Identification and molecular mapping of Rps14, a gene conferring broad-spectrum resistance to Phytophthora sojae in soybean.

KEY MESSAGE: A soybean landrace carries broad-spectrum resistance to Phytophthora sojae, which is conferred by a single gene, designated Rps14, on the short arm of chromosome 3. Phytophthora sojae is the causative agent for Phytophthora root and stem rot in soybean [Glycine max (L.) Merr.] and can be managed by deployment of resistance to P. sojae (Rps) genes. PI 340,029 is a soybean landrace carrying broad-spectrum resistance to the pathogen. Analysis of an F2 population derived from a cross between PI 340,029 and a susceptible cultivar 'Williams' reveals that the resistance to P. sojae race 1 is conferred by a single gene, designated Rps14, which was initially mapped to a 4.5-cM region on the short arm of chromosome 3 by bulked segregant analysis (BSA), and subsequently narrowed to a 1.48 cM region corresponding to 229-kb in the Williams 82 reference genome (Wm82 v2.a1), using F3:4 families derived from the F2 population. Further analysis indicates that the broad-spectrum resistance carried by PI 340,029 is fully attributable to Rps14. The genomic sequences corresponding to the defined Rps14 region from a set of diverse soybean varieties exhibit drastic NBS-LRR gene copy number variation, ranging from 3 to 17 copies. Ultimate isolation of Rps14 would be critical for precise selection and deployment of the gene for soybean protection.

RNA sequencing analysis of salt tolerance in soybean (Glycine max).

Salt stress causes foliar chlorosis and scorch, plant stunting, and eventually yield reduction in soybean. There are differential responses, namely tolerance (excluder) and intolerance (includer), among soybean germplasm. However, the genetic and physiological mechanisms for salt tolerance is complex and not clear yet. Based on the results from the screening of the RA-452 x Osage mapping population, two F4:6 lines with extreme responses, most tolerant and most sensitive, were selected for a time-course gene expression study in which the 250 mM NaCl treatment was initially imposed at the V1 stage and continued for 24 h (hrs). Total RNA was isolated from the leaves harvested at 0, 6, 12, 24 h after the initiation of salt treatment, respectively. The RNA-Seq analysis was conducted to compare the salt tolerant genotype with salt sensitive genotype at each time point using RNA-Seq pipeline method. A total of 2374, 998, 1746, and 630 differentially expressed genes (DEGs) between salt-tolerant line and salt-sensitive line, were found at 0, 6, 12, and 24 h, respectively. The expression patterns of 154 common DEGs among all the time points were investigated, of which, six common DEGs were upregulated and seven common DEGs were downregulated in salt-tolerant line. Moreover, 13 common DEGs were dramatically expressed at all the time points. Based on Log2 (fold change) of expression level of salt-tolerant line to salt-sensitive line and gene annotation, Glyma.02G228100, Glyma.03G226000, Glyma.03G031000, Glyma.03G031400, Glyma.04G180300, Glyma.04G180400, Glyma.05 g204600, Glyma.08G189600, Glyma.13G042200, and Glyma.17G173200, were considered to be the key potential genes involving in the salt-tolerance mechanism in the soybean salt-tolerant line.

Innovation of a Regulatory Mechanism Modulating Semi-determinate Stem Growth through Artificial Selection in Soybean.

It has been demonstrated that Terminal Flowering 1 (TFL1) in Arabidopsis and its functional orthologs in other plants specify indeterminate stem growth through their specific expression that represses floral identity genes in shoot apical meristems (SAMs), and that the loss-of-function mutations at these functional counterparts result in the transition of SAMs from the vegetative to reproductive state that is essential for initiation of terminal flowering and thus formation of determinate stems. However, little is known regarding how semi-determinate stems, which produce terminal racemes similar to those observed in determinate plants, are specified in any flowering plants. Here we show that semi-determinacy in soybean is modulated by transcriptional repression of Dt1, the functional ortholog of TFL1, in SAMs. Such repression is fulfilled by recently enabled spatiotemporal expression of Dt2, an ancestral form of the APETALA1/FRUITFULL orthologs, which encodes a MADS-box factor directly binding to the regulatory sequence of Dt1. In addition, Dt2 triggers co-expression of the putative SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (GmSOC1) in SAMs, where GmSOC1 interacts with Dt2, and also directly binds to the Dt1 regulatory sequence. Heterologous expression of Dt2 and Dt1 in determinate (tfl1) Arabidopsis mutants enables creation of semi-determinacy, but the same forms of the two genes in the tfl1 and soc1 background produce indeterminate stems, suggesting that Dt2 and SOC1 both are essential for transcriptional repression of Dt1. Nevertheless, the expression of Dt2 is unable to repress TFL1 in Arabidopsis, further demonstrating the evolutionary novelty of the regulatory mechanism underlying stem growth in soybean.

Dt2 is a gain-of-function MADS-domain factor gene that specifies semideterminacy in soybean.

Similar to Arabidopsis thaliana, the wild soybeans (Glycine soja) and many cultivars exhibit indeterminate stem growth specified by the shoot identity gene Dt1, the functional counterpart of Arabidopsis TERMINAL FLOWER1 (TFL1). Mutations in TFL1 and Dt1 both result in the shoot apical meristem (SAM) switching from vegetative to reproductive state to initiate terminal flowering and thus produce determinate stems. A second soybean gene (Dt2) regulating stem growth was identified, which, in the presence of Dt1, produces semideterminate plants with terminal racemes similar to those observed in determinate plants. Here, we report positional cloning and characterization of Dt2, a dominant MADS domain factor gene classified into the APETALA1/SQUAMOSA (AP1/SQUA) subfamily that includes floral meristem (FM) identity genes AP1, FUL, and CAL in Arabidopsis. Unlike AP1, whose expression is limited to FMs in which the expression of TFL1 is repressed, Dt2 appears to repress the expression of Dt1 in the SAMs to promote early conversion of the SAMs into reproductive inflorescences. Given that Dt2 is not the gene most closely related to AP1 and that semideterminacy is rarely seen in wild soybeans, Dt2 appears to be a recent gain-of-function mutation, which has modified the genetic pathways determining the stem growth habit in soybean.

Identification and molecular mapping of Rps11, a novel gene conferring resistance to Phytophthora sojae in soybean.

KEY MESSAGE: Rps11 confers excellent resistance to predominant Phytophthora sojae isolates capable of defeating major Rps genes deployed into soybean production, representing a novel source of resistance for soybean cultivar enhancement. ABSTRACT: Phytophthora root and stem rot (PRSR), caused by the soil-borne pathogen Phytophthora sojae, is a devastating disease of soybean [Glycine max (L.) Merr.] throughout the world. Deploying resistant soybean cultivars is the most effective and environmentally friendly approach to managing this disease. The soybean landrace PI 594527 was found to carry excellent resistance to all P. sojae isolates examined, some of which were capable of overcoming the major Rps genesp, such as Rps1-k, Rps1-c, and Rps3-a, predominantly used for soybean protection in the past decades. A mapping population consisting of 58 F2 individuals and 209 F2:3 families derived from a cross between PI 594527 and the susceptible cultivar 'Williams' was used to characterize the inheritance pattern of the resistance to P. soja (Rps) in PI 594527. It was found that the resistance was conferred by a single Rps gene, designated Rps11, which was initially defined as an ~5 Mb genomic region at the beginning of chromosome 7 by bulked segregant analysis (BSA) with a nucleotide polymorphism (SNP) chip comprising 7039 SNP markers. Subsequently, simple sequence repeat (SSR) markers in the defined region were used to genotype the F2:3 mapping population to map Rps11 to a 225.3 kb genomic region flanked by SSR markers BARCSOYSSR_07_0286 and BARCSOYSSR_07_0300, according to the soybean reference genome sequence. Particularly, an SSR marker (i.e., BARCSOYSSR_07_0295) was found to tightly co-segregate with Rps11 in the mapping population and can be effectively used for marker-assisted selection of this gene for development of resistant soybean cultivars.

Fine mapping and candidate gene analysis of two loci conferring resistance to Phytophthora sojae in soybean.

KEY MESSAGE: RpsUN1 and RpsUN2 were fine mapped to two genomic regions harboring disease resistance-like genes. The haplotypes and instability of the regions and candidate genes for the two resistance loci were characterized. Phytophthora root and stem rot caused by Phytophthora sojae, is one of the most destructive diseases of soybean. Deploying soybean cultivars carrying race-specific resistance conferred by Rps genes is the most practical approach to managing this disease. Previously, two Rps genes, RpsUN1 and RpsUN2 were identified in a landrace PI 567139B and mapped to a 6.5 cM region on chromosome 3 and a 3.0 cM region on chromosome 16, corresponding to 1387 and 423 kb of the soybean reference genome sequences. By analyzing recombinants defined by genotypic and phenotypic screening of the 826 F2:3 families derived from two reciprocal crosses between the two parental lines, RpsUN1 and RpsUN2, were further narrowed to a 151 kb region that harbors five genes including three disease resistance (R)-like genes, and a 36 kb region that contains four genes including five R-like genes, respectively, according to the reference genome. Expressional changes of these nine genes before and after inoculation with the pathogen, as revealed by RNA-seq, suggest that Glyma.03g034600 in the RpsUN1 region and Glyma.16g215200 and Glyma.16g214900 in the RpsUN2 region of PI 567139B may be associated with the resistance to P. sojae. It is also suggested that unequal recombination between/among R-like genes may have occurred, resulting in the formation of two recombinants with inconsistent genotypic and phenotypic observations. The haplotype variation of genomic regions where RpsUN1 and RpsUN2 reside in the entire soybean germplasm deposited in the US soybean germplasm collection suggests that RpsUN1 and RpsUN2 are most likely novel genes.

Molecular response to the pathogen Phytophthora sojae among ten soybean near isogenic lines revealed by comparative transcriptomics.

BACKGROUND: Phytophthora root and stem rot (PRR) of soybean, caused by Phytophthora sojae, is controlled by Rps genes. However, little is known regarding the Rps-induced molecular responses to P. sojae and how they actually overlap. We thus sequenced, analyzed, and compared the transcriptomes of 10 near isogenic lines (NILs), each with a unique Rps gene/allele, and the susceptible parent Williams, pre- and post-inoculation with the pathogen. RESULTS: A total of 4,330 differentially expressed genes (DEGs) were identified in Williams versus 2,014 to 5,499 DEGs in individual NILs upon inoculation with the pathogen. Comparisons of the DEGs between the NILs and Williams identified incompatible interaction genes (IIGs) and compatible interaction genes (CIGs). Hierarchical cluster and heatmap analyses consistently grouped the NILs into three clusters: Cluster I (Rps1-a), Cluster II (Rps1-b, 1-c and 1-k) and Cluster III (Rps3-a, 3-b, 3-c, 4, 5, and 6), suggesting an overlap in Rps-induced defense signaling among certain NILs. Gene ontology (GO) analysis revealed associations between members of the WRKY family and incompatible reactions and between a number of phytohormone signaling pathways and incompatible/compatible interactions. These associations appear to be distinguished according to the NIL clusters. CONCLUSIONS: This study characterized genes and multiple branches of putative regulatory networks associated with resistance to P. sojae in ten soybean NILs, and depicted functional "fingerprints" of individual Rps-mediated resistance responses through comparative transcriptomic analysis. Of particular interest are dramatic variations of detected DEGs, putatively involved in ethylene (ET)-, jasmonic acid (JA)-, (reactive oxygen species) ROS-, and (MAP-kinase) MAPK- signaling, among these soybean NILs, implicating their important roles of these signaling in differentiating molecular defense responses. We hypothesize that different timing and robustness in defense signaling to the same pathogen may be largely responsible for such variations.

Molecular mapping of two genes conferring resistance to Phytophthora sojae in a soybean landrace PI 567139B.

Phytophthora root and stem rot (PRR), caused by the soil-borne oomycete pathogen Phytophthora sojae, is one of the most destructive diseases of soybean. PRR can be effectively controlled by race-specific genes conferring resistance to P. sojae (Rps). However, the Rps genes are usually non-durable, as populations of P. sojae are highly diverse and quick to adapt, and can be overcome 8-15 years after deployment. Thus, it is important to identify novel Rps genes for development of resistant soybean cultivars. PI 567139B is a soybean landrace carrying excellent resistance to nearly all predominant P. sojae races in Indiana. A mapping population consisting of 245 F2 individuals and 403 F2:3 families was developed from a cross between PI 567139B and the susceptible cultivar 'Williams', and used to dissect the resistance carried by PI 567139B. We found that the resistance in PI 567139B was conferred by two independent Rps genes, designated RpsUN1 and RpsUN2. The former was mapped to a 6.5 cM region between SSR markers Satt159 and BARCSOYSSR_03_0250 that spans the Rps1 locus on chromosome 3, while the latter was mapped to a 3.0 cM region between BARCSOYSSR_16_1275 and Sat_144, approximately 3.0-3.4 cM upstream of Rps2 on chromosome 16. According to the 'Williams 82' reference genome sequence, both regions are highly enriched with NBS-LRR genes. Marker assisted resistance spectrum analyses of these genes with 16 isolates of P. sojae, in combination with the mapping results, suggested that RpsUN1 was likely to be a novel allele at the Rps1 locus, while RpsUN2 was more likely to be a novel Rps gene.