Ichinohe and Sclerotinia sclerotiorum (Lib.) de Bary. However, limited information is available on the genomes of these perennial Glycine species. To generate molecular resources for gene mapping and identification, high-density linkage maps were constructed for G. latifolia using single nucleotide polymorphism (SNP) markers generated by genotyping by sequencing and evaluated in an F2 population and confirmed in an F5 population. In each population, greater than 2,300 SNP markers were selected for analysis and segregated to form 20 large linkage groups. Marker orders were similar in the F2 and F5 populations. The relationships between G. latifolia linkage groups and G. max and common bean (Phaseolus vulgaris L.) chromosomes were examined by aligning SNP-containing sequences from G. latifolia to the genome sequences of G. max and P. vulgaris. Twelve of the 20 G. latifolia linkage groups were nearly collinear with G. max chromosomes. The remaining eight G. latifolia linkage groups appeared to be products of multiple interchromosomal translocations relative to G. max. Large syntenic blocks also were observed between G. latifolia and P. vulgaris. These experiments are the first to compare genome organizations among annual and perennial Glycine species and common bean. The development of molecular resources for species closely related to G. max provides information into the evolution of genomes within the genus Glycine and tools to identify genes within perennial wild relatives of cultivated soybean that could be beneficial to soybean production.
Citation: Chang S, Thurber CS, Brown PJ, Hartman GL, Lambert KN, Domier LL (2014) Comparative Mapping of the Wild Perennial Glycine latifolia and Soybean (G. max) Reveals Extensive Chromosome Rearrangements in the Genus Glycine. PLoS ONE 9(6): e99427. https://doi.org/10.1371/journal.pone.0099427
Editor: Matthew N. Nelson, The University of Western Australia, Australia
Received: February 21, 2014; Accepted: May 14, 2014; Published: June 17, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This study was supported by funding from the National Sclerotinia Initiative and the United States Department of Agriculture-Agricultural Research Service. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Soybean (Glycine max L. Mer.) is a major source of dietary protein and oil in animal production and for human consumption worldwide . With increasing utilization of soybean for animal feed in countries like China, there is added demand for soybean production . Most of the increased demand for soybean products has been met by expanding the land area devoted to soybean production . However, it is not clear if the expansion of soybean production areas alone will be able to keep pace with this growing demand. In addition, the global movement of soybean pathogens and pests and the emergence of new pathogens, as illustrated by the recent identification of soybean aphids (Aphis glycines Matsumura), soybean rust (Phakopsora pachyrhizi Syd. & P. Syd) and Soybean vein necrosis virus in North America –, necessitates the identification of novel genes that will enable producers to meet the ever increasing demand for soybean production in the face of changing abiotic and biotic stresses.
Because of its narrow genetic base, soybean, like many cultivated crops, lacks diversity found in some of its wild relatives. The genus Glycine consists of 28 species split between two subgenera, Glycine Willd. and Soja (Moench) F. J. Hermann. The subgenus Soja contains two annual species, G. max, the domesticated species in the genus, and G. soja Sieb. & Zucc., both of which are native to Asia. The Glycine subgenus contains 26 species, including G. latifolia (Benth.) Newell & Hymowitz, that are native to Australia and surrounding islands, and have been shown to possess genes for agronomically valuable traits, such as resistance to Heterodera glycines Ichinohe and tolerance to Sclerotinia sclerotiorum (Lib.) de Bary. and drought –.
To date however, it has not been possible to utilize genes from the perennial Glycine species for soybean improvement even though G. max and the perennial Glycine species share a relatively recent whole genome duplication that occurred between 5 and 13 million years ago , . Attempts to hybridize G. max and Glycine perennials have, with the exception of G. tomentella (2n = 78) Hayata, been unsuccessful, even with in vitro embryo rescue , . Cytogenetic observations have shown aberrant chromosome paring in F1 hybrids leading to embryo abortion . Sequence data from G. latifolia and G. tomentella suggest that differences in intergenic and pericentromeric sequences, including sequences of widely dispersed retrotransposons, have reduced chromosomal pairing during hybridization –.
Advances in molecular biology provide tools to circumvent the genetic barriers to capturing the biological diversity present in the perennial relatives of G. max. In addition to the genome sequence of G. max , the genome sequence of the wild annual species G. soja was recently determined , and shed light on similarities and differences between the two interfertile species. Using the genomic information, high-throughput sequencing and virus-based gene silencing techniques, multiple genes have been identified in soybean –. Even though gene mapping resources developed for G. max have not been directly useful in perennial Glycine species, the identification of large syntenic blocks between G. max and other legume species – suggests that high levels of synteny will be observed between annual and perennial Glycine species. The development of methods for cost-efficient discovery and mapping of single nucleotide polymorphism (SNP) markers through methods like genotyping by sequencing (GBS) ,  have made it possible to fine map genes in plant species for which genetic resources are lacking, as is the case with the perennial Glycine species. Here, we describe the construction of high-density linkage maps for a perennial relative, G. latifolia, of cultivated soybean and compare the orders of mapped SNP markers to their positions in the genome sequences of G. max and Phaseolus vulgaris L.
Materials and Methods
Reciprocal crosses were performed between G. latifolia plant introduction (PI) 559298 and PI 559300 (obtained from the USDA Soybean Germplasm Collection in Urbana, Illinois; http://www.ars-grin.gov/npgs/urbana.html) as previously described . Populations were advanced by selfing the F2 generation to the F5 generation.
DNA was extracted from leaf tissue of PI 559298, PI 559300, 146 F2 plants and 89 F5 plants using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). DNA samples were digested with BfaI and PstI-HF restriction enzymes (New England Biolabs, Ipswich, MA) as described by Thurber et al. . For these experiments, BfaI was selected because it did not produce strong banding patterns in preliminary restriction enzyme digestions of G. latifolia DNA and PstI was selected because G. latifolia sequence data  contained an intermediate number of PstI recognition sites. For example, the previously determined G. latifolia sequence data were predicted to contain 2.1×104 MluI sites, 8.9×104 PstI sites and 3.1×105 HindIII sites. Up to 96 samples were sequenced per lane of a HiSeq2000 (Illumina Inc., San Diego, CA) at the W. M. Keck Center at the University of Illinois, Urbana, IL, USA to produced 100-nt single-end reads. In both experiments, DNA from each of the parental lines was independently processed twice to serve as a control for SNP identification. The barcode splitter from TASSEL  was used to assign sequence reads to individual lines and remove barcode sequences, which produced 90-nucleotide sequence reads that were analyzed for SNPs. The parsed sequence data for the F2 and F5 populations have been deposited in the NCBI Short Read Archive as part of project SRP013346. Next, three Perl scripts were used to analyze the sequence reads for the bi-parental populations. First, sequence reads for each individual/line in the F2 and F5 populations and from the parental lines, PI 559298 and PI 559300, were aligned using Bowtie  to a G. latifolia pseudo-reference sequence, which was generated by sequencing 180-bp, 500-bp paired-end and 3-kb, 8-kb, and 15-kb mate-pair libraries prepared from G. latifolia PI 559298 DNA, sequenced on an Illumina HiSeq 2000, and de novo assembled using ALLPATHS-LG  (Chang et al., manuscript in preparation). The resulting assembly contained 16,423 scaffolds representing 1,069 Mbp, with an N50 of 235 Kb. Bowtie2 , which allows for insertions and deletions (indels), was also evaluated for read mapping, but at the high stringencies for matching employed, few indels were detected and the output from Bowtie was parsed more directly to SNP calls than output from Bowtie2. Second, SNPs were called when at least three reads from both replications of PI 559298 differed from both replications of PI 559300. Finally, Bowtie output files for each individual/line were used to assess allelic frequencies for each SNP using a custom Perl script, which ignored SNPs in low quality sequence reads (average quality scores of 40 or less). Based on allelic frequencies at each locus for each line, the Perl script then created a genotype matrix file for linkage analysis. Markers with less than 30% missing data and whose segregation did not differ significantly (P>0.05) from expected segregation ratios were selected for de novo linkage map construction.
Linkage maps were constructed using MSTMap  with a P-value = 1.0−9, and visualized using MapDraw . Consensus linkage maps were constructed for G. latifolia from the F2 and F5 data using MergeMap . A weight of 5.0 was assigned to the F5 linkage maps and a weight of 1.0 to F2 linkage maps to reflect the higher confidence in the quality of the maps because of the reduced potential for errors in calling of heterozygous genotypes in the F5 population relative to the F2 population. To assess the synteny between G. latifolia linkage groups (LGs) and G. max chromosomes, SNP-containing sequences from G. latifolia were aligned to the G. max genome sequence  using BLAST . For comparisons with P. vulgaris chromosomes, G. latifolia SNP-containing sequences and G. max gene models  were aligned to P. vulgaris chromosomes (http://www.phytozome.net/commonbean.php) using BLAST and visualized with MizBee . When G. latifolia sequences aligned at more than one location, the most syntenic location was chosen for these analyses.
Mapping GBS SNP markers in G. latifolia populations
Genotyping by sequencing of the F2 population produced a total of 4.00×108 100-nt reads, of which 1.70×108 passed all quality controls and uniquely aligned to PI 559300 sequences. After barcodes were removed, 90 nt were used for SNP discovery. In the F2 population, 5,160 markers could be reliably scored between the parental lines PI 559298 and PI 559300. Linkage maps constructed from that initial data set represented over 13,000 centimorgans (cM), which was significantly larger than G. max (2,296 to 2,550 cM) and previous G. latifolia (1972 cM) linkage maps , – and likely resulted from errors in calling heterozygous genotypes because of low coverage at some loci. The data set was reprocessed to exclude markers with more than 30% missing data and with segregation ratios that differed significantly from 1∶2∶1 (P>0.05), which resulted in 2,377 markers (Table S1). The average depth of coverage for the selected SNPs was 32 reads per locus and ranged from 0 to 270 reads. The markers formed 20 large LGs (Figure S1), with an average of 119 markers per LG (Table 1), and a total length of 2,305 cM. To confirm marker orders, an F5 population was analyzed by the same procedures. The analysis produced a total of 1.92×108 100-nt reads, of which 1.05×108 passed all quality controls and uniquely aligned to PI 559300 sequences. The data produced 7,081 SNPs between the parental lines, from which 3,110 GBS markers (Table S2) were selected using similar criteria and analyzed in an F5 population. Average depth of coverage for the selected SNPs was 21 reads and ranged from 0 to 264 reads. As with the F2 population, most of the markers formed 20 large LGs (Figure S2), with an average of 155 markers per LG and a total map length of 3,110 cM. A total of 1,777 markers were shared between the two populations with 600 markers unique to the F2 population and 1,333 markers unique to the F5 population. The orders of shared markers were very similar in linkage maps constructed from the F2 and F5 populations (Figure 1). In some cases, markers that appeared to segregate in the F2 population did not segregate in the F5, presumably caused by errors in calling heterozygous loci in the F2 population. The shared markers were used as a framework to construct consensus linkage maps for G. latifolia (Figures 1 & S3). The merged consensus maps contained 3,710 markers (Table 1).
Orders of SNP markers were very similar between the F2 and F5 populations. In some cases, markers that segregated in the F2 population co-localized in the F5 population, which may have resulted from errors in calling heterozygous loci in the F2 population. While linkage group 1 showed a high level of collinearity with G. max chromosome 1, linkage group 20 had regions of collinearity with multiple G. max chromosomes. Even so, there was good agreement in marker order between the F2 and F5 populations for linkage group 20. Markers were named for the G. max chromosome and the nucleotide position on the chromosome (×10−6) to which the SNP-containing sequences aligned. Markers that did not align to a G. max chromosome were named for the G. latifolia scaffold containing the SNP.
Synteny between G. latifolia linkage groups and G. max chromosomes
Because little information is available on G. latifolia chromosomes, or chromosomes of any other perennial Glycine species, G. latifolia LGs were named for the G. max chromosomes to which G. latifolia SNP-containing sequences predominantly aligned. When mapped orders of G. latifolia SNPs were compared to positions of their sequences in the G. max genome, G. latifolia LGs 1, 3, 4, 6, 9, 10, 11, 12, 14, 15, 17, and 18 showed a high degree of collinearity with the corresponding G. max chromosomes and no interchromosomal rearrangements (Figure 2). In contrast, the remaining eight chromosomes each had at least one interchromosomal rearrangement relative to G. max (LG 13: one rearrangement; LG16: two rearrangements; LGs 2, 5, and 8: three rearrangements; LGs 7 and 20: four rearrangements and LG 19: five rearrangements). For example, G. latifolia LG 13 contained regions syntenic with G. max chromosomes 13 and 20 and G. latifolia LG 2 contained regions syntenic to G. max chromosomes 1, 2, 8, 13 and 19 (Figure 2). Glycine latifolia chromosome 8 appeared to be the product of two translocations between G. max chromosomes 2 and 8. Similarly, G. latifolia LG 20 appeared to contain a reciprocal translocation between G. max chromosomes 2 and 20. Syntenic blocks in rearranged LGs corresponded to between 0.3 Mb and 30 Mb (between G. latifolia LG 8 and G. max chromosome 8) in the G. max genome with an average of 6.9 Mb. Even though we described the structure of G. latifolia linkage groups as products for rearrangements relative to G. max, the structures of the ancestral chromosomes is not known. Hence, in some cases, G. latifolia linkage groups may have under gone fewer rearrangements than G. max chromosomes. Singleton markers (single G. latifolia SNP markers that aligned to a G. max chromosome without at least a second proximal collinear marker) were ignored for these analyses.
Sequences containing mapped G. latifolia SNPs were aligned to the G. max genome sequence. Glycine latifolia linkage groups (top) and physical maps for each G. max chromosome (bottom) are displayed as linear arrays. Vertical and diagonal lines connect genetic and physical locations of SNP markers between the two species.
As with molecular markers in G. max, comparison of the genetic distances between GBS markers in G. latifolia and the physical distances between positions to which the SNP-containing sequences aligned on G. max chromosomes, indicated that there was reduced recombination in regions that corresponded to G. latifolia centromeres (Figure 3). Points deviating from the main line may represent mis-aligned, or mis-mapped sequences or intrachromosomal rearrangements. No G. latifolia markers were identified that aligned to 13.7 Mb and 15.0 Mb in the central regions of G. max chromosomes 5 and 20, respectively, which may have been caused by low GBS marker density (i.e., lack of PstI cutting sites) or low sequence conservation in highly repetitive pericentromeric regions.
Glycine latifolia linkage groups 1 and 18 showed a high degree of collinearity with the corresponding G. max chromosomes. The genetic distances in G. latifolia were plotted against the physical locations of the SNP markers on the G. max chromosomes 1 and 18. As in G. max, the predicted ratios of genetic and physical distances varied along G. latifolia linkage groups. The slopes were steeper near the ends of linkage groups and flatter near the center in regions predicted to correspond to centromeres, where recombination is lower.
Similarities between G. latifolia linkage groups and Phaseolus vulgaris chromosomes
The progenitor of P. vulgaris diverged from the Glycine about 19 million years ago , . While 3,664 SNP-containing sequences from G. latifolia aligned to the G. max genome, 2,063 G. latifolia sequences aligned to P. vulgaris chromosomes. For G. max, the sequences of 30,327 gene models aligned to P. vulgaris chromosomes. As reported for comparisons between G. max and P. vulgaris , extensive blocks of synteny were observed between G. latifolia LGs and P. vulgaris chromosomes (Figure 4). McClean et al. observed that the pericentromeric regions of G. max chromosomes 10, 12, 14, 17, 18, and 20 had extensive syntenic blocks with P. vulgaris chromosomes. Glycine latifolia LGs 7 and 20 appeared to have larger syntenic blocks with single P. vulgaris chromosomes than with G. max chromosomes. Both G. latifolia LGs 10 and 20 showed large blocks of synteny with P. vulgaris chromosome 7, as did G. max chromosome 10. The results suggest that G. max chromosome 7 and 20 have been reorganized after the whole genome duplication event, but G. max chromosome 10 and G. latifolia LGs 7, 10 and 20 appear to have retained gene orders more similar to shared ancestral chromosomes.
Glycine latifolia linkage groups (A & B) and G. max chromosomes (C) are placed at the top of each circle with colored lines connecting positions of G. latifolia SNP markers (A & B) or G.max gene model (C) sequences to positions in G. max (Gm01 – Gm20) or P. vulgaris (PV01 – PV11) chromosomes, represented by gray boxes. Glycine latifolia linkage groups 7 and 20 appeared to have larger syntenic blocks with single P. vulgaris chromosomes than G. max chromosomes. Both G. latifolia linkage groups 10 and 20 showed large blocks of synteny with P. vulgaris chromosome 7, as did G. max chromosome 10. Synteny maps were constructed using MizBee .
Glycine latifolia is a perennial relative of soybean that is native to eastern Australia with a trailing or twining growth habit . Like G. max, the genome of G. latifolia contains 2n = 40 morphologically similar chromosomes . In this study, we used GBS to construct high-density linkage maps for G. latifolia and showed that eight of the 20 G. latifolia LGs were rearranged relative to G. max chromosomes. Linkage maps were constructed de novo from F2 and F5 populations to confirm marker orders. Genotyping by sequencing has been applied to several plant species with and without reference genome sequences. For example, Sonah et al.  reported the application of GBS to a set of eight diverse soybean genotypes and identified from 4,028 to 5,807 SNPs between pairs of soybean lines by aligning sequence reads to the G. max genome sequence. Similar to results reported here, Ward et al.  produced genetic maps for raspberry (Rubus idaeus L.) without a reference sequence consisting of 2,391 and 4,521 markers. Russell et al.  used 1,901 SNPs identified using GBS without a reference sequence to map quantitative trait loci in blackcurrant (Ribes nigrum L.). Similar to this study, Ma et al.  identified 3,745 SNP by GBS in Miscanthus sinensis Anderss, a potential bioenergy crop , and used them to compare the genome organization of M. sinensis to those of Brachypodium distachyon L., Oryza sativa L., Sorghum bicolor (L.) Moench, and Zea mays L. by aligning the SNP-containing sequences to the heterologous genome sequences.
Mahama et al.  genetically and cytologically identified seven different chromosomal translocation lines in soybean and reported that crossing a soybean line homozygous for a single translocation with a wild-type soybean line resulted in significant levels of pollen abortion, ovule abortion, and reduction in seed set. Hence, it is not surprising that crosses between G. latifolia and G. max that involve eight chromosomes with multiple translocations do not produce fertile progeny . Following embryo rescue and colchicine treatment to double chromosome numbers , it may be possible to recover lines that retain unrearranged G. latifolia chromosomes from wide-hybridization experiments between G. latifolia and G. max, but lines containing chromosomes with multiple translocations would likely not be fertile.
Using fluorescence in situ hybridization–based karyotyping of the seven soybean translocation lines, Findley et al.  identified reciprocal translocations between G. max chromosomes 1 and 8; 2 and 8; 2 and 11; 4 and 13; and 5 and 13. It is interesting that G. latifolia LGs also showed reciprocal exchanges between chromosomes 2 and 8, which may indicate the presence of recombination hotspots in the rearranged chromosomes. It has been difficult to identify translocations in soybean because of its small and morphologically similar chromosomes . Consequently, it is possible that other recombination hotspots remain to be identified.
Based on hybridization success, hybrid seed viability, fertility of F1 plants in intra- and interspecific hybrids and degree of meiotic chromosome pairing, species within the genus Glycine have been assigned genome types . Glycine latifolia, along with G. microphylla Tindale, and G. tabacina (Labill.), Benth (all 2n = 40) contain B genome types. In interspecific crosses, G. latifolia, G. microphylla and G. tabacina produce vigorous F1 plants with normal seed set –, which suggests that G. microphylla and G. tabacina, other than paracentric inversions , have chromosome structures very similar to those found in G. latifolia. In contrast, F1 plants from crosses between D-genome perennial species (i.e., G. tomentella) show seedling lethality , . It has been possible to recover plants from crosses between G. tomentella and G. max, using G. tomentella lines with 78 chromosomes , . The 2n = 78 G. tomentella lines may contain a set of 20 chromosomes that have fewer rearrangements than G. latifolia that facilitate chromosome pairing during crossing with G. max.
Generally, nuclear genomes of closely related species show high degrees of collinearity that degrade with increasing phylogenetic distance, but the rates at which chromosomes diverge vary widely among taxa . In comparisons of genetic maps between Arabidopsis thaliana (L.) Heynh. and Brassica nigra (L.) W.D.J. Koch, Lagercrantz  estimated that there had been about three chromosomal rearrangements per million years since the two species had diverged 11 to 35 million years ago . In contrast, Koch et al.  estimated that Arabidopsis lyrata L. and Capsella rubella Reut. had undergone less than 0.09 rearrangements per million years since the two lineages diverged 10 to 14 million years ago. Like G. tomentella, G. latifolia diverged from the progenitors of G. max between 5 and 7 million years ago , , which would mean that the genomes of G. latifolia and G. max have undergone up to five interchromosomal rearrangements per million years. This number is higher than in the studies mentioned above and may indicate a higher rate of chromosomal instability or simply that a higher density of markers was used which permitted the detection of a larger number of chromosomal rearrangements. In addition to being geographically isolated from the progenitors of G. max, G. latifolia, like other 2n = 40 perennial Glycine species, often produces seed from cleistogamous flowers , which reduces its opportunities for outcrossing and possibly removing some of the selection pressure against chromosomal rearrangements. As a consequence, more translocations may have been preserved in Glycine species than in other non-Glycine species examined that outcross more readily.
As functions are assigned to G. max genes, the perennial Glycine species become important sources of new alleles that can be isolated and moved into G. max by standard transformation or developing gene replacement technologies –. Jones et al.  recently demonstrated the feasibility of this approach by using Agrobacterium-mediated transformation to transfer a functional gene for resistance to late blight (caused by Phytophthora infestans (Mont.) DeBary) from Solanum venturii Hawkes & Hjerting to cultivated potato (Solanum tuberosum L.). Gene identification in perennial Glycines species would be greatly aided by determining the genomic sequences for at least a subset of the species. Because many of the chromosomes of perennial Glycine species are collinear with their G. max homologues, the G. max genome sequence could be used as a reference to assemble genome sequences of perennial Glycine species. Using the heterologous G. max reference sequence will reduce the depth of sequence coverage needed for genome assembly compared to de novo genome sequencing , . Even though genetic hybridization may not be possible by standard means, methods for high-throughput gene mapping and identification afforded by next-generation sequencing provide tools to capture the variation present in the wild species.
Genetic linkage maps of GBS markers in a Glycine latifolia F2 population.
Genetic linkage maps of GBS markers in a Glycine latifolia F5 population.
Consensus genetic linkage maps for Glycine latifolia derived from 3710 GBS markers.
GBS SNP Genotype data for the F2 mapping population.
We thank Nancy McCoppin for assistance in maintenance of mapping populations. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the United States Department of Agriculture or the University of Illinois and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
Conceived and designed the experiments: SC PJB GLH LLD. Performed the experiments: SC CST GLH. Analyzed the data: SC LLD PJB. Contributed reagents/materials/analysis tools: SC CST PJB GLH KNL LLD. Wrote the paper: SC LLD GLH PJB CST KNL.
- 1. Hartman GL, West ED, Herman TK (2011) Crops that feed the World 2. Soybean-worldwide production, use, and constraints caused by pathogens and pests. Food Security 3: 5–17.
- 2. Simelton E (2011) Food self-sufficiency and natural hazards in China. Food Security 3: 35–52.
- 3. Brown LR (2012) Full Planet, Empty Plates: The new geopolitics of food scarcity. New York, New York: W. W. Norton & Company.
- 4. Schneider RW, Hollier CA, Whitam HK, Palm ME, McKemy JM, et al. (2005) First report of soybean rust caused by Phakopsora pachyrhizi in the continental United States. Plant Disease 89: 774–774.
- 5. Ragsdale DW, Voegtlin DJ, O'Neil RJ (2004) Soybean aphid biology in North America. Annals of the Entomological Society of America 97: 204–208.
- 6. Zhou J, Kantartzi SK, Wen RH, Newman M, Hajimorad MR, et al. (2011) Molecular characterization of a new tospovirus infecting soybean. Virus Genes 43: 289–295.
- 7. Lim SM, Hymowitz T (1987) Reactions of perennial wild species of genus Glycine to Septoria glycines. Plant Disease 71: 891–893.
- 8. Riggs RD, Wang S, Singh RJ, Hymowitz T (1998) Possible transfer of resistance to Heterodera glycines from Glycine tomentella to soybean. Journal of Nematology 30: 547–552.
- 9. Hartman GL, Gardner ME, Hymowitz T, Naidoo GC (2000) Evaluation of perennial Glycine species for resistance to soybean fungal pathogens that cause Sclerotinia stem rot and sudden death syndrome. Crop Science 40: 545–549.
- 10. Hartman GL, Wang TC, Hymowitz T (1992) Sources of resistance to soybean rust in perennial Glycine species. Plant Disease 76: 396–399.
- 11. Burdon JJ, Marshall DR (1981) Evaluation of Australian native species of Glycine for resistance to soybean rust. Plant Disease 65: 44–45.
- 12. Horlock CM, Teakle DS, Jones RM (1997) Natural infection of the native pasture legume, Glycine latifolia, by alfalfa mosaic virus in Queensland. Australasian Plant Pathology 26: 115–116.
- 13. Jones RM, Brown AHD, Coote JN (1996) Variation in growth and forage quality of Glycine latifolia (Benth.) (Newell and Hymowitz). Genetic Resources Communication No. 26. Australia: Division of Tropical Crops and Pastures, CSIRO.
- 14. Doyle JJ, Egan AN (2010) Dating the origins of polyploidy events. New Phytologist 186: 73–85.
- 15. Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, et al. (2010) Genome sequence of the palaeopolyploid soybean. Nature 463: 178–183.
- 16. Singh RJ, Kollipara KP, Hymowitz T (1998) Monosomic alien addition lines derived from Glycine max (L) Merr and G. tomentella Hayata: production, characterization, and breeding behavior. Crop Science 38: 1483–1489.
- 17. Singh RJ (2010) Methods for producing fertile crosses between wild and domestic soybean species. In: USPO, editor. USA.
- 18. Hymowitz T, Singh RJ, Kollipara KP (1998) The genomes of the Glycine. Plant Breeding Reviews 16: 289–231.
- 19. Chang S, Hartman GL, Singh RJ, Lambert KN, Hobbs HA, et al. (2013) Identification of high-quality single-nucleotide polymorphisms in Glycine latifolia using a heterologous reference genome sequence. Theoretical and Applied Genetics 126: 1627–1638.
- 20. Innes RW, Ameline-Torregrosa C, Ashfield T, Cannon E, Cannon SB, et al. (2008) Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean. Plant Physiology 148: 1740–1759.
- 21. Wawrzynski A, Ashfield T, Chen NWG, Mammadov J, Nguyen A, et al. (2008) Replication of nonautonomous retroelements in soybean appears to be both recent and common. Plant Physiology 148: 1760–1771.
- 22. Kim MY, Lee S, Van K, Kim TH, Jeong SC, et al. (2010) Whole-genome sequencing and intensive analysis of the undomesticated soybean (Glycine soja Sieb. and Zucc.) genome. Proceedings of the National Academy of Sciences of the United States of America 107: 22032–22037.
- 23. Meyer JDF, Silva DCG, Yang C, Pedley KF, Zhang C, et al. (2009) Identification and analyses of candidate genes for Rpp4-mediated resistance to Asian soybean rust in soybean. Plant Physiology 150: 295–307.
- 24. Cook DE, Lee TG, Guo XL, Melito S, Wang K, et al. (2012) Copy number variation of multiple genes at Rhg1 mediates nematode resistance in soybean. Science 338: 1206–1209.
- 25. Liu SM, Kandoth PK, Warren SD, Yeckel G, Heinz R, et al. (2012) A soybean cyst nematode resistance gene points to a new mechanism of plant resistance to pathogens. Nature 492: 256–+.
- 26. Xia ZJ, Watanabe S, Yamada T, Tsubokura Y, Nakashima H, et al. (2012) Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering. Proceedings of the National Academy of Sciences of the United States of America 109: E2155–E2164.
- 27. Boutin SR, Young ND, Olson TC, Yu ZH, Shoemaker RC, et al. (1995) Genome conservation among three legume genera detected with DNA markers. Genome 38: 928–937.
- 28. Lee JM, Grant D, Vallejos CE, Shoemaker RC (2001) Genome organization in dicots. II. Arabidopsis as a ‘bridging species’ to resolve genome evolution events among legumes. Theoretical and Applied Genetics 103: 765–773.
- 29. Muchero W, Diop NN, Bhat PR, Fenton RD, Wanamaker S, et al. (2009) A consensus genetic map of cowpea Vigna unguiculata (L) Walp. and synteny based on EST-derived SNPs. Proceedings of the National Academy of Sciences of the United States of America 106: 18159–18164.
- 30. Young ND, Debelle F, Oldroyd GED, Geurts R, Cannon SB, et al. (2011) The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480: 520–524.
- 31. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, et al. (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLOS ONE 6: e19379.
- 32. Poland JA, Bradbury PJ, Buckler ES, Nelson RJ (2011) Genome-wide nested association mapping of quantitative resistance to northern leaf blight in maize. Proceedings of the National Academy of Sciences of the United States of America 108: 6893–6898.
- 33. Thurber CS, Ma JM, Higgins RH, Brown PJ (2013) Retrospective genomic analysis of sorghum adaptation to temperate-zone grain production. Genome Biology 14: 12.
- 34. Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, et al. (2007) TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23: 2633–2635.
- 35. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology 10: R25.
- 36. Gnerre S, MacCallum I, Przybylski D, Ribeiro FJ, Burton JN, et al. (2011) High-quality draft assemblies of mammalian genomes from massively parallel sequence data. Proceedings of the National Academy of Sciences of the United States of America 108: 1513–1518.
- 37. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nature Methods 9: 357–359.
- 38. Wu YH, Bhat PR, Close TJ, Lonardi S (2008) Efficient and accurate construction of genetic linkage maps from the minimum spanning tree of a graph. PLOS Genetics 4: e1000212.
- 39. Liu RH, Meng JL (2003) MapDraw: a Microsoft Excel macro for drawing genetic linkage maps based on given genetic linkage data. Heraditas 25: 317–321.
- 40. Wu Y, Close TJ, Lonardi S (2008) On the accurate construction of consensus genetic maps. Comput Syst Bioinformatics Conf 7: 285–296.
- 41. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. Journal of Molecular Biololgy 215: 403–410.
- 42. Meyer M, Munzner T, Pfister H (2009) MizBee: a multiscale synteny browser. Ieee Transactions on Visualization and Computer Graphics 15: 897–904.
- 43. Choi IY, Hyten DL, Matukumalli LK, Song QJ, Chaky JM, et al. (2007) A soybean transcript map: Gene distribution, haplotype and single-nucleotide polymorphism analysis. Genetics 176: 685–696.
- 44. Hyten DL, Choi IY, Song QJ, Specht JE, Carter TE, et al. (2010) A high density integrated genetic linkage map of soybean and the development of a 1536 universal soy linkage panel for quantitative trait locus mapping. Crop Science 50: 960–968.
- 45. Song QJ, Marek LF, Shoemaker RC, Lark KG, Concibido VC, et al. (2004) A new integrated genetic linkage map of the soybean. Theoretical and Applied Genetics 109: 122–128.
- 46. Cregan PB, Jarvik T, Bush AL, Shoemaker RC, Lark KG, et al. (1999) An integrated genetic linkage map of the soybean genome. Crop Science 39: 1464–1490.
- 47. Lavin M, Herendeen PS, Wojciechowski MF (2005) Evolutionary rates analysis of Leguminosae implicates a rapid diversification of lineages during the tertiary. Systematic Biology 54: 575–594.
- 48. Stefanović S, Pfeil BE, Palmer JD, Doyle JJ (2009) Relationships among phaseoloid legumes based on sequences from eight chloroplast regions. Systematic Botany 34: 115–128.
- 49. McClean PE, Mamidi S, McConnell M, Chikara S, Lee R (2010) Synteny mapping between common bean and soybean reveals extensive blocks of shared loci. BMC Genomics 11: 184 doi10.1186/1471-2164-1111-1184.
- 50. Newell CA, Hymowitz T (1980) A taxonomic revision in the genus Glycine subgenus Glycine (Leguminosae). Brittonia 32: 63–69.
- 51. Sonah H, Bastien M, Iquira E, Tardivel A, Legare G, et al. (2013) An improved genotyping by sequencing (GBS) approach offering increased versatility and efficiency of SNP discovery and genotyping. PLOS ONE 8: e54603.
- 52. Ward JA, Bhangoo J, Fernandez-Fernandez F, Moore P, Swanson JD, et al. (2013) Saturated linkage map construction in Rubus idaeus using genotyping by sequencing and genome-independent imputation. BMC Genomics 14: 2
- 53. Russell J, Hackett C, Hedley P, Liu H, Milne L, et al. (2013) The use of genotyping by sequencing in blackcurrant (Ribes nigrum): developing high-resolution linkage maps in species without reference genome sequences. Molecular Breeding doi:https://doi.org/10.1007/s11032-11013-19996-11038.
- 54. Ma XF, Jensen E, Alexandrov N, Troukhan M, Zhang LP, et al. (2012) High resolution genetic mapping by genome sequencing reveals genome duplication and tetraploid genetic structure of the diploid Miscanthus sinensis. PLOS ONE 7: e33821.
- 55. Graham-Rowe D (2011) Agriculture: Beyond food versus fuel. Nature 474: S6–S8.
- 56. Mahama AA, Deaderick LM, Sadanaga K, Newhouse KE, Palmer RG (1999) Cytogenetic analysis of translocations in soybean. Journal of Heredity 90: 648–653.
- 57. Chung GH, Kim KS (1991) Obtaining intersubgeneric hybridization between Glycine max and Glycine latifolia through embryo culture. Korean Journal of Plant Tissue Culture 18: 39–45.
- 58. Singh RJ, Kollipara KP, Hymowitz T (1993) Backcross (BC2-BC4)-derived fertile plants from Glycine max and Glycine tomentella intersubgeneric hybrids. Crop Science 33: 1002–1007.
- 59. Findley SD, Cannon S, Varala K, Du JC, Ma JX, et al. (2010) A fluorescence in situ hybridization system for karyotyping soybean. Genetics 185: 727–744.
- 60. Hymowitz T (2004) Speciation and cytogenetics. In: Boerma HR, Specht JE, editors. oybeans: Improvement, Production, and Uses. Third ed. Madison, WI: ASA-CSSA-SSSA. pp. 97–136.
- 61. Singh RJ, Hymowitz T (1985) Intra- and interspecific hybridization in the genus Glycine, subgenus Glycine Willd.: chromosome-pairing and genome relationships. Zeitschrift für Pflanzenzuchtung 95: 289–310.
- 62. Singh RJ, Hymowitz T (1985) An intersubgeneric hybrid between Glycine tomentella Hayata and the soybean, Glycine max (L.) Merr. Euphytica 34: 187–192.
- 63. Singh RJ, Kollipara KP, Hymowitz T (1988) Further data on the genomic relationships among wild perennial species (2n = 40) of the genus Glycine Willd. Genome 30: 166–176.
- 64. Doyle JJ, Doyle JL, Grace JP, Brown AHD (1990) Reproductively isolated polyploid races of Glycine tabcina (Leguminosae) had different chloroplast genome donors. Systematic Botany 15: 173–181.
- 65. Singh RJ, Hymowitz T (1985) The genomic relationships among six wild perennial species of the genus Glycine subgenus Glycine Willd. Theoretical and Applied Genetics 71: 221–230.
- 66. Kellogg EA, Bennetzen JL (2004) The evolution of nuclear genome structure in seed plants. American Journal of Botany 91: 1709–1725.
- 67. Lagercrantz U (1998) Comparative mapping between Arabidopsis thaliana and Brassica nigra indicates that Brassica genomes have evolved through extensive genome replication accompanied by chromosome fusions and frequent rearrangements. Genetics 150: 1217–1228.
- 68. Muller J (1981) Fossil pollen records of extant angiosperms. Botanical Review 47: 1–&.
- 69. Koch MA, Kiefer M (2005) Genome evolution among cruciferous plants: A lecture from the comparison of the genetic maps of three diploid species - Capsella rubella, Arabidopsis lyrata subsp Petraea, and A. thaliana. American Journal of Botany 92: 761–767.
- 70. Gill N, Findley S, Walling JG, Hans C, Ma JX, et al. (2009) Molecular and chromosomal evidence for allopolyploidy in soybean. Plant Physiology 151: 1167–1174.
- 71. Dinkins RD, Collins GB (2008) Agrobacterium-mediated genetic transformation of soybean. In: Kirti PD, editor. Handbook of New Technologies for Genetic Improvement of Legumes. Boca Raton, Florida: CRC Press. pp. 89–102.
- 72. Rech EL, Vianna GR, Aragao FJL (2008) High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nature Protocols 3: 410–418.
- 73. Weinthal DM, Taylor RA, Tzfira T (2013) Nonhomologous end joining-mediated gene replacement in plant cells. Plant Physiology 162: 390–400.
- 74. Jones JDG, Witek K, Verweij W, Jupe F, Cooke D, et al. (2014) Elevating crop disease resistance with cloned genes. Philosophical Transactions of the Royal Society B: Biological Sciences 369.
- 75. Kim J, Larkin DM, Cai QL, Asan, Zhang YF, et al. (2013) Reference-assisted chromosome assembly. Proceedings of the National Academy of Sciences of the United States of America 110: 1785–1790.
- 76. Schneeberger K, Ossowski S, Ott F, Klein JD, Wang X, et al. (2011) Reference-guided assembly of four diverse Arabidopsis thaliana genomes. Proceedings of the National Academy of Sciences of the United States of America 108: 10249–10254.