Genome-wide identification and expression analysis of sulphate transporter (<Emphasis Type="Italic">SULTR</Emphasis>) genes under sulfur deficiency in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>

Sulphur is an important mineral element for plant growth and development. It involves in a number of metabolic processes with crucial functions. This study has performed a genome-wide analysis of sulfate transporter (SULTR) genes in Brachypodium distachyon. Ten putative SULTR genes were identified in Brachypodium genome. BdSULTR genes included 6–17 exons encoding a protein of 647–693 residues with basic nature. BdSULTR proteins included both sulfate_transp (PF00916) and STAS (PF01740) domains. BdSULTRs were classified into 4 groups based on the phylogenetic distribution. Promoter regions of all BdSULTR genes, except for BdSULTR3;3 and 3;5 included the SURECOREATSULTR11 elements. A considerable structural overlap was identified between superimposed SULTR1;3 and 3;1 proteins, indicating that SULTR1 members may also involve in plant stress response/tolerance like SULTR3 members. Microarray and RNA-Seq analyses also revealed the differential expression of SULTR 1 and 3 genes under different biotic/abiotic stresses. Protein–protein interaction partners of BdSULTRs were mainly related with adenylyl-sulfate kinases, 5′-adenylylsulfate reductases, ATP sulfurylases, and acyl carrier proteins. Moreover, expression profiles of identified BdSULTR genes under S-deficiency were analyzed using RT-qPCR. It was revealed that BdSULTR1;1 and 3;1 are highly expressed in plant roots as ~tenfold and ~fivefold, respectively, while BdSULTR2 (~15-fold) and 3;1 (~twofold) are abundantly expressed in leaf tissues.     

Rapid and efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis>) employing standard binary vectors and <Emphasis Type="Italic">bar</Emphasis> gene as a selectable marker

Key message

A rapid and efficient Agrobacterium -mediated transformation system in sorghum has been developed employing standard binary vectors and bar gene as a selectable marker.

Abstract

Sorghum (Sorghum bicolor) is an important food and biofuel crop worldwide, for which improvements in genetic transformation are needed to study its biology and facilitate agronomic and commercial improvement. Here, we report optimization of regeneration and transformation of public sorghum genotype P898012 using standard binary vectors and bar gene as a selectable marker. The tissue culture regeneration time frame has been reduced to 7–12 weeks with a yield of over 18 plants per callus, and the optimized transformation system employing Agrobacterium tumefaciens strain AGL1 and the bar with a MAS promoter achieved an average frequency over 14 %. Of randomly analyzed independent transgenic events, 40–50 % carry single copy of integrated T-DNA. Some independent transgenic events were derived from the same embryogenic callus lines, but a 3:1 Mendelian segregation ratio was found in all transgenic events with single copy as estimated by Southern blots. The system described here should facilitate studies of sorghum biology and agronomic improvement.

     

Molecular tagging and validation of microsatellite markers linked to the low germination stimulant gene (<Emphasis Type="Italic">lgs</Emphasis>) for <Emphasis Type="Italic">Striga</Emphasis> resistance in sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench]

Striga is a devastating parasitic weed in Africa and parts of Asia. Low Striga germination stimulant activity, a well-known resistance mechanism in sorghum, is controlled by a single recessive gene (lgs). Molecular markers linked to the lgs gene can accelerate development of Striga-resistant cultivars. Using a high density linkage map constructed with 367 markers (DArT and SSRs) and an in vitro assay for germination stimulant activity towards Striga asiatica in 354 recombinant inbred lines derived from SRN39 (low stimulant) × Shanqui Red (high stimulant), we precisely tagged and mapped the lgs gene on SBI-05 between two tightly linked microsatellite markers SB3344 and SB3352 at a distance of 0.5 and 1.5 cM, respectively. The fine-mapped lgs region was delimited to a 5.8 cM interval with the closest three markers SB3344, SB3346 and SB3343 positioned at 0.5, 0.7 and 0.9 cM, respectively. We validated tightly linked markers in a set of 23 diverse sorghum accessions, most of which were known to be Striga resistant, by genotyping and phenotyping for germination stimulant activity towards both S. asiatica and S. hermonthica. The markers co-segregated with Striga germination stimulant activity in 21 of the 23 tested lines. The lgs locus similarly affected germination stimulant activity for both Striga species. The identified markers would be useful in marker-assisted selection for introgressing this trait into susceptible sorghum cultivars. Examination of the sorghum genome sequence and comparative analysis with the rice genome suggests some candidate genes in the fine-mapped region (400 kb) that may affect strigolactone biosynthesis or exudation. This work should form a foundation for map-based cloning of the lgs gene and aid in elucidation of an exact mechanism for resistance based on low Striga germination stimulant activity.     

Development of marker-free transgenic sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench] using standard binary vectors with <Emphasis Type="Italic">bar</Emphasis> as a selectable marker

We report an Agrobacterium-mediated transformation system that can generate marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] from a public line [P898012] using standard binary vectors with bar as a selectable marker. Eight co-cultivation conditions were examined for their effect on transformation. The average transformation frequencies were 0.4 and 0.7% for pZY101-TC2 and pZY101-SKRS, respectively, derived from binary vector pZY102 and containing bar and target gene(s) in separate T-DNA regions. A low selection pressure (2.5 mg l⁻¹ DL-phosphinothrithin, PPT) was deployed during callus induction in combination with rapid selection to generate plants from 80 independent events, all but three of which were fertile and set seed. PCR and Southern analyses showed that 36 out of 80 events contained both bar and the target gene(s) (an average co-transformation frequency of 45%). Seedlings of the T1 generation transmitted T-DNAs with target gene(s) and bar gene independently, generating a fraction of progeny with only the target gene(s).     

Fine Mapping of <Emphasis Type="Italic">qDor7</Emphasis>, a Major QTL Affecting Seed Dormancy in Sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench)

Seed dormancy is a key domestication trait for major crops, which is acquired in long-term systems development processes and enables the survival of plants in adverse natural conditions. It is a complex trait under polygenic control and is affected by endogenous and environmental factors. In the present study, a major seed dormancy QTL in sorghum (Sorghum bicolor (L.) Moench), qDor7, detected previously, was fine mapped using a large, multi-generational population. The qDor7 locus was delimited to a 96-kb region which contains 16 predicted gene models. These results lay a solid foundation for cloning qDor7. In addition, the functional markers tightly linked to the seed dormancy QTL may be used in marker-assisted selection for seed dormancy in sorghum.     

Genetic dissection of early-season cold tolerance in sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench)

Soil temperatures at 15°C or below limit germination and seedling establishment for warm season cereal crops such as sorghum (Sorghum bicolor (L.) Moench) during early-season planting. To better understand the genetics of early-season cold tolerance in sorghum, mapping of quantitative trait loci (QTL) associated with germination, emergence and vigor using a recombinant inbred mapping population was carried out. A mapping population consisting of 171 F₇–F₈ recombinant inbred lines (RILs) derived from the cross between RTx430 (cold-sensitive) and PI610727 (cold-tolerant) was developed and a genetic map was constructed using 141 microsatellites or simple sequence repeat (SSR) markers. The RILs were evaluated for cold and optimal temperature germinability in the laboratory, field emergence, and seedling vigor in two locations during early-season planting. Two or more QTL were detected for all traits, except for seedling vigor, with only one QTL was detected in the population. A QTL for cold germinability (Germ 12-2.1) showed the highest LOD value and was also associated with optimal germinability. One of the QTL for field emergence, Fearlygerm-9.3, a contribution from PI610727, was found significant in both locations used for the study. This study showed alignment of QTL in SBi1 (Fearlygerm-1.2 and FGerm30-1.2) with previously reported QTL associated with late field emergence identified from a different mapping population. This indicates that PI617027 shares some common loci with other known early-season cold-tolerant sorghum germplasm but also harbors novel QTL that could be useful in introgression of enhanced laboratory germination and early-season field emergence.     

High-density AFLP map of nonbrittle rachis 1 (<Emphasis Type="Italic">btr1</Emphasis>) and 2 (<Emphasis Type="Italic">btr2</Emphasis>) genes in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Wild relatives of barley disperse their seeds at maturity by means of their brittle rachis. In cultivated barley, brittleness of the rachis was lost during domestication. Nonbrittle rachis of occidental barley lines is controlled by a single gene (btr1) on chromosome 3H. However, nonbrittle rachis of oriental barley lines is controlled by a major gene (btr2) on chromosome 3H and two quantitative trait loci on chromosomes 5HL and 7H. This result suggests multiple mutations of the genes involved in the formation of brittle rachis in oriental lines. The btr1 and btr2 loci did not recombine in the mapping population analyzed. This result agrees with the theory of tight linkage between the two loci. A high-density amplified fragment-length polymorphism (AFLP) map of the btr1/btr2 region was constructed, providing an average density of 0.08 cM/locus. A phylogenetic tree based on the AFLPs showed clear separation of occidental and oriental barley lines. Thus, barley consists of at least two lineages as far as revealed by molecular markers linked to nonbrittle rachis genes.Electronic Supplementary Material Supplementary material is available for this article at An erratum to this article can be found at     

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.     

Evaluation of salinity tolerance in sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> L.) using ion accumulation,proline and peroxidase criteria

Salt tolerance of sorghum varieties in terms of fresh weight, ion accumulations, proline content and peroxidase activity was analyzed in this study. Three sorghum varieties, Payam, Kimia, and Jambo, differing in salt tolerance, were grown in a greenhouse-hydroponic culture with a complete nutrition solution to which 0, 50, 100, 150 and 200 mM NaCl was added. Plant roots and leaves were harvested at 15 and 30 days after treatment and subjected to analysis. Clear decline in K⁺ and Ca²⁺ concentrations and increase in Na⁺ and proline contents were observed in the root and leaf tissues at each NaCl concentration in all varieties during the NaCl treatment. The Ca²⁺ concentration in leaves was higher than in roots, and had the following order in the tested cultivars: Jambo, Kimia, and Payam. Total peroxidase activity increased under salinity stress and it was proportional with the salt concentration. Payam had the largest decrease (46.95%) in fresh weight caused by NaCl, while Jambo had the lowest decrease, 28.63%. Linear regression analysis revealed significant relationships between the estimated factors and fresh weight. The profiles of isoperoxidases were modified under stress conditions. Two isoforms, A1 and A2, were detected in all three varieties with different intensities. Under NaCl stress, isoperoxidases were strongly expressed and a third isoform, A3, was specifically found in variety Jambo suggesting that A3 is implicated in salt adaptation of this variety.     

Mannose binding lectin (<Emphasis Type="Italic">MBL</Emphasis>) copy number polymorphism in Zebrafish (<Emphasis Type="Italic">D. rerio</Emphasis>) and identification of haplotypes resistant to <Emphasis Type="Italic">L. anguillarum</Emphasis>

We describe a novel extension of the Genomic Matching Technique (GMT) that defines haplotypes of the mannose binding lectin (MBL) region in Zebrafish (D. rerio). Four ancestral haplotypes have been identified to date, with at least one of these demonstrating a significant increase in resistance to L. anguillarum. MBL activates the lectin pathway of the complement system and stimulates the development of the complement cascade and the Membrane Attack Complex. Polymorphisms in humans have been associated with increased susceptibility and severity to a number of pathogenic organisms. As teleosts have a relatively immature acquired immune system, polymorphisms within MBL and other innate defence genes are likely to be critical in defining their susceptibility/resistance to various pathogenic organisms. We report multiple copies of MBL-like genes in D. rerio, with up to three copies tightly linked within a cluster spanning ∼15 kb on chromosome 2. Genomic analysis suggests that duplication, retroviral insertion and possibly gene mutation and/or deletion have been key factors in the evolution of this cluster. Molecular analysis has revealed extensive polymorphism, including at least five distinct amplicons and haplospecific gene copy number variation. This study demonstrates polymorphism within a critical component of the teleost innate immune system. The polymorphisms and the haplotypes encoding the unique variants are likely to be informative in defining susceptibility/resistance to infectious agents commonly encountered within aquatic environments. Future investigations will define other important haplotypes and transfer the knowledge to other finfish species, thereby enabling selection of broodstock for the aquaculture industry. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome-wide association mapping of resistance to eyespot disease (<Emphasis Type="Italic">Pseudocercosporella herpotrichoides</Emphasis>) in European winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) and fine-mapping of <Emphasis Type="Italic">Pch1</Emphasis>

Key message

Genotypes with recombination events in the Triticum ventricosum introgression on chromosome 7D allowed to fine-map resistance gene Pch1, the main source of eyespot resistance in European winter wheat cultivars.

Abstract

Eyespot (also called Strawbreaker) is a common and serious fungal disease of winter wheat caused by the necrotrophic fungi Oculimacula yallundae and Oculimacula acuformis (former name Pseudocercosporella herpotrichoides). A genome-wide association study (GWAS) for eyespot was performed with 732 microsatellite markers (SSR) and 7761 mapped SNP markers derived from the 90 K iSELECT wheat array using a panel of 168 European winter wheat varieties as well as three spring wheat varieties and phenotypic evaluation of eyespot in field tests in three environments. Best linear unbiased estimations (BLUEs) were calculated across all trials and ranged from 1.20 (most resistant) to 5.73 (most susceptible) with an average value of 4.24 and a heritability of H ² = 0.91. A total of 108 SSR and 235 SNP marker–trait associations (MTAs) were identified by considering associations with a ?log₁₀ (P value) ≥3.0. Significant MTAs for eyespot-score BLUEs were found on chromosomes 1D, 2A, 2D, 3D, 5A, 5D, 6A, 7A and 7D for the SSR markers and chromosomes 1B, 2A, 2B, 2D, 3B and 7D for the SNP markers. For 18 varieties (10.5%), a highly resistant phenotype was detected that was linked to the presence of the resistance gene Pch1 on chromosome 7D. The identification of genotypes with recombination events in the introgressed genomic segment from Triticum ventricosum harboring the Pch1 resistance gene on chromosome 7DL allowed the fine-mapping of this gene using additional SNP markers and a potential candidate gene Traes_7DL_973A33763 coding for a CC-NBS-LRR class protein was identified.

     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

Genetic diversity assessment in Somali sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench) accessions using microsatellite markers

In the north-western region of Somalia, bordering Ethiopia, sorghum represents an important resources for human and animal nutrition. The critical situation of Somalia is threatening the preservation of this valuable resource and it becomes urgent to develop a strategy of correct evaluation of the sorghum germplasm in order to promote conservation and preservation programs. Microsatellites, also known as Simple Sequence Repeats (SSRs), are reproducible molecular markers useful in assessing the level of genetic diversity of plants. A total of 5 sorghum SSR-specific primer pairs were used to assess the genetic diversity of Somali sorghum landraces. Extensive variation was found at the microsatellite loci analysed, except for a locus that resulted in a monomorphic for some accessions. Considerable differences were found between total and effective number of alleles indicating non uniform allele frequency. Moreover allele frequency at a single locus significantly changed among accessions. Total gene diversity calculated for each locus ranged from 0.44 to 0.79. Most of the genetic diversity occurred within accessions demonstrating that accessions are not under selection processes and/or there is a continuous exchange of genes between sorghum populations. In any case, the patterns of clustering were significantly affected by the presence/absence of some alleles with high discriminant weight. Accessions Carabi, Abaadiro, Masego Cas and Masego Cad represent distinct genotypes confirming finding observed in previous phenotypic studies. The results highlight the central role of local farmers in maintaining and shaping local germplasm.     

Arbuscular mycorrhizal fungi-parasite-host interaction for the control of<Emphasis Type="Italic"> Striga hermonthica</Emphasis> (Del.) Benth. in sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench]

Five Glomus species (G. intraradices, G. albidum, G. mosseae, G. fasciculatum, and G. etunicatum) were compared against a check [without arbuscular mycorrhizal (AM) fungi, plus Striga] and control (without AM fungi or Striga) treatments for the control of Striga in a tolerant sorghum variety (War-wara bashi) in an experiment carried out in 12-cm-diameter clay pots. The experiment was carried out in a controlled growth chamber. G. mosseae significantly reduced the number of Striga emerging per plant, increased plant growth, shoot and total dry matter yield of sorghum, did not affect the root dry matter compared with the other AM fungi species, but had a comparable effect to the control treatment. All the AM fungi except G. mosseae, and also the Striga-infested treatment, increased the root:shoot ratio compared to the control treatment. The percent reduction (62%) of Striga emergence after G. mosseae inoculation resulted in about a 30% increase in total dry matter yield of sorghum over the control, while the total loss in dry matter yield of sorghum due to Striga infestation was 36%. Root colonization of sorghum by AM fungi was highest for G. mosseae (44%) followed by G. intraradices (24%) and G. albidum (23%) then G. fasciculatum (18%), with the lowest recorded for G. etunicatum (14%). No colonization of Striga roots was observed. The potential of AM fungi to reduce or to compensate for Striga infestation could be important for soil management, especially in the tropics, and for the reduction of Striga-resistant varieties of sorghum which are mycorrhiza-responsive.     

Identification of quantitative trait loci for resistance to shoot fly in sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench]

The shoot fly is one of the most destructive insect pests of sorghum at the seedling stage. Deployment of cultivars with improved shoot fly resistance would be facilitated by the use of molecular markers linked to QTL. The objective of this study was to dissect the genetic basis of resistance into QTL, using replicated phenotypic data sets obtained from four test environments, and a 162 microsatellite marker-based linkage map constructed using 168 RILs of the cross 296B (susceptible) × IS18551 (resistant). Considering five component traits and four environments, a total of 29 QTL were detected by multiple QTL mapping (MQM) viz., four each for leaf glossiness and seedling vigor, seven for oviposition, six for deadhearts, two for adaxial trichome density and six for abaxial trichome density. The LOD and R ² (%) values of QTL ranged from 2.6 to 15.0 and 5.0 to 33%, respectively. For most of the QTL, IS18551 contributed resistance alleles; however, at six QTL, alleles from 296B also contributed to resistance. QTL of the related component traits were co-localized, suggesting pleiotropy or tight linkage of genes. The new morphological marker Trit for trichome type was associated with the major QTL for component traits of resistance. Interestingly, QTL identified in this study correspond to QTL/genes for insect resistance at the syntenic maize genomic regions, suggesting the conservation of insect resistance loci between these crops. For majority of the QTL, possible candidate genes lie within or very near the ascribed confidence intervals in sorghum. Finally, the QTL identified in the study should provide a foundation for marker-assisted selection (MAS) programs for improving shoot fly resistance in sorghum.