Mutation and sequencing-based cloning and functional studies of a rust resistance gene in sunflower (Helianthus annuus).

Rust, caused by the fungus Puccinia helianthi Schwein., is one of the most devastating diseases of sunflower (Helianthus annuus L.), affecting global production. The rust R gene R11 in sunflower line HA-R9 shows broad-spectrum resistance to P. helianthi virulent races and was previously mapped to an interval on sunflower chromosome 13 encompassing three candidate genes annotated in the XRQr1.0 reference genome assembly. In the current study, we combined ethyl methane sulfonate (EMS) mutagenesis with targeted region capture and PacBio long-read sequencing to clone the R11 gene. Sequencing of a 60-kb region spanning the R11 locus from the R11 -HA-R9 rust-resistant line and three EMS-induced susceptible mutants facilitated the identification of R11 and definition of induced mutations. The R11 gene is predicted to have a single 3996-bp open reading frame and encodes a protein of 1331 amino acids with CC-NBS-LRR domains typical of genes conferring plant resistance to biotrophic pathogens. Point mutations identified in the R11 rust-susceptible mutants resulted in premature stop codons, consistent with loss of function leading to rust susceptibility. Additional functional studies using comparative RNA sequencing of the resistant line R11 -HA-R9 and R11 -susceptible mutants revealed substantial differences in gene expression patterns associated with R11 -mediated resistance at 7 days post-inoculation with rust, and uncovered the potential roles of terpenoid biosynthesis and metabolism in sunflower rust resistance.

Genetic Insight into Disease Resistance Gene Clusters by Using Sequencing-Based Fine Mapping in Sunflower (Helianthus annuus L.).

Rust and downy mildew (DM) are two important sunflower diseases that lead to significant yield losses globally. The use of resistant hybrids to control rust and DM in sunflower has a long history. The rust resistance genes, R13a and R16, were previously mapped to a 3.4 Mb region at the lower end of sunflower chromosome 13, while the DM resistance gene, Pl33, was previously mapped to a 4.2 Mb region located at the upper end of chromosome 4. High-resolution fine mapping was conducted using whole genome sequencing of HA-R6 (R13a) and TX16R (R16 and Pl33) and large segregated populations. R13a and R16 were fine mapped to a 0.48 cM region in chromosome 13 corresponding to a 790 kb physical interval on the XRQr1.0 genome assembly. Four disease defense-related genes with nucleotide-binding leucine-rich repeat (NLR) motifs were found in this region from XRQr1.0 gene annotation as candidate genes for R13a and R16. Pl33 was fine mapped to a 0.04 cM region in chromosome 4 corresponding to a 63 kb physical interval. One NLR gene, HanXRQChr04g0095641, was predicted as the candidate gene for Pl33. The diagnostic SNP markers developed for each gene in the current study will facilitate marker-assisted selections of resistance genes in sunflower breeding programs.

Map and sequence-based chromosome walking towards cloning of the male fertility restoration gene Rf5 linked to R11 in sunflower.

The nuclear fertility restorer gene Rf5 in HA-R9, originating from the wild sunflower species Helianthus annuus, is able to restore the widely used PET1 cytoplasmic male sterility in sunflowers. Previous mapping placed Rf5 at an interval of 5.8 cM on sunflower chromosome 13, distal to a rust resistance gene R11 at a 1.6 cM genetic distance in an SSR map. In the present study, publicly available SNP markers were further mapped around Rf5 and R11 using 192 F2 individuals, reducing the Rf5 interval from 5.8 to 0.8 cM. Additional SNP markers were developed in the target region of the two genes from the whole-genome resequencing of HA-R9, a donor line carrying Rf5 and R11. Fine mapping using 3517 F3 individuals placed Rf5 at a 0.00071 cM interval and the gene co-segregated with SNP marker S13_216392091. Similarly, fine mapping performed using 8795 F3 individuals mapped R11 at an interval of 0.00210 cM, co-segregating with two SNP markers, S13_225290789 and C13_181790141. Sequence analysis identified Rf5 as a pentatricopeptide repeat-encoding gene. The high-density map and diagnostic SNP markers developed in this study will accelerate the use of Rf5 and R11 in sunflower breeding.

High-Density Mapping and Candidate Gene Analysis of Pl18 and Pl20 in Sunflower by Whole-Genome Resequencing.

Downy mildew (DM) is one of the severe biotic threats to sunflower production worldwide. The inciting pathogen, Plasmopara halstedii, could overwinter in the field for years, creating a persistent threat to sunflower. The dominant genes Pl18 and Pl20 conferring resistance to known DM races have been previously mapped to 1.5 and 1.8 cM intervals on sunflower chromosomes 2 and 8, respectively. Utilizing a whole-genome resequencing strategy combined with reference sequence-based chromosome walking and high-density mapping in the present study, Pl18 was placed in a 0.7 cM interval on chromosome 2. A candidate gene HanXRQChr02g0048181 for Pl18 was identified from the XRQ reference genome and predicted to encode a protein with typical NLR domains for disease resistance. The Pl20 gene was placed in a 0.2 cM interval on chromosome 8. The putative gene with the NLR domain for Pl20, HanXRQChr08g0210051, was identified within the Pl20 interval. SNP markers closely linked to Pl18 and Pl20 were evaluated with 96 diverse sunflower lines, and a total of 13 diagnostic markers for Pl18 and four for Pl20 were identified. These markers will facilitate to transfer these new genes to elite sunflower lines and to pyramid these genes with broad-spectrum DM resistance in sunflower breeding.

Genetic Dissection of Phomopsis Stem Canker Resistance in Cultivated Sunflower Using High Density SNP Linkage Map.

Phomopsis stem canker (PSC) caused by Diaporthe helianthi is increasingly becoming a global threat for sunflower production. In this study, the genetic basis of PSC resistance was investigated in a recombinant inbred line (RIL) population developed from a cross between HA 89 (susceptible) and HA-R3 (resistant). The RIL population was evaluated for PSC disease incidence (DI) in seven screening trials at multiple locations during 2016-2018. The distribution of PSC DI in the RIL population was continuous, confirming a polygenic inheritance of the trait. A moderately high broad-sense heritability (H2, 0.76) was estimated for the trait across environments. In the combined analysis, both the genotype and the genotype × environment interactions were highly significant. A linkage map spanning 1505.33 cM was constructed using genotyping-by-sequencing derived markers. Marker-trait association analysis identified a total of 15 quantitative trait loci (QTL) associated with PSC resistance on 11 sunflower chromosomes, each explaining between 5.24 and 17.39% of the phenotypic variation. PSC resistance QTL were detected in two genomic regions each on chromosomes 3, 5, 13, and 17, while one QTL each was detected in the remaining seven chromosomes. Tightly linked single nucleotide polymorphism (SNP) markers flanking the PSC resistance QTL will facilitate marker-assisted selection in PSC resistance sunflower breeding.

Marker-Assisted Gene Pyramiding and the Reliability of Using SNP Markers Located in the Recombination Suppressed Regions of Sunflower (Helianthus annuus L.).

Rust caused by the fungus Puccinia helianthi and downy mildew (DM) caused by the obligate pathogen Plasmopara halstedii are two of the most globally important sunflower diseases. Resistance to rust and DM is controlled by race-specific single dominant genes. The present study aimed at pyramiding rust resistance genes combined with a DM resistance gene, using molecular markers. Four rust resistant lines, HA-R3 (carrying the R4 gene), HA-R2 (R5), HA-R8 (R15), and RHA 397 (R13b), were each crossed with a common line, RHA 464, carrying a rust gene R12 and a DM gene PlArg. An additional cross was made between HA-R8 and RHA 397. Co-dominant simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers linked to the target genes were used to discriminate between homozygotes and heterozygotes in F2 populations. Five pyramids with different combinations of rust resistance genes were selected in the homozygous condition through marker-assisted selection, and three of them were combined with a DM resistance gene PlArg: R4/R12/PlArg, R5/R12/PlArg, R13b/R12/PlArg, R15/R12, and R13b/R15. The pyramiding lines with the stacking of two rust and one DM genes were resistant to all known races of North American sunflower rust and all known races of the pathogen causing DM, potentially providing multiple and durable resistance to both rust and DM. A cluster of 12 SNP markers spanning a region of 34.5 Mb on chromosome 1, which co-segregate with PlArg, were tested in four populations. Use of those markers, located in a recombination suppressed region in marker selection, is discussed.

Molecular dissection of resistance gene cluster and candidate gene identification of Pl17 and Pl19 in sunflower by whole-genome resequencing.

Sunflower (Helianthus annuus L.) production is challenged by different biotic and abiotic stresses, among which downy mildew (DM) is a severe biotic stress that is detrimental to sunflower yield and quality in many sunflower-growing regions worldwide. Resistance against its infestation in sunflower is commonly regulated by single dominant genes. Pl17 and Pl19 are two broad-spectrum DM resistance genes that have been previously mapped to a gene cluster spanning a 3.2 Mb region at the upper end of sunflower chromosome 4. Using a whole-genome resequencing approach combined with a reference sequence-based chromosome walking strategy and high-density mapping populations, we narrowed down Pl17 to a 15-kb region flanked by SNP markers C4_5711524 and SPB0001. A prospective candidate gene HanXRQChr04g0095641 for Pl17 was identified, encoding a typical TNL resistance gene protein. Pl19 was delimited to a 35-kb region and was approximately 1 Mb away from Pl17, flanked by SNP markers C4_6676629 and C4_6711381. The only gene present within the delineated Pl19 locus in the reference genome, HanXRQChr04g0095951, was predicted to encode an RNA methyltransferase family protein. Six and eight SNP markers diagnostic for Pl17 and Pl19, respectively, were identified upon evaluation of 96 diverse sunflower lines, providing a very useful tool for marker-assisted selection in sunflower breeding programs.

Bioinspired Metamaterials: Multibands Electromagnetic Wave Adaptability and Hydrophobic Characteristics.

Although various photonic devices inspired by natural materials have been developed, there is no research focusing on multibands adaptability, which is not conducive to the advancement of materials science. Herein, inspired by the moth eye surface model, state-of-the-art hierarchical metamaterials (MMs) used as tunable devices in multispectral electromagnetic-waves (EMWs) frequency range, from microwave to ultraviolet (UV), are designed and prepared. Experimentally, the robust broad bandwidth of microwave absorption greater than 90% (reflection loss (RL) < -10 dB) covering almost entire X and Ku bands (8.04-17.88 GHz) under a deep sub-wavelength thickness (1 mm) is demonstrated. The infrared emissivity is reduced and does not affect the microwave absorption simultaneously, further realizing anti-reflection and camouflage via the strong visible light scattering by the microstructure, and can prevent degradation by reducing the transmittance to less than 10% over the whole near UV band, as well as having hydrophobic abilities. The mechanism explored via simulation model is that topological effects are found in the bio-structure. This discovery points to a pathway for using natural models to overcome physical limits of MMs and has promising prospect in novel photonic materials.

Linkage Mapping and Genome-Wide Association Studies of the Rf Gene Cluster in Sunflower (Helianthus annuus L.) and Their Distribution in World Sunflower Collections.

Commercial hybrid seed production in sunflower currently relies on a single cytoplasmic male sterility (CMS) source, PET1 and the major fertility restoration gene, Rf1, leaving the crop highly vulnerable to issues with genetic bottlenecks. Therefore, having multiple CMS/Rf systems is important for sustainable sunflower production. Here, we report the identification of a new fertility restoration gene, Rf7, which is tightly linked to a new downy mildew (DM) resistance gene, Pl34 , in the USDA sunflower inbred line, RHA 428. The Rf7 gene was genetically mapped to an interval of 0.6 cM on the lower end of linkage group (LG) 13, while Pl34 was mapped 2.1 cM proximal to the Rf7. Both the genes are located in a cluster of Rf and Pl genes. To gain further insights into the distribution of Rf genes in the sunflower breeding lines, we used a genome-wide association study (GWAS) approach to identify markers associated with the fertility restoration trait in a panel of 333 sunflower lines genotyped with 8,723 single nucleotide polymorphism (SNP) markers. Twenty-four SNP markers on the lower end of LG13 spanning a genomic region of 2.47 cM were significantly associated with the trait. The significant markers were surveyed in a world collection panel of 548 sunflower lines and validated to be associated with the Rf1 gene. The SNP haplotypes for the Rf1 gene are different from Rf5 and the Rf7gene located in the Rf gene cluster on LG13. The SNP and SSR markers tightly flanking the Rf7 gene and the Pl34 gene would benefit the sunflower breeders in facilitating marker assisted selection (MAS) of Rf and Pl genes.

Cloning and characterization of the homoeologous genes for the Rec8-like meiotic cohesin in polyploid wheat.

BACKGROUND: Meiosis is a specialized cell division critical for gamete production in the sexual reproduction of eukaryotes. It ensures genome integrity and generates genetic variability as well. The Rec8-like cohesin is a cohesion protein essential for orderly chromosome segregation in meiotic cell division. The Rec8-like genes and cohesins have been cloned and characterized in diploid models, but not in polyploids. The present study aimed to clone the homoeologous genes (homoeoalleles) for Rec8-like cohesin in polyploid wheat, an important food crop for humans, and to characterize their structure and function under a polyploid condition. RESULTS: We cloned two Rec8-like homoeoalleles from tetraploid wheat (TtRec8-A1 and TtRec8-B1) and one from hexaploid wheat (TaRec8-D1), and performed expression and functional analyses of the homoeoalleles. Also, we identified other two Rec8 homoeoalleles in hexaploid wheat (TaRec8-A1 and TaRec8-B1) and the one in Aegilops tauschii (AetRec8-D1) by referencing the DNA sequences of the Rec8 homoeoalleles cloned in this study. The coding DNA sequences (CDS) of these six Rec8 homoeoalleles are all 1,827 bp in length, encoding 608 amino acids. They differed from each other primarily in introns although single nucleotide polymorphisms were detected in CDS. Substantial difference was observed between the homoeoalleles from the subgenome B (TtRec8-B1 and TaRec8-B1) and those from the subgenomes A and D (TtRec8-A1, TaRec8-A1, and TaRec8-D1). TtRec8-A1 expressed dominantly over TtRec8-B1, but comparably to TaRec8-D1, in polyploid wheat. In addition, we developed the antibody against wheat Rec8 and used the antibody to detect Rec8 cohesin in the Western blotting and subcellular localization analyses. CONCLUSIONS: The Rec8 homoeoalleles from the subgenomes A and D are transcriptionally more active than the one from the subgenome B in polyploid wheat. The structural variation and differential expression of the Rec8 homoeoalleles indicate a unique cross-genome coordination of the homoeologous genes in polyploid wheat, and imply the distinction of the wheat subgenome B from the subgenomes A and D in the origin and evolution.

Molecular cytogenetic and genomic analyses reveal new insights into the origin of the wheat B genome.

KEY MESSAGE: This work pinpointed the goatgrass chromosomal segment in the wheat B genome using modern cytogenetic and genomic technologies, and provided novel insights into the origin of the wheat B genome. Wheat is a typical allopolyploid with three homoeologous subgenomes (A, B, and D). The donors of the subgenomes A and D had been identified, but not for the subgenome B. The goatgrass Aegilops speltoides (genome SS) has been controversially considered a possible candidate for the donor of the wheat B genome. However, the relationship of the Ae. speltoides S genome with the wheat B genome remains largely obscure. The present study assessed the homology of the B and S genomes using an integrative cytogenetic and genomic approach, and revealed the contribution of Ae. speltoides to the origin of the wheat B genome. We discovered noticeable homology between wheat chromosome 1B and Ae. speltoides chromosome 1S, but not between other chromosomes in the B and S genomes. An Ae. speltoides-originated segment spanning a genomic region of approximately 10.46 Mb was detected on the long arm of wheat chromosome 1B (1BL). The Ae. speltoides-originated segment on 1BL was found to co-evolve with the rest of the B genome. Evidently, Ae. speltoides had been involved in the origin of the wheat B genome, but should not be considered an exclusive donor of this genome. The wheat B genome might have a polyphyletic origin with multiple ancestors involved, including Ae. speltoides. These novel findings will facilitate genome studies in wheat and other polyploids.

A new mechanism for improving electromagnetic properties based on tunable crystallographic structures of FeCoNiSi x Al0.4 high entropy alloy powders.

Mechanical grinding method was employed to prepare FeCoNiSi x Al0.4 high entropy alloy powders, which present a simple solid solution structure (FCC and BCC). After annealing at 673 K, a large amount of BCC phase precipitate and a small amount of CoFe2O4 phase generate. The change of crystal structure may lead to an increase in M s (from 100.3 emu g-1 to 124.2 emu g-1) and a decrease in H c (from 107 Oe to 59.5 Oe for FeCoNiSi0.3Al0.4). The silica content has a significant effect on the electromagnetic parameters of the as-milled and as-annealed alloy powders, presenting the trend of first increase and then decrease. And the dielectric constant is obviously improved after annealing (e.g. from 8.48 to 11.21 and from 0.15 to 2.84 for the ε' and ε'' of FeCoNiSi0.3Al0.4 at 18 GHz, respectively), while the permeability is reduced. Compared with those of the as-milled samples, the µ' of as-annealed FeCoNiSi x Al0.4 (x = 0.1, 0.3, 0.4) remain unchanged or even increase due to the formation of CoFe2O4. Meanwhile, the relative content of the precipitated BCC to FCC for FeCoNiSi0.3Al0.4 enhance with the annealing temperature increase from 573 K to 773 K, and then reduce. And the ε' and µ' at 2 GHz present the same trend as the content ratio (A BCC/A FCC), while the ε'' improve obviously after annealing, corresponding to the elevation of conductivity.

SNP Discovery and QTL Mapping of Sclerotinia Basal Stalk Rot Resistance in Sunflower using Genotyping-by-Sequencing.

Basal stalk rot (BSR), caused by the ascomycete fungus (Lib.) de Bary, is a serious disease of sunflower ( L.) in the cool and humid production areas of the world. Quantitative trait loci (QTL) for BSR resistance were identified in a sunflower recombinant inbred line (RIL) population derived from the cross HA 441 × RHA 439. A genotyping-by-sequencing (GBS) approach was adapted to discover single nucleotide polymorphism (SNP) markers. A genetic linkage map was developed comprised of 1053 SNP markers on 17 linkage groups (LGs) spanning 1401.36 cM. The RILs were tested in five environments (locations and years) for resistance to BSR. Quantitative trait loci were identified in each environment separately and also with integrated data across environments. A total of six QTL were identified in all five environments: one of each on LGs 4, 9, 10, 11, 16, and 17. The most significant QTL, and , were identified at multiple environments on LGs 10 and 17, explaining 31.6 and 20.2% of the observed phenotypic variance, respectively. The remaining four QTL, , , , and , were detected in only one environment on LGs 4, 9, 11, and 16, respectively. Each of these QTL explains between 6.4 and 10.5% of the observed phenotypic variation in the RIL population. Alleles conferring increased resistance were contributed by both parents. The potential of the and in marker-assisted selection (MAS) breeding are discussed.

Combination use of platelets and recombinant activated factor VII for increased hemostasis during acute type a dissection operations.

BACKGROUND: Refractory blood loss is a common problem in surgeries for acute type A aortic dissections. Significant evidence has supported the benefit of using recombinant activated factor VII (rFVIIa) to control of intractable bleeding in patients after cardiac surgery. In this prospective clinical study, we present a novel method to achieve intraoperative hemostasis by using a combination of platelets and rFVIIa during operations for acute type A aortic dissections. METHODS: Between May 2009 and August 2012, 71 patients with acute type A dissections who underwent emergency surgery were prospectively included and allocated to one of the following two intervention groups for hemostasis: 3 units platelets combined with 2.4 mg rFVIIa (n = 25), and conventional methods (n = 46). RESULTS: The patients who received the combination of platelets and rFVIIa required fewer transfusions of red blood cells (6.2 ± 3.1 units vs 9.8 ± 2.8 units; p < 0.05), fresh frozen plasma (736.9 ± 178.3 ml vs 1264.3 ± 245.2 ml, p < 0.05), platelet concentrates (3 units vs 5.0 ± 1.8 units, p < 0.001), and cryoprecipitate (2.8 ± 0.9 units vs 8.2 ± 2.3 units, p < 0.05). These patients also required less time for sternal closure (76.9 ± 17.2 min vs 102.3 ± 10.7 min, p < 0.05) compared with the conventional therapy patients. There was no statistically significant difference in the incidence of serious adverse events between these two groups. CONCLUSIONS: Using a combination of platelets and rFVIIa is an effective strategy for achieving hemostasis during acute type A dissection surgery. This hemostatic strategy does not appear to be associated with an increase in postoperative adverse events.

Cloning and characterization of cotton GhBG gene encoding beta-glucosidase.

Beta-1,4-glucosidase (BG, EC3.2.1.21), one of three cellulases, is a widespread family of enzymes involved in the metabolism of cell wall polysaccharides in both prokaryocytes and eukaryotes. Here, we report the isolation of a full-length cDNA encoding beta-1,4-glucosidase protein (designated as GhBG) and its putative function in the process of fiber development and in yeast. Through random sequencing of the cotton fiber cDNA library from 7235 germplasm line, with elite fiber quality in Gossypium hirsutum L. and utilizing the 5' rapid amplification of cDNA ends (RACE) technique, a 2133 bp cDNA clone encoding a cotton fiber specifically expressed protein (accession number: DQ103699) was isolated. GhBG was composed of a 1884 bp open reading frame (ORF) encoding 627 amino acid residues. This putative protein had an isoelectric point of 8.17, a calculated molecular weight of 68.78 KD and a signal peptide with 23 amino acid residues at the N-terminal. RT-PCR analysis indicated GhBG was specifically expressed in fiber cells and was highly abundant in 5-17 day post anthesis (DPA). It was not, however, expressed in root, hypocotyls or leaves. Southern blotting analysis showed there were two copies of GhBG in the upland cotton genome; most likely contained in sub-genome A and sub-genome D. GhBG was then integrated into a yeast expression vector, pREP-5N and electro-transformed into fission yeast Schizosaccharomyces pombe Q-01. The results demonstrated that GhBG led to a significant increase in cell length and width and a remarkable decrease of the length/width ratio. Compared to vector control transformants, cells were significantly larger and rounder and their growth velocity was also reduced.