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HS-182 and HS-183 are food-grade soybean lines [Glycine max (L.) Merr.] with distinct seed protein profiles and food processing quality. HS-182 is a 7S β-conglycinin α’ and 11S glycinin A4 null with a high protein concentration of 45.7% and good processing quality. HS-183 is a 7S β-conglycinin α’ and 11S glycinin null with a protein concentration of 42.7% and poor tofu processing quality. They are adapted to areas of southwestern Ontario with 3100 or more crop heat units and have relative maturity groups of 2.5 and 2.4, respectively.

SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

A single point-mutation in GmHMA3 (Glycine max heavy metal-associated ATPase; a wild type allele cloned from a low Cd-accumulated soybean) is closely associated with seed cadmium (Cd) concentration. It is linked to Cd transportation in yeast, and is primarily expressed in the roots of plants. We hypothesized that the function of GmHMA3w in soybean would be akin to that of OsHMA3 in rice, which expresses in the root tonoplast and sequestrates Cd into the root vacuole to reduce Cd translocation to the shoots and limit its accumulation in the seeds. In this study, the transient expression of the GmHMA3w-GFP fusion protein in rice mesophyll protoplasts indicated that the subcellular localization of GmHAM3w was in the endoplasmic reticulum (ER). Overexpression of GmHMA3w increased the Cd concentration in the roots, decreased the Cd concentration in the stems, and did not affect the Cd concentration in the leaves. Additionally, its overexpression did not alter the Cd concentration across the whole plant. These findings indicated that GmHMA3w does not influence the Cd uptake, but limits the translocation of Cd from the roots to the stems. GmHMA3w thus acts in metal transportation. Assessment of the subcellular distribution of Cd indicated that GmHMA3w facilitated transport of Cd from the cell wall fraction to the organelle fraction, and then sequestrated Cd into the root ER, thus limiting its translocation to the stems. Additionally, the results also suggested that the ER constitutes a site of particularly high Cd sensitively in plants.

AAC Big Ben is a high yielding, soybean cyst nematode resistant food-grade soybean [Glycine max (L.) Merr.] cultivar with yellow hilum and acceptable processing quality for foreign and domestic tofu, soymilk, and miso markets. AAC Big Ben was developed at the Agriculture and Agri-Food Canada (AAFC) Harrow Research and Development Centre (Harrow-RDC), Harrow, ON. AAC Big Ben is adapted to areas of southwestern Ontario with 3300 or more crop heat units and has a relative maturity group of 2.3 (MG 2.3).

AAC Wigle is a high yielding, high-protein, large-seeded, soybean cyst nematode resistant food-grade soybean [Glycine max (L.) Merr.] cultivar with yellow hilum and acceptable processing quality for foreign and domestic tofu, soymilk, and miso markets. It was developed at the Agriculture and Agri-Food Canada (AAFC) Harrow Research and Development Centre (Harrow-RDC), Harrow, ON. AAC Wigle is adapted to areas of southwestern Ontario with 3200 or more crop heat units and has a relative maturity of 2.2 (MG 2.2) and protein content of 45.8%.

Endocytosis and endosomal trafficking play essential roles in diverse biological processes including responses to pathogen attack. It is well established that animal viruses enter host cells through receptor-mediated endocytosis for infection. However, the role of endocytosis in plant virus infection still largely remains unknown. Plant dynamin-related proteins 1 (DRP1) and 2 (DRP2) are the large, multidomain GTPases that participate together in endocytosis. Recently, we have discovered that DRP2 is co-opted by Turnip mosaic virus (TuMV) for infection in plants. We report here that DRP1 is also required for TuMV infection. We show that overexpression of DRP1 from Arabidopsis thaliana (AtDRP1A) promotes TuMV infection, and AtDRP1A interacts with several viral proteins including VPg and cylindrical inclusion (CI), which are the essential components of the virus replication complex (VRC). AtDRP1A colocalizes with the VRC in TuMV-infected cells. Transient expression of a dominant negative (DN) mutant of DRP1A disrupts DRP1-dependent endocytosis and supresses TuMV replication. As adaptor protein (AP) complexes mediate cargo selection for endocytosis, we further investigated the requirement of AP in TuMV infection. Our data suggest that the medium unit of the AP2 complex (AP2β) is responsible for recognizing the viral proteins as cargoes for endocytosis, and knockout of AP2β impairs intracellular endosomal trafficking of VPg and CI and inhibits TuMV replication. Collectively, our results demonstrate that DRP1 and AP2β are two proviral host factors of TuMV and shed light into the involvement of endocytosis and endosomal trafficking in plant virus infection.

Rsv1, a single dominant resistance locus in soybean, confers extreme resistance to the majority of Soybean mosaic virus (SMV) strains, but is susceptible to the G7 strain. In Rsv1-genotype soybean, G7 infection provokes a lethal systemic hypersensitive response (LSHR), a delayed host defence response. The Rsv1-mediated LSHR signalling pathway remains largely unknown. In this study, we employed a genome-wide investigation to gain an insight into the molecular interplay between SMV G7 and Rsv1-genotype soybean. Small RNA (sRNA), degradome and transcriptome sequencing analyses were used to identify differentially expressed genes (DEGs) and microRNAs (DEMs) in response to G7 infection. A number of DEGs, DEMs and microRNA targets, and the interaction network of DEMs and their target mRNAs responsive to G7 infection, were identified. Knock-down of one of the identified DEGs, the eukaryotic translation initiation factor 5A (eIF5A), diminished the LSHR and enhanced viral accumulation, suggesting the essential role of eIF5A in the G7-induced, Rsv1-mediated LSHR signalling pathway. This work provides an in-depth genome-wide analysis of high-throughput sequencing data, and identifies multiple genes and microRNA signatures that are associated with the Rsv1-mediated LSHR.

Additional file 5: Table S2. Summary of GFP-positive hairy roots at puncture sites.

SummarySPT6 is a conserved transcription regulator that is generally viewed as an elongation factor. However, emerging evidence show its potential role in the control of transcription initiation at genic and intragenic promoters. Here we first present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNA Polymerase II (RNAPII) occupancy across transcribed genes. Further, we show that SPT6L enrichment is shifted, unexpectedly, from gene body to the transcription starting site (TSS) when its association with RNAPII is disrupted. Finally, we demonstrate that recruitment of SPT6L starts at TSS, and then spreads to the gene body during transcription. These findings refine the mechanisms underlying SPT6L recruitment in transcription and shed light on the role of SPT6L in transcription initiation.

It is well known that animal viruses enter host cells via endocytosis, whereas plant viruses require physical assistance, such as human and insect activities, to penetrate the host cell to establish their infection. In this study, we report that the endocytosis pathway is also involved in virus infection in plants. We show that plant potyviruses recruit endocytosis dynamin-like proteins to support their infection. Depletion of them by knockout of the corresponding genes suppresses virus replication, whereas overexpression of them enhances virus replication and intercellular movement. We also demonstrate that the dynamin-like proteins interact with several viral proteins that are essential for virus replication and cell-to-cell movement. We further show that treatment of a dynamin-specific inhibitor disrupts endocytosis and inhibits virus replication and intercellular movement. Therefore, the dynamin-like proteins are novel host factors of potyviruses. The corresponding genes may be manipulated using advanced biotechnology to control potyviral diseases., Endocytosis and endosomal trafficking regulate the proteins targeted to the plasma membrane and play essential roles in diverse cellular processes, including responses to pathogen attack. Here, we report the identification of Glycine max (soybean) endocytosis dynamin-like protein 5A (GmSDL5A) associated with purified soybean mosaic virus (SMV) virions from soybean using a bottom-up proteomics approach. Knockdown of GmSDL5A and its homologous gene GmSDL12A inhibits SMV infection in soybean. The role of analogous dynamin-like proteins in potyvirus infection was further confirmed and investigated using the Arabidopsis/turnip mosaic virus (TuMV) pathosystem. We demonstrate that dynamin-related proteins 2A and 2B in Arabidopsis thaliana (AtDRP2A, AtDRP2B), homologs of GmSDL5A, are recruited to the virus replication complex (VRC) of TuMV. TuMV infection is inhibited in both A. thaliana drp2a (atdrp2a) and atdrp2b knockout mutants. Overexpression of AtDRP2 promotes TuMV replication and intercellular movement. AtRDP2 interacts with TuMV VPg, CP, CI, and 6K2. Of these viral proteins, VPg, CP, and CI are essential for viral intercellular movement, and 6K2, VPg, and CI are critical components of the VRC. We reveal that VPg and CI are present in the punctate structures labeled by the endocytic tracer FM4-64, suggesting that VPg and CI can be endocytosed. Treatment of plant leaves with a dynamin-specific inhibitor disrupts the delivery of VPg and CI to endocytic structures and suppresses TuMV replication and intercellular movement. Taken together, these data suggest that dynamin-like proteins are novel host factors of potyviruses and that endocytic processes are involved in potyvirus infection. IMPORTANCE It is well known that animal viruses enter host cells via endocytosis, whereas plant viruses require physical assistance, such as human and insect activities, to penetrate the host cell to establish their infection. In this study, we report that the endocytosis pathway is also involved in virus infection in plants. We show that plant potyviruses recruit endocytosis dynamin-like proteins to support their infection. Depletion of them by knockout of the corresponding genes suppresses virus replication, whereas overexpression of them enhances virus replication and intercellular movement. We also demonstrate that the dynamin-like proteins interact with several viral proteins that are essential for virus replication and cell-to-cell movement. We further show that treatment of a dynamin-specific inhibitor disrupts endocytosis and inhibits virus replication and intercellular movement. Therefore, the dynamin-like proteins are novel host factors of potyviruses. The corresponding genes may be manipulated using advanced biotechnology to control potyviral diseases.

The Polycomb Group (PcG) proteins form two protein complexes, PcG Repressive Complex 1 (PRC1) and PRC2, which are key epigenetic regulators in eukaryotes. PRC2 represses gene expression by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3). In Arabidopsis (Arabidopsis thaliana), CURLY LEAF (CLF) and SWINGER (SWN) are two major H3K27 methyltransferases and core components of PRC2, playing essential roles in plant growth and development. Despite their importance, genome‐wide binding profiles of CLF and SWN have not been determined and compared yet. In this study, we generated transgenic lines expressing GFP‐tagged CLF/SWN under their respective native promoters and used them for ChIP‐seq analyses to profile the genome‐wide distributions of CLF and SWN in Arabidopsis seedlings. We also profiled and compared the global H3K27me3 levels in wild‐type (WT) and PcG mutants (clf, swn, and clf swn). Our data show that CLF and SWN bind to almost the same set of genes, except that SWN has a few hundred more targets. Two short DNA sequences, the GAGA‐like and Telo‐box‐like motifs, were found enriched in the CLF and SWN binding regions. The H3K27me3 levels in clf, but not in swn, were markedly reduced compared with WT; and the mark was undetectable in the clf swn double mutant. Further, we profiled the transcriptomes in clf, swn, and clf swn, and compared that with WT. Thus this work provides a useful resource for the plant epigenetics community for dissecting the functions of PRC2 in plant growth and development.

HS-181 is an 11S glycinin null food-grade soybean line [Glycine max (L.) Merr.] with yellow hilum. The absence of 11S subunits would result in a relatively higher concentration of 7S β-conglycinin in soy food. β-conglycinins suppress food intake, which can help to fight obesity. HS-181 is adapted to areas of southwest Ontario with 3100 or more crop heat units and has a relative maturity of 2.5 (MG 2.5).

SnRK2 are plant-specific serine/threonine kinases that are involved in plant responses to abiotic stresses. In this study, four novel genes SnRK2s:TpSnRK2.11, TpSnRK2.2, TpSnRK2.5, and TpSnRK2.10 from dwarf Polish wheat (Triticum polonicum L.) were characterized and classified into three groups. TpSnRK2.5 and TpSnRK2.11 were members of group 1; TpSnRK2.2 was member of group 2; TpSnRK2.10 belonged to group 3. The expression of TpSNRK2.2 was strongly regulated by polyethylene glycol (PEG), NaCl, and cold in roots and leaves, as well as by ABA in leaves. The transcript of TpSnRK2.5 was intensely induced by all the treatments in roots and leaves. The distinct expression patterns of TpSnRK2.10 indicate that this gene was very sensitive to ABA and NaCl, less sensitive to cold and PEG. The transcript of TpSnRK2.11 was activated significantly by PEG, NaCl, and cold, but weakly by ABA. Our results indicate that these four genes were probably involved in wheat responses to different abiotic stresses in different tissues.

Four soybean storage protein subunit QTLs were mapped using bulked segregant analysis and an F2 population, which were validated with an F5 RIL population. The storage protein globulins β-conglycinin (7S subunit) and glycinin (11S subunits) can affect the quantity and quality of proteins found in soybean seeds and account for more than 70% of the total soybean protein. Manipulating the storage protein subunits to enhance soymeal nutrition and for desirable tofu manufacturing characteristics are two end-use quality goals in soybean breeding programs. To aid in developing soybean cultivars with desired seed composition, an F2 mapping population (n = 448) and an F5 RIL population (n = 180) were developed by crossing high protein cultivar ‘Harovinton’ with the breeding line SQ97-0263_3-1a, which lacks the 7S α′, 11S A1, 11S A2, 11S A3 and 11S A4 subunits. The storage protein composition of each individual in the F2 and F5 populations were profiled using SDS-PAGE. Based on the presence/absence of the subunits, genomic DNA bulks were formed among the F2 plants to identify genomic regions controlling the 7S α′ and 11S protein subunits. By utilizing polymorphic SNPs between the bulks characterized with Illumina SoySNP50K iSelect BeadChips at targeted genomic regions, KASP assays were designed and used to map QTLs causing the loss of the subunits. Soybean storage protein QTLs were identified on Chromosome 3 (11S A1), Chromosome 10 (7S α′ and 11S A4), and Chromosome 13 (11S A3), which were also validated in the F5 RIL population. The results of this research could allow for the deployment of marker-assisted selection for desired storage protein subunits by screening breeding populations using the SNPs linked with the subunits of interest.

HS-172 is a black-seeded food-grade soybean line [Glycine max (L.) Merr.] suitable for use to make douchi or fermented, salt-preserved black soybean. It is adapted to the areas of southwest Ontario with 3100 or more crop heat units and has a relative maturity rating of 2.5.

HS-171 is a large-seeded edamame food-grade soybean genotype [Glycine max. (L.) Merr.] with yellow hilum, low oil, and high protein and sucrose concentrations. It is adapted to the areas of southwest Ontario with 3100 or more crop heat units and has a relative maturity rating of 2.6.

Common bacterial blight (CBB) is a major disease of common bean (Phaseolus vulgaris L.) worldwide. Genetic resistance is the most effective and environmentally safe approach for controlling CBB, and identification of resistance quantitative trait loci (QTL) can improve response to selection when breeding for CBB resistance. Interactions of CBB resistance QTL and pathogen isolates with different levels of aggressiveness were studied using an F 4:5 recombinant inbreed line (RIL) population, derived from a cross between the susceptible cultivar “Sanilac” and the resistant breeding line “OAC 09-3.” Disease phenotyping was performed under field and growth room conditions using multiple bacterial isolates with differential levels of aggressiveness. QTL analysis was performed with 237 molecular markers. The effect of pathogen isolate on the average phenotypic value in the RIL population and the interaction of RILs and the pathogen isolates were highly significant. Two QTL underlying CBB resistance were detected on Pv08 and Pv03. A major QTL (R 2 p between 15 and 56%) was identified in a 5-cM (380 kbp) interval in the distal end of the long arm of Pv08. This genomic region was significantly associated with multiple disease evaluation traits in field and growth room assays and against different isolates of the pathogen, which included the previously known CBB marker SU91. A new QTL on Pv03 (Xa3.3SO), associated with the PvSNP85p745405 allele from the susceptible parent, Sanilac, appeared to be an isolate-specific QTL against the aggressive fuscans isolate ISO118. Interaction between the SU91 and Xa3.3SO QTL resulted in a significant reduction in mean disease severity for almost all disease evaluation traits after plants were challenged with the isolate ISO118. The 7.92 and 7.79% diseased areas in RILs with both QTL, compared with 14.92 and 13.81% in RILs without either in test1 and in test2 quantified by image analysis, showed a 44 and 47% reduction of percent diseased areas, indicating that the two QTL interact to limit the expansion of CBB symptoms after infection by ISO118. The information obtained in this study indicates that while the broad-spectrum SU91 QTL is useful in breeding programs, isolate-specific QTL, such as Xa3.3SO, will aid in breeding bean varieties with enhanced resistance against aggressive regional isolates.

HS-151 is a lipoxygenase free food grade soybean line (Glycine max. L.) with large seed size, high protein content, yellow hilum, and satisfactory food processing quality. It is adapted to areas of southwest Ontario with 3100 or more crop heat units and has a relative maturity of 2.2 (MG 2.2).

Single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels) are valuable molecular markers for genomics and genetics studies and molecular breeding. The advent of next-generation sequencing techniques has enabled researchers to approach high-throughput and cost-effective SNP and InDel discovery on a genomic scale. In this report, 36 common bean genotypes grown in Canada were used to construct reduced representation libraries for next-generation sequencing. Using 76 million sequence reads generated by the Illumina HiSeq 2000 Sequencing System, we identified a total of 43,698 putative SNPs and 1,267 putative InDels. Of the SNPs, 43,504 were bi-allelic and 194 were tri-allelic, and the InDels comprised 574 insertions and 693 deletions. The putative bi-allelic SNPs were distributed across all 11 chromosomes with the highest number of SNPs observed in chromosome 2 (4,788), and the lowest in chromosome 10 (2,941). With the aid of the recent release of the first chromosome-scale version of Phaseolus vulgaris, 24,907 bi-allelic SNPs, 79 tri-allelic SNPs, 315 insertions, and 377 deletions were located in 8,758, 77, 273, and 364 genes, respectively. Among these 24,907 bi-allelic SNPs, 7,168 nonsynonymous bi-allelic SNPs were identified within 36 common bean genotypes that were located in 4,303 genes. A total of 113 putative SNPs were randomly chosen for validation using high-resolution melt analysis. Of the 113 candidate SNPs, 105 (92.9 %) contained the predicted SNPs.

Boersma, J. G., Conner, R. L., Balasubramanian, P. M., Yu, K. and Hou, A. 2013. Marker-assisted dissection of anthracnose resistance in the dry bean cultivar Morden003. Can. J. Plant Sci. 93: 1115–1123. The dry bean cultivar Morden003 is resistant to anthracnose races 73 and 105, the two most prevalent races in western Canada. Previous studies found that Morden003 carried markers OF10530r, SCAreoli and SAS13 that are linked to the Co-1, Co-2 and Co-4 resistance genes on chromosomes Pv01, Pv11 and Pv08, respectively. Morden003 had a reported resistance spectrum similar to three other cultivars that carry the Co-15resistance gene. Using F2and F2:3populations from the reciprocal crosses of Morden003/OAC Rex, we mapped two race-specific resistance gene loci. An examination of known anthracnose resistance and other core markers showed no evidence of resistance being associated with the Co-1, Co-2, or Co-4 loci. Instead, the resistance genes were co-located in the vicinity of the Co-3 locus on Pv04. They were 2 cM apart and flanked by markers SAH181100and BM161. The map generated in this research also showed strong linkage of the anthracnose resistance loci to markers SW12, PVctt001 and SF10, which were associated with the Co-3 and Co-10 loci by previous researchers. A weak, distant linkage of marker SB12 to the Co-3 locus was also detected.

Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli, is an important disease of common bean (Phaseolus vulgaris L.). Genetic resistance is the most economically efficient, environmentally friendly and socially acceptable approach to control plant diseases including CBB. To examine the main and interaction effects of the previously identified CBB resistance quantitative trait loci (QTL) associated with markers BC420 (B) on Pv06, SAP6 (S) on Pv10 and PVctt001 (P) on Pv04, in presence of the major QTL associated with the marker SU91 (Su) on Pv08, a resistant F4:5 recombinant inbred line population of the reciprocal crosses between OAC Rex (bb/ss/PP/SuSu) and HR45 (BB/SS/pp/SuSu) was evaluated under artificial field inoculation in disease nurseries in 2009 and 2010. While, in presence of the CBB QTL on Pv08, the QTL on Pv06 of HR45 accounted for 37–46% of phenotypic variation in the field, the effect of QTL on Pv04 and Pv10 were not significant under field conditions, even in the absence of the Pv06 QTL. Broad-sense heritability estimates of CBB resistance and the QTL associated with BC420 were high for CBB severity and the area under the disease progress curve, promoting the continued efforts in pyramiding the QTL on Pv06 and Pv08 in common background, which provide high levels of resistance.

Navabi A., Rupert, T., Park, S. J, Yu, K., Smith, T. H. and Pauls, K. P. 2013. Apex common bean. Can. J. Plant Sci. 93: 131–135. Apex (CFIA registration no. 7052) is an indeterminate, full-season navy bean (Phaseolus vulgaris L.) cultivar with an upright plant architecture, suitable for direct harvest, high yield potential, moderate resistance to common bacterial blight (CBB; caused by Xanthomonas axonopodis pv. phaseoli) and superior cooking quality. Apex is adapted to and recommended for the dry bean growing areas with higher than 2700 CHU in southwestern Ontario.

HS-162 is a 7S β-conglycinin α′ and 11S glycinin A4 null food-grade soybean (Glycine max L. Merr.) line with high protein concentration, large seed, yellow hilum, and excellent food processing qual...

HS-161 is a 7S β-conglycinin α′ and 11S glycinin A3 null food-grade soybean line (Glycine max (L.) Merr.) with high protein concentration, yellow hilum, and fair food processing quality. It is adap...

Pulse crops are considered minor on a global scale despite their nutritional value for human consumption. Therefore, they are relatively less extensively studied in comparison with the major crops. The need to improve pulse crop production and quality will increase with the increasing global demand for food security and people's awareness of nutritious food. The improvement of pulse crops will require fully utilizing all their genetic resources. Bacterial artificial chromosome (BAC) libraries of pulse crops are essential genomic resources that have the potential to accelerate gene discovery and enhance molecular breeding in these crops. Here, we review the availability, characteristics, applications, and potential applications of the BAC libraries of pulse crops.

Soybean mutant lines that differ in 11S glycinin and 7S β-conglycinin seed storage protein subunit compositions were developed. These proteins have significant influence on tofu quality. The molecular mechanisms underlying the mutant lines are unknown. In this study, gene-specific markers for five of the glycinin genes (Gy1 to Gy5) were developed using three 11S null lines, two A(4) null Japanese cultivars, Enrei and Raiden, and a control cultivar, Harovinton. Whereas gene-specific primers produced the appropriate products in the control cultivar for the Gy1, Gy2, Gy3 and Gy5 genes, they did not amplify in mutants missing the A(1a)B(2), A(2)B(1a), A(1b) B(1b), and A(3)B(4) subunits. However, ecotype targeting induced local lesions in genomes (EcoTILLING) and sequencing analysis revealed that the absence of the A(4) peptide in the mutants is due to the same point mutation as that in Enrei and Raiden. Selection efficiency of the gene-specific primer pairs was tested using a number of breeding lines segregating for the different subunits. Primer pairs specific to each of the Gy1, Gy2, Gy3, and Gy5 genes can be used to detect the presence or absence of amplification in normal or mutant lines. The Gy4 null allele can be selected for by temperature-switch PCR (TS-PCR) for identification of the A(4) (G4) null genotypes. In comparison to protein analysis by SDS-PAGE, gene-specific markers are easier, faster and more accurate for analysis, they do not have to use seed, and can be analyzed at any plant growth stage for marker-assisted selection.

Accurate normalization of gene expression with qRT-PCR depends on the use of appropriate reference genes (RGs) for the species under a given set of experimental conditions. Multiple RGs for gene expression analysis of soybean exposed to heavy metal stress treatment have not been reported in the literature. In this study, we evaluated the expression stability of ten candidate RGs in leaves, roots and stems of two soybean cultivars exposed to cadmium (Cd). Based on the geNorm and NormFinder analysis, ACT3, PP2A, ELF1B and F-box were the most stable RGs in these gene expression studies. In contrast, G6PD, UBC2, TUB, and ELF1A were the most variable ones and should not be used as RGs in these experimental conditions.

The Polycomb Group (PcG) proteins form two protein complexes, PcG Repressive Complex 1 (PRC1) and PRC2, which are key epigenetic regulators in eukaryotes. PRC2 represses gene expression by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3). In Arabidopsis (Arabidopsis thaliana), CURLY LEAF (CLF) and SWINGER (SWN) are two major H3K27 methyltransferases and core components of PRC2, playing essential roles in plant growth and development. Despite their importance, genome-wide binding profiles of CLF and SWN have not been determined and compared yet. In this study, we generated transgenic lines expressing GFP-tagged CLF/SWN under their respective native promoters and used them for ChIP-seq analyses to profile the genome-wide distributions of CLF and SWN in Arabidopsis seedlings. We also profiled and compared the global H3K27me3 levels in wild-type (WT) and PcG mutants (clf, swn, and clf swn). Our data show that CLF and SWN bind to almost the same set of genes, except that SWN has a few hundred more targets. Two short DNA sequences, the GAGA-like and Telo-box-like motifs, were found enriched in the CLF and SWN binding regions. The H3K27me3 levels in clf, but not in swn, were markedly reduced compared with WT; and the mark was undetectable in the clf swn double mutant. Further, we profiled the transcriptomes in clf, swn, and clf swn, and compared that with WT. Thus this work provides a useful resource for the plant epigenetics community for dissecting the functions of PRC2 in plant growth and development.

Phytophthora root rot (PRR) of soybean (Glycine max (L.) Merr.) is the second most important cause of yield loss by disease in North America, surpassed only by soybean cyst nematode (Wrather et al. in Can J Plant Pathol 23:115-121, 2001). Tolerance can provide economically useful disease control, conditioning partial resistance of soybean to PRR. The aims of this study were to identify new quantitative trait loci (QTL) underlying tolerance to PRR, and to evaluate the effects of pyramided or stacked loci on the level of tolerance. A North American cultivar 'Conrad' (tolerant to PRR) was crossed with a northeastern China cultivar 'Hefeng 25' (tolerant to PRR). Through single-seed descent, 140 F2:5 and F2:6 recombinant inbred lines were advanced. A total of 164 simple sequence repeat (SSR) markers were used to construct a genetic linkage map. The percentage of seedling death was measured over 2 years (2007 and 2008) in the field at four naturally infested locations in Canada and China following additional soil infestation and in the greenhouse following inoculation with Phytophthora sojae isolate. A total of eight QTL underlying tolerance to PRR were identified, located in five linkage groups (F, D1b+w, A2, B1, and C2). The phenotypic variation contributed by the loci ranged from 4.24 to 27.98%. QPRR-1 (anchored in the interval of SSR markers Satt325 and Satt343 of LG F), QPRR-2 (anchored in the interval of Satt005 and Satt600 of LG D1b+w), and QPRR-3 (anchored in the interval of Satt579 and Sat_089 of LG D1b+w) derived their beneficial allele from 'Conrad'. They were located at chromosomal locations known to underlie PRR tolerance in diverse germplasm. Five QTL that derived beneficial alleles from 'Hefeng 25' were identified. The QTL (QPRR-1 to QPRR-7) that were detected across at least three environments were selected for loci stacking and to analyze the relationship between number of tolerance loci and disease loss percentage. The accumulation of tolerance loci was positively correlated with decreases in disease loss percentage. The pyramid of loci underlying tolerance to PRR provided germplasm useful for crop improvement by marker-assisted selection and may provide durable cultivar tolerance against the PRR disease.

Common bacterial blight (CBB), incited by Xanthomonas axonopodis pv. phaseoli (Xap), is a serious seed-borne disease of common bean (Phaseolus vulgaris L.) in both temperate and tropical production zones. The line HR45 is highly resistant to Xap infection on leaves and pods in both field and greenhouse. To understand the molecular mechanisms underlying CBB resistance in HR45, cDNA-amplified fragment length polymorphism (AFLP) technique was used to identify the genes that are differentially expressed in the leaves of HR45 at different time-periods after inoculation. Selective amplifications with 34 primer combinations allowed the visualization of 2,448 transcript-derived fragments (TDFs) in infected leaves, and 259 (10.6%) of them were differentially expressed TDFs (DE-TDFs). Seventy-seven of the DE-TDFs were cloned and sequenced. Thirty-nine of the 77 (50.6%) DE-TDFs representing bean transcripts were not previously reported in any EST database. The expression patterns of 10 representative DE-TDFs were further confirmed by real-time RT-PCR. BLAST analysis suggested that 40% (31 of 77) of the DE-TDFs were homologous to the genes related to metabolism, photosynthesis, and cellular transport, whereas 28% (22 of 77) of the DE-TDFs showed homology to the genes involved in defence response, response to stimulus, enzyme regulation, and transcription regulation. Thus, the 22 pathogenesis-related DE-TDFs were selected as potential functional candidate genes (FCGs) in association with CBB resistance. Meanwhile, six of the DE-TDFs (1FCG and five other DE-TDFs) were in silico mapped to the distal region of the bean linkage group B6 (the genomic location containing the major CBB resistance QTL in HR45) and, therefore, were considered as positional candidate genes (PCGs). This study represents a first step towards the discovery of bean genes expressed upon Xap infection. This information will be useful for elucidating the molecular basis of the resistance response process and identifying the genes that underlie the CBB-resistance.

Daily consumption of cadmium (Cd) contaminated foods poses a risk to human health. Cultivar selection is an important method to limit Cd uptake and accumulation; however, analyzing grain Cd concentration is costly and time-consuming. Developing markers for low Cd accumulation will facilitate marker assisted selection (MAS). Inheritance studies using a threshold value of 0.2 mg kg(-1) for low and high and an F(2:3) population showed that low Cd accumulation in soybean seed is under the control of a major gene (Cda1, proposed name) with the allele for low accumulation being dominant. A recombinant inbred line (RIL) population (F(6:8)) derived from the cross AC Hime (high Cd accumulation) and Westag-97 (low Cd accumulation) was used to identify the DNA markers linked to Cda gene(s) or quantitative trait loci (QTLs) controlling low Cd accumulation. We screened 171 simple sequence repeat (SSR) primers that showed polymorphism between parents on the 166 RILs. Of these, 40 primers were newly developed from the soybean genomic DNA sequence. Seven SSR markers, SatK138, SatK139, SatK140 (0.5 cM), SatK147, SacK149, SaatK150 and SattK152 (0.3 cM), were linked to Cda1 in soybean seed. All the linked markers were mapped to the same linkage group (LG) K. The closest flanking SSR markers linked to Cda1 were validated using a parallel population (RILs) involving Leo x Westag-97. Linked markers were also validated with diverse soybean genotypes differing in their seed Cd concentration and showed that SSR markers SatK147, SacK149, and SattK152 clearly differentiated the high and low Cd accumulating genotypes tested. To treat Cd uptake as a quantitative trait, QTL analysis using a linkage map constructed with 161 markers identified a major QTL associated with low Cd concentration in the seeds. The QTL was also mapped to the same location as Cda1 on LG-K. This QTL accounted for 57.3% of the phenotypic variation. Potential candidate genes (genes with known or predicted function that could influence the seed Cd concentration) like protein kinase, putative Adagio-like protein, and plasma membrane H(+)-ATPase were found to be located in the locus of interest. Of the four SSR markers located in the region, SattK152 was localized in the plasma membrane H(+)-ATPase gene. SSR markers closely linked to Cda1 in seeds of soybean were identified and have potential to be used for MAS to develop low Cd accumulating cultivars in a breeding program.

A possible solution to stability problems is to genetically reduce the content of linolenic acid in soybean seed. RG10 is a low linolenic acid line (< 25 g/kg) produced by ethyl methane sulfonate (EMS) treatment of the low linolenic acid EMS mutant line C1640. The objective of this study was to determine the molecular basis of the low linolenic acid trait in RG10. Sequence analyses of mutant RG10 and wild-type OX948 ω-fatty acid desaturase (Fad3) genes showed that the low level of linolenic acid in RG10 is likely a result of mutations in two Fad3 genes. A mutation in the Fad3A gene introduces a stop codon in exon 6 that would prematurely terminate translation and a second mutation in the 5' splice site of intron 5 of the Fad3B gene may result in abnormal mRNA splicing products. Both mutations would result in a non-functional enzyme. Molecular markers developed for these mutations should simplify and accelerate introgression of the RG10-based low linolenic acid trait into elite soybean cultivars.

Previously described random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) markers were used to identify specific genes for anthracnose resistance in 20 dry bean (Phaseolus vulgaris) cultivars and one breeding line. These included six navy bean, five black bean, three kidney bean, two great northern bean, two pinto bean, one cranberry bean, one pink bean and one small red bean cultivars. The reactions of these bean cultivars to five races (i.e., races 23, 31, 73, 81, 105) of Colletotrichum lindemuthianum were used to verify the results of the molecular marker tests. The combined analysis of genotypes estimated with markers and race reactions showed that navy bean cultivar Envoy carried gene Co-12, possibly in combination with other resistance genes, which conferred resistance to all the races except race 105. The bean cultivars Morden003, Messina and CDC Nordic carried Co-15, which provided resistance to only races 73 and 105. Resistance in AC Mariner appeared to be controlled by gene Co-2 either alone or possibly in combination with Co-11. The kidney bean cultivars Chardonnay and ROG 802 carried resistance gene Co-1. Eight other cultivars were resistant to races 23 and 31, which indicated that they carried resistance gene Co-3 or another unidentified resistance gene. Six dry bean cultivars were susceptible to all five anthracnose races. Genotype estimates were based primarily on the virulence pattern of the races on specific cultivars. The results from the molecular tests sometimes supported the proposed genotypes based on the results from the race inoculations, but false positive results often occurred with molecular markers for genes Co-2 and Co-4. Virulence pattern information allowed the detection of specific resistance genes in certain cultivars, but did not always rule out the presence of other genes. Key words: Disease resistance, gene identification, anthracnose (Colletotrichum lindemuthianum), dry bean (Phaseolus vulgaris), molecular marker

Acetyl-coenzyme A (acetyl-CoA) is a central metabolite and the acetyl source for protein acetylation, particularly histone acetylation that promotes gene expression. However, the effect of acetyl-CoA levels on histone acetylation status in plants remains unknown. Here, we show that malfunctioned cytosolic acetyl-CoA carboxylase1 (ACC1) in Arabidopsis leads to elevated levels of acetyl-CoA and promotes histone hyperacetylation predominantly at lysine 27 of histone H3 (H3K27). The increase of H3K27 acetylation (H3K27ac) is dependent on adenosine triphosphate (ATP)-citrate lyase which cleaves citrate to acetyl-CoA in the cytoplasm, and requires histone acetyltransferase GCN5. A comprehensive analysis of the transcriptome and metabolome in combination with the genome-wide H3K27ac profiles of acc1 mutants demonstrate the dynamic changes in H3K27ac, gene transcripts and metabolites occurring in the cell by the increased levels of acetyl-CoA. This study suggests that H3K27ac is an important link between cytosolic acetyl-CoA level and gene expression in response to the dynamic metabolic environments in plants.

Rsv1, a single dominant resistance locus in soybean, confers extreme resistance to the majority of Soybean mosaic virus (SMV) strains, but is susceptible to the G7 strain. In Rsv1‐genotype soybean, G7 infection provokes a lethal systemic hypersensitive response (LSHR), a delayed host defence response. The Rsv1‐mediated LSHR signalling pathway remains largely unknown. In this study, we employed a genome‐wide investigation to gain an insight into the molecular interplay between SMV G7 and Rsv1‐genotype soybean. Small RNA (sRNA), degradome and transcriptome sequencing analyses were used to identify differentially expressed genes (DEGs) and microRNAs (DEMs) in response to G7 infection. A number of DEGs, DEMs and microRNA targets, and the interaction network of DEMs and their target mRNAs responsive to G7 infection, were identified. Knock‐down of one of the identified DEGs, the eukaryotic translation initiation factor 5A (eIF5A), diminished the LSHR and enhanced viral accumulation, suggesting the essential role of eIF5A in the G7‐induced, Rsv1‐mediated LSHR signalling pathway. This work provides an in‐depth genome‐wide analysis of high‐throughput sequencing data, and identifies multiple genes and microRNA signatures that are associated with the Rsv1‐mediated LSHR.

MicroRNAs (miRNAs) play key roles in a variety of cellular processes through regulation of their target gene expression. Accumulated experimental evidence has demonstrated that infections by viruses are associated with the altered expression profile of miRNAs and their mRNA targets in the host. However, the regulatory network of miRNA-mRNA interactions during viral infection remains largely unknown. In this study, we performed small RNA (sRNA)-seq, degradome-seq and as well as a genome-wide transcriptome analysis to profile the global gene and miRNA expression in soybean following infections by three different Soybean mosaic virus (SMV) isolates, L (G2 strain), LRB (G2 strain) and G7 (G7 strain). sRNA-seq analyses revealed a total of 253 soybean miRNAs with a two-fold or greater change in abundance compared with the mock-inoculated control. 125 transcripts were identified as the potential cleavage targets of 105 miRNAs and validated by degradome-seq analyses. Genome-wide transcriptome analysis showed that total 2679 genes are differentially expressed in response to SMV infection including 71 genes predicted as involved in defense response. Finally, complex miRNA-mRNA regulatory networks were derived using the RNAseq, small RNAseq and degradome data. This work represents a comprehensive, global approach to examining virus-host interactions. Genes responsive to SMV infection are identified as are their potential miRNA regulators. Additionally, regulatory changes of the miRNAs themselves are described and the regulatory relationships were supported with degradome data. Taken together these data provide new insights into molecular SMV-soybean interactions and offer candidate miRNAs and their targets for further elucidation of the SMV infection process.

Broad tolerance to phytophthora root rot (PRR) caused by Phytophthora sojae has become an important goal for the improvement of soybean (Glycine max) because of the rapid spread of races that defeat the available resistance genes. The aim of this research was to identify the location of quantitative trait loci (QTL) in ‘Conrad’, a soybean cultivar with broad tolerance to many races of P. sojae. A PRR susceptible breeding line ‘OX760-6-1’was crossed with Conrad. Through single-seed-descent, 112, F2 derived, F7 recombinant inbred lines (RILs) were advanced. A total of 39 random amplified polymorphic DNA bands (RAPDs) and 89 type 1 microsatellite (simple sequence repeat; SSR) markers were used to construct a genetic linkage map. In the greenhouse, RILs were inoculated with four P. sojae isolates (three from China and one from Canada). Disease was measured as the percent of dead plants 20 days after germination in P. sojae inoculated vermiculite in the greenhouse. Three QTLs (QGP1, QGP2, QGP3) for PRR tolerance in the greenhouse were detected using WinQTLCart 2.0 with a log-likelihood (LOD) score 27.14 acquired through permutations (1,000 at P ≤ 0.05). QGP1 (near Satt509) was located at linkage group F and explained 13.2%, 5.9%, and 6.7% of the phenotypic variance for tolerance to the JiXi, JianSanJiang and ShuangYaShan isolates, respectively. QGP2 (near Satt334) was located in a different interval on linkage group F and explained 5.1% and 2.4% of the phenotypic variance for JiXi and ShuangYaShan isolates, respectively. QGP3 was located on linkage group D1b + W (near OPL18800/SCL18659) and explained 10.2% of the phenotypic variance for Woodslee isolate. QGP1 and QGP2 appeared to be associated with PRR tolerance across a range of isolates but QGP3 was active only against the Woodslee isolate. At Woodslee and Weaver (in Ontario) in 2000, the interval associated with QGP3 explained 21.6% and 16.7% of phenotypic variance in resistance to PRR, respectively and was referred as QFP1. The identified QTLs would be beneficial for marker assistant selection of PRR tolerance varieties against both China and North America P. sojae races.

‘Conrad’, a soybean cultivar tolerant to Phytophthora root rot (PRR), and ‘OX760-6-1’, a breeding line with low tolerance to PRR, were crossed. F2 derived recombinant inbred lines were advanced to F6 to generate a population through single-seed descent. This population was used to identify quantitative trait loci (QTLs) influencing PRR tolerance in ‘Conrad’. A total of 99 simple sequence repeat (SSR), or microsatellite, markers that were polymorphic and clearly segregated in the F6 mapping population were used for QTL detection. Based on the data of PRR in the field at two planting locations, Woodslee and Weaver, for the years 2000 and 2001, one putative QTL, designated as Qsatt414-596, was detected using MapMaker/QTL. Qsatt414-596 was flanked by two SSR markers from the linkage group MLG J, Satt414 and Satt596. Satt414 and Satt596 were also detected to be significantly (P

Root rot, caused by Fusarium solani f. sp. phaseoli, is one of the main root diseases impacting production of common beans throughout the world. Because resistance of common beans to root rot is a quantitative trait that is strongly influenced by environmental factors, reproducible methods to screen bean plants for resistance to root rot are critical to the selection process. In this study, we adapted the inoculum layer method (ILM) developed for screening soybeans for resistance to Phytophthora rot and compared it with the traditional liquid inoculum method (LIM) for screening common beans for resistance to Fusarium root rot. In addition, two methods of evaluating resistance using the ILM were compared. The most significant Pearson correlation coefficient between trials involving 80 recombinant inbred lines was achieved with the ILM and counting discoloured vascular bundles in the lower stem (rp = 0.7113***) compared to rating the discoloration on root and hypocotyl (rp = 0.5555***). The traditional (LIM) screening method and rating the discolouration on roots resulted in a non-significant correlation between trials (rp = 0.1084).

Tofu was made, using two coagulants, from soybean lines which lacked specific glycinin and β-conglycinin protein subunits and the quality evaluated to determine the effects of specific protein subunits. The group IIb (A3) glycinin subunit played the major role in contributing to tofu firmness, regardless of coagulant, while the group IIa (A4) subunit had a negative effect on tofu quality in 2002. Soybeans with the group I (A1A2) subunit resulted in tofu with textural properties about one-third higher, expressed as a percent of Harovinton’s values, than tofu prepared from soybeans without the group I subunit. The individual components of group I had contradictory effects on GDL tofu quality in 2002, with the A1 subunit having a negative effect and A2 having a major positive effect. Lack of the α′ subunit of β-conglycinin increased gel hardness relative to the complete 7S protein.

A simple sequence repeat (SSR) marker composed of a tetra nucleotide repeat is tightly linked to a major gene of common bean (Phaseolus vulgaris L.) conferring resistance to common bacterial blight (CBB) incited by Xanthomonas axonopodis pv. phasoli (Xap). This SSR is located in the third intron region of the common bean nitrate reductase (NR) gene, which is mapped to linkage group (LG) H7, corresponding to LG B7 of the bean Core map. Co-segregation analysis between the SSR marker and CBB resistance in a recombinant inbred line (RIL) population demonstrated a tight linkage between the NR gene-specific marker and the major gene for CBB resistance. In total, the marker explained approximately 70% of the phenotypic variation in the population. Because it is co-dominant, this SSR marker should be more efficient for marker-assisted selection (MAS) than dominant/recessive random amplified polymorphic DNA (RAPD) or sequence characterized amplified region (SCAR) markers that have been developed, especially for early generation selection.

The simple sequence repeat (SSR) or microsatellite marker is currently the preferred molecular marker due to its highly desirable properties. The aim of this study was to develop and characterize more SSR markers because the number of SSR markers currently available in tomato is very limited. Five hundred DNA sequences of tomato were searched for SSRs and analyzed for the design of PCR primers. Of the 158 pairs of SSR primers screened against a set of 19 diverse tomato cultivars, 129 pairs produced the expected DNA fragments in their PCR products, and 65 of them were polymorphic with the polymorphism information content (PIC) ranging from 0.09 to 0.67. Among the polymorphic loci, 2-6 SSR alleles were detected for each locus with an average of 2.7 alleles per locus; 49.2% of these loci had two alleles and 33.8% had three alleles. The vast majority (93.8%) of the microsatellite loci contained di- or tri-nucleotide repeats and only 6.2% had tetra- and penta-nucleotide repeats. It was also found that TA/AT was the most frequent type of repeat, and the polymorphism information content (PIC) was positively correlated with the number of repeats. The set of 19 tomato cultivars were clustered based on the banding patterns generated by the 65 polymorphic SSR loci. Since the markers developed in this study are primarily from expressed sequences, they can be used not only for molecular mapping, cultivar identification and marker-assisted selection, but for identifying gene-trait relations in tomato.

Hou, A., Balasubramanian, P., Conner, R.L., Park, S., Yu, K., Kiehn, F.A. and Navabi, A. 2011. Portage common bean. Can. J. Plant Sci. 91: 523–525. Portage (CFIA registration no. 6881) is a high-yielding, early-maturing navy bean (Phaseolus vulgaris L.) cultivar with moderate resistance to common bacterial blight, developed at the Agriculture and Agri-Food Canada, Research Station, Morden, MB, and the Greenhouse and Processing Crops Research Centre at Harrow, ON. Portage is adapted to the dry bean producing regions of Manitoba. Portage has similar cooking and canning quality as the check cultivars.