Genetic mapping and development of co-segregating markers of <Emphasis Type="Italic">RpsQ</Emphasis>, which provides resistance to <Emphasis Type="Italic">Phytophthora sojae</Emphasis> in soybean

Key message

The RpsQ Phytophthora resistance locus was finely mapped to a 118-kb region on soybean chromosome 3. A best candidate gene was predicted and three co-segregating gene markers were developed.

Abstract

Phytophthora root rot (PRR), caused by Phytophthora sojae, is a major threat to sustainable soybean production. The use of genetically resistant cultivars is considered the most effective way to control this disease. The Chinese soybean cultivar Qichadou 1 exhibited a broad spectrum resistance, with a distinct resistance phenotype, following inoculation with 36 Chinese P. sojae isolates. Genetic analyses indicated that the disease resistance in Qichadou 1 is controlled by a single dominant gene. This gene locus was designated as RpsQ and mapped to a 118-kb region between BARCSOYSSR_03_0165 and InDel281 on soybean chromosome 3, and co-segregated with Insert11, Insert144 and SNP276. Within this region, there was only one gene Glyma.03g27200 encoding a protein with a typical serine/threonine protein kinase structure, and the expression pattern analysis showed that this gene induced by P. sojae infection, which was suggested as a best candidate gene of RpsQ. Candidate gene specific marker Insert144 was used to distinguish RpsQ from the other known Rps genes on chromosome 3. Identical polymerase chain reaction amplification products were produced for cultivars Qichadou 1 (RpsQ) and Ludou 4 (Rps9). All other cultivars carrying Rps genes on chromosome 3 produced different PCR products, which all lacked a 144-bp fragment present in Qichadou 1 and Ludou 4. The phenotypes of the analyzed cultivars combined with the physical position of the PRR resistance locus, candidate gene analyses, and the candidate gene marker test revealed RpsQ and Rps9 are likely the same gene, and confer resistance to P. sojae.

     

Fine mapping of a <Emphasis Type="Italic">Phytophthora</Emphasis>-resistance gene <Emphasis Type="Italic">RpsWY</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) by high-throughput genome-wide sequencing

Key message

Using a combination of phenotypic screening, genetic and statistical analyses, and high-throughput genome-wide sequencing, we have finely mapped a dominant Phytophthora resistance gene in soybean cultivar Wayao.

Abstract

Phytophthora root rot (PRR) caused by Phytophthora sojae is one of the most important soil-borne diseases in many soybean-production regions in the world. Identification of resistant gene(s) and incorporating them into elite varieties are an effective way for breeding to prevent soybean from being harmed by this disease. Two soybean populations of 191 F₂ individuals and 196 F_7:8 recombinant inbred lines (RILs) were developed to map Rps gene by crossing a susceptible cultivar Huachun 2 with the resistant cultivar Wayao. Genetic analysis of the F₂ population indicated that PRR resistance in Wayao was controlled by a single dominant gene, temporarily named RpsWY, which was mapped on chromosome 3. A high-density genetic linkage bin map was constructed using 3469 recombination bins of the RILs to explore the candidate genes by the high-throughput genome-wide sequencing. The results of genotypic analysis showed that the RpsWY gene was located in bin 401 between 4466230 and 4502773 bp on chromosome 3 through line 71 and 100 of the RILs. Four predicted genes (Glyma03g04350, Glyma03g04360, Glyma03g04370, and Glyma03g04380) were found at the narrowed region of 36.5 kb in bin 401. These results suggest that the high-throughput genome-wide resequencing is an effective method to fine map PRR candidate genes.

     

First report of an atypical new <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> isolates with nucleotide insertion in <Emphasis Type="Italic">aflR</Emphasis> gene resembling to <Emphasis Type="Italic">A. sojae</Emphasis>

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using β-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B₁, B₂, G₁, and G₂ and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153–1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.     

Over-Expression of <Emphasis Type="Italic">PmHSP17.9</Emphasis> in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Confers Thermotolerance

Small heat shock proteins (sHSPs) have been shown to be involved in stress tolerance. However, their functions in Prunus mume under heat treatment are poorly characterized. To improve our understanding of sHSPs, we cloned a sHSP gene, PmHSP17.9, from P. mume. Sequence alignment and phylogenetic analysis indicated that PmHSP17.9 was a member of plant cytosolic class III sHSPs. Besides heat stress, PmHSP17.9 was also upregulated by salt, dehydration, oxidative stresses and ABA treatment. Leaves of transgenic Arabidopsis thaliana that ectopically express PmHSP17.9 accumulated less O₂ ^? and H₂O₂ compared with wild type (WT) after 42 °C treatment for 6 h. Over-expression of PmHSP17.9 in transgenic Arabidopsis enhanced seedling thermotolerance by decreased relative electrolyte leakage and MDA content under heat stress treatment when compared to WT plants. In addition, the induced expression of HSP101, HSFA2, and delta 1-pyrroline-5-carboxylate synthase (P5CS) under heat stress was more pronounced in transgenic plants than in WT plants. These results support the positive role of PmHSP17.9 in response to heat stress treatment.     

Next-generation sequencing to identify candidate genes and develop diagnostic markers for a novel Phytophthora resistance gene, <Emphasis Type="Italic">RpsHC18</Emphasis>, in soybean

Key message

A novel Phytophthora sojae resistance gene RpsHC18 was identified and finely mapped on soybean chromosome 3. Two NBS–LRR candidate genes were identified and two diagnostic markers of RpsHC18 were developed.

Abstract

Phytophthora root rot caused by Phytophthora sojae is a destructive disease of soybean. The most effective disease-control strategy is to deploy resistant cultivars carrying Phytophthora-resistant Rps genes. The soybean cultivar Huachun 18 has a broad and distinct resistance spectrum to 12 P. sojae isolates. Quantitative trait loci sequencing (QTL-seq), based on the whole-genome resequencing (WGRS) of two extreme resistant and susceptible phenotype bulks from an F_2:3 population, was performed, and one 767-kb genomic region with ΔSNP-index ≥ 0.9 on chromosome 3 was identified as the RpsHC18 candidate region in Huachun 18. The candidate region was reduced to a 146-kb region by fine mapping. Nonsynonymous SNP and haplotype analyses were carried out in the 146-kb region among ten soybean genotypes using WGRS. Four specific nonsynonymous SNPs were identified in two nucleotide-binding sites–leucine-rich repeat (NBS–LRR) genes, RpsHC18-NBL1 and RpsHC18-NBL2, which were considered to be the candidate genes. Finally, one specific SNP marker in each candidate gene was successfully developed using a tetra-primer ARMS-PCR assay, and the two markers were verified to be specific for RpsHC18 and to effectively distinguish other known Rps genes. In this study, we applied an integrated genomic-based strategy combining WGRS with traditional genetic mapping to identify RpsHC18 candidate genes and develop diagnostic markers. These results suggest that next-generation sequencing is a precise, rapid and cost-effective way to identify candidate genes and develop diagnostic markers, and it can accelerate Rps gene cloning and marker-assisted selection for breeding of P. sojae-resistant soybean cultivars.

     

Isolation and identification of cultivated bacteria associated with soybeans and their biocontrol activity against <Emphasis Type="Italic">Phytophthora sojae</Emphasis>

One hundred bacteria, isolated from rhizospheric soil and rhizoplane of healthy soybean plants, were assayed for antifungal activity against six Phytophthora sojae isolates. Nine of the tested bacteria inhibited the hyphal growth of P. sojae in vitro. They were subsequently evaluated for their in vitro traits and identified using the 16S rRNA gene sequences. Four of them (Paenibacillus sp.,—S1; Streptomyces sp.,—S9, S10 and S11) were further selected on the basis of their strongest antagonistic activity in vitro against P. sojae race 4, the predominant race in most growing soybean areas in Canada, and tested for their beneficial effects on soybean plants in the greenhouse. Results showed that application of bacterial strain S11 as seed coating reduced the disease severity by 57.1% and increased the root and shoot weight by and 140 and 108% respectively, in comparison to the diseased control. Overall, a positive correlation was recorded between the in vitro and in planta effects of the selected bacteria. This is promising for further application as select environmentally safe biological control agents in the protection of soybean against root rot diseases.     

Over-expression of <Emphasis Type="Italic">JrsHSP17.3</Emphasis> gene from <Emphasis Type="Italic">Juglans regia</Emphasis> confer the tolerance to abnormal temperature and NaCl stresses

Small heat shock proteins (sHSPs) are an HSP subgroup and involved in environmental stress response. In the current study, to understand the role of sHSP protein in a widely distribution nut woody tree, a sHSP gene was cloned from Juglans regia (JrsHSP17.3, GeneBank No.: KT277704). Compared with control condition, the expression of JrsHSP17.3 was induced to 58.1-fold (6 h) in the roots, 86.8-fold in the stems (9 h), 50.9-fold in the leaves (6 h) under 10°C; and was up-regulated to 2.9- ～ 79.9-fold response to 40°C for 3～9 h; meanwhile, it was transcribed to 5.9 - ～39.7-fold under 9 h NaCl treatment, suggesting the potential role of JrsHSP17.3 to cold, heat and NaCl stimulus. Further, JrsHSP17.3 transgenic yeasts showed improved tolerance to freezing, heat and salt stresses compared with control yeast. JrsHSP17.3 was transient over-expressed in J. regia leaves. The leaves non-transgenic (NT) and vector prokII transgenic (empty, PT) were used as control. The expression of JrsHSP17.3 was 81.6-, 125.4-, and 54.2-fold of the control lines under normal conditions, indicating the success over-expression of JrsHSP17.3. Cell damage staining and physiological index determination showed that JrsHSP17.3 transformed lines, NT and PT displayed no obvious difference under control conditions, however, after treated with 16°C, 40°C and NaCl, JrsHSP17.3 transformed lines displayed weaker cell damage, lower level of electrolyte leakages (EL) rate, malondialdehyde (MDA) and H₂O₂ content, and higher activities of catalase (CAT), glutathione transferase (GST), superoxide dismutase (SOD) and peroxidase (POD) as well as more accumulation of proline than NT and PT. Meanwhile, NT and PT were similar and showed no significant difference under all conditions. All of these results indicated that JrsHSP17.3 can improve plant tolerance to abnormal temperatures and NaCl stresses, it represents a potential candidate gene for molecular breeding to enhance stress tolerance in plants.     

A <Emphasis Type="Italic">Vitis vinifera</Emphasis> xanthine dehydrogenase gene, <Emphasis Type="Italic">VvXDH</Emphasis>, enhances salinity tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xanthine dehydrogenase (EC1.1.1.204; XDH) plays an important role in purine catabolism that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Long attributed to its role in recycling and remobilization of nitrogen, recently, XDH is implicated in plant stress responses and acclimation, such research efforts, however, have thus far been restricted to Arabidopsis XDH-knockdown/knockout studies. This study, using an ectopic overexpression approach, is expected to provide novel findings. In this study, a XDH gene from Vitis vinifera, named VvXDH, was synthesized and overexpressed in Arabidopsis, the transgenic Arabidopsis showed enhanced salt tolerance. The VvXDH gene was investigated and the results demonstrated the explicit role of VvXDH in conferring salt stress by increasing allantoin accumulation and activating ABA signaling pathway, enhancing ROS scavenging in transgenic Arabidopsis. In addition, the water loss and chlorophyll content loss were reduced in transgenic plants; the transgenic plants showed higher proline level and lower MDA content than that of wild-type Arabidopsis, respectively. In conclusion, the VvXDH gene has the potential to be applied in increasing allantoin accumulation and enhancing the tolerance to abiotic stresses in Arabidopsis and other plants.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Improving tobacco freezing tolerance by co-transfer of stress-inducible <Emphasis Type="Italic">CbCBF</Emphasis> and <Emphasis Type="Italic">CbICE53</Emphasis> genes

Cold stress is one of the major limitations to crop productivity worldwide. We investigated the effects of multiple gene expression from cold tolerant Capsella bursa-pastoris in transgenic tobacco (Nicotiana tabaccum) plants. We combined CblCE53 and CbCBF into a reconstruct vector by isocaudomers. Plant overexpression of CbICE53 under the stress inducible CbCOR15b promoter and CbCBF under a constitutive promoter showed increased tolerance to both chilling and freezing temperatures in comparison to wild-type plants, according to the electrolyte leakage and relative water content. The expressions of endogenous cold-responsive genes in transgenic tobacco (NtDREB1, NtDREB3, NtERD10a and NtERD10b) were obviously upregulated under normal and low temperature conditions. These results suggest that the CbICE53 + CbCBF transgenic plants showed a much greater cold tolerance as well as no dwarfism and delayed flowering. Thus they can be considered as a potential candidate for transgenic engineering for cold tolerant tobacco.     

Cloning <Emphasis Type="Italic">PIP</Emphasis> genes in drought-tolerant vetiver grass and responses of transgenic <Emphasis Type="Italic">VzPIP2;1</Emphasis> soybean plants to water stress

Vetiver grass [Vetiveria zizanioides (L.) Nash] displays comprehensive abiotic stress tolerance closely related to fine maintenance of plant water relation mediated by plasma membrane intrinsic proteins (PIPs). Two open reading frame sequences of PIPs (867 and 873 bp) were cloned from vetiver grass and named as VzPIP1;1 and VzPIP2;1, respectively. Expression of green fluorescent protein revealed only subcellular localization of VzPIP2;1 in the plasma membrane. Agrobacterium tumefaciens mediated transgenic (VzPIP2;1) soybean plants had a higher water content in above-ground parts under sufficient water supply through enhancing transpiration as compared to the non-transgenic plants but displayed a more severe drought injury because of a lower photosynthesis and a higher transpiration rate. However, A. rhizogenes mediated transgenic soybean plants kept a higher water content in above-ground parts by improving root water transport and kept a more effective photosynthesis under normal and drought conditions.     

Overexpression of the ABC transporter gene <Emphasis Type="Italic">TsABCG11</Emphasis> increases cuticle lipids and abiotic stress tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The cuticle, composed primarily of wax and cutin, covers most plant aerial surfaces and plays a vital role in interactions between plants and their environment. Some ATP-binding cassette G subfamily (ABCG) members are involved in cuticular lipid molecule exportation to outside in the plant surface. Thellungiella salsugineum, a relative of Arabidopsis thaliana with a heavy cuticle, has extreme stress tolerance. TsABCG11, an ABCG transporter was cloned (GenBank accession number JQ389853), and its structure was studied. qRT-PCR showed that TsABCG11 expression varied in different organs of T. salsugineum and was upregulated under ABA, NaCl, drought and cold conditions. The rosette leaves from 4-week-old TsABCG11 overexpressed (OE) Arabidopsis plants displayed lower rates of water loss and decreased chlorophyll-extracted rates compared to wild-type plants. TsABCG11-OE plants also exhibited significantly increased total cuticular wax and cutin monomer amounts, mainly due to prominent changes in the C₂₉, C₃₁, and C₃₃ alkanes in the wax and C18:2 dioic in cutin monomers, respectively. TsABCG11-OE seedlings exhibit lower root growth inhibition under 100 mM of NaCl or 1 µM of ABA than the wild type. Four-week-old TsABCG11-OE plants exhibited higher photosynthetic rates and water-use efficiency under cold stress (4 °C) than control plants. These results indicate that TsABCG11 plays an important role in cuticle lipid exportation and is involved in abiotic stresses, probably having a close relationship with extreme stress tolerance in T. salsugineum.     

Overexpression of <Emphasis Type="Italic">AlTMP2</Emphasis> gene from the halophyte grass <Emphasis Type="Italic">Aeluropus littoralis</Emphasis> in transgenic tobacco enhances tolerance to different abiotic stresses by improving membrane stability and deregulating some stress-related genes

Herein, we report isolation of the AlTMP2 gene from the halophytic C4 grass Aeluropus littoralis. The subcellular localization suggested that AlTMP2 is a plasma membrane protein. In A. littoralis exposed to salt and osmotic stresses, the AlTMP2 gene was induced early and at a high rate, but was upregulated relatively later in response to abscisic acid and cold treatments. Expression of AlTMP2 in tobacco conferred improved tolerance against salinity, osmotic, H₂O₂, heat, and freezing stresses at the germination and seedling stages. Under control conditions, no growth or yield penalty were mentioned in transgenic plants due to the constitutive expression of AlTMP2. Interestingly, under greenhouse conditions, the seed yield of transgenic plants was significantly higher than that of non-transgenic (NT) plants grown under salt or drought stress. Furthermore, AlTMP2 plants had less electrolyte leakage, higher membrane stability, and lower Na⁺ and higher K⁺ accumulation than NT plants. Finally, six stress-related genes were shown to be deregulated in AlTMP2 plants relative to NT plants under both control and stress conditions. Collectively, these results indicate that AlTMP2 confers abiotic stress tolerance by improving ion homeostasis and membrane integrity, and by deregulating certain stress-related genes.