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 Meiotic Genes Rec8 and Cdc5 and Subcellular Analysis of Kinetochore Orientation in Wheat

Meiosis is a specialized cell division that halves chromosomes and generates haploid gametes in eukaryotes. It is a dynamic cellular process governed by a complex genetic network. Two key players of this network, Rec8 and Cdc5, were cloned and analyzed using comparative genomics and subcellular immunolocalization methodologies in wheat (Triticum turgidum L., genome AABB). TtRec8 and TtCdc5 were localized to group 1 and 5 chromosomes, with two homoeoalleles in sub-genome A and B, respectively. One of the two TtRec8 homoeoalleles, TtRec8-A1, contains 20 exons in a 6.5 kb-genomic DNA fragment, and the coding region encodes 608 amino acids. Two homoeoalleles of TtCdc5 separately encode 1,081 and 1,084 amino acids. The expression profilings of TtRec8 and TtCdc5 were meiotic tissue dominant in LDN, and the highest levels of TtRec8 and TtCdc5 were at interphase through early prophase I and at pachytene stage of meiosis, separately, and then decreased as meiosis proceeded. TtRec8 protein was detected along the entire chromosomes through the early stages of prophase I. Thereafter, TtRec8 protein was mostly removed from the chromosomes. The DNA sequences and conserved domains of TtRec8 and TtCdc5 as well as their kinetics through the meiotic process in LDN were very similar as the cohesion protein Rec8 and polo-like kinase Cdc5 in models, suggesting their specific roles in meiosis. Chromosome pairing (or synapsis) may play a role in kinetochore orientation during meiosis. Special genotypes that contained both paired (bivalents) and unpaired (univalent) chromosomes in the LDN background were constructed to determine the orientation of sister kinetochores in the univalent and bivalent chromosomes in meiosis I. Among the special genotypes included the hybrids from the crosses of the disomic LDN D-genome substitution lines LDN 1D(1B), LDN 2D(2A), LDN 2D(2B), LDN 3D(3A), LDN 4D(4B), LDN 6D(6A), LDN 6D(6B), LDN 7D(7A), and LDN 7D(7B) with LDN, LDN 1D(1A) with rye (Secale cereale L., genome RR) `Gazelle', LDN with Aegilops tauschii (genome DD) RL5286, and LDN 1D(1B) with Ae. tauschii RL5286. All univalents were found amphitelically orientated and all bivalents syntelically orientated at metaphase I, suggesting meiotic pairing mediates kinetochore orientation and subsequently chromosome segregation in LDN

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

Tool-life prediction under multi-cycle loading during metal forming: a feasibility study

In the present research, the friction and wear behaviour of a hard coating were studied by using ball-on-disc tests to simulate the wear process of the coated tools for sheet metal forming process. The evolution of the friction coefficient followed a typical dual-plateau pattern, i.e. at the initial stage of sliding, the friction coefficient was relatively low, followed by a sharp increase due to the breakdown of the coatings after a certain number of cyclic dynamic loadings. This phenomenon was caused by the interactive response between the friction and wear from a coating tribo-system, which is often neglected by metal forming researchers, and constant friction coefficient values are normally used in the finite element (FE) simulations to represent the complex tribological nature at the contact interfaces. Meanwhile, most of the current FE simulations consider single-cycle loading processes, whereas many metal-forming operations are conducted in a form of multi-cycle loading. Therefore, a novel friction/wear interactive friction model was developed to, simultaneously, characterise the evolutions of friction coefficient and the remaining thickness of the coating layer, to enable the wear life of coated tooling to be predicted. The friction model was then implemented into the FE simulation of a sheet metal forming process for feasibility study

Tool life prediction under multi-cycle loading conditions: A feasibility study

In the present research, the friction and wear behaviour of a hard coating were studied by using ball-on-disc tests to simulate the wear process of the coated tools for sheet metal forming process. The evolution of the friction coefficient followed a typical dual-plateau pattern, i.e. at the initial stage of sliding, the friction coefficient was relatively low, followed by a sharp increase due to the breakdown of the coatings after a certain number of cyclic dynamic loadings. This phenomenon was caused by the interactive response between the friction and wear from a coating tribo-system, which has not been addressed so far by metal forming researchers, and constant friction coefficient values are normally used in the FE simulations to represent the complex tribological nature at the contact interfaces. Meanwhile, most of the current FE simulations are single cycle, whereas most sheet metal forming operations are conducted as multi-cycle. Therefore, a novel friction/wear interactive friction model was developed to, simultaneously, characterise the evolutions of friction coefficient and the remaining thickness of the coating layer, to enable the wear life of coated tooling to be predicted. The friction model was then implemented into the FE simulation of a sheet metal forming process for feasibility study

Tool life prediction under multi-cycle loading conditions: A feasibility study

In the present research, the friction and wear behaviour of a hard coating were studied by using ball-on-disc tests to simulate the wear process of the coated tools for sheet metal forming process. The evolution of the friction coefficient followed a typical dual-plateau pattern, i.e. at the initial stage of sliding, the friction coefficient was relatively low, followed by a sharp increase due to the breakdown of the coatings after a certain number of cyclic dynamic loadings. This phenomenon was caused by the interactive response between the friction and wear from a coating tribo-system, which has not been addressed so far by metal forming researchers, and constant friction coefficient values are normally used in the FE simulations to represent the complex tribological nature at the contact interfaces. Meanwhile, most of the current FE simulations are single cycle, whereas most sheet metal forming operations are conducted as multi-cycle. Therefore, a novel friction/wear interactive friction model was developed to, simultaneously, characterise the evolutions of friction coefficient and the remaining thickness of the coating layer, to enable the wear life of coated tooling to be predicted. The friction model was then implemented into the FE simulation of a sheet metal forming process for feasibility study

Map and sequence-based chromosome walking towards cloning of the male fertility restoration gene Rf5 linked to R 11 in sunflower

Abstract 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 R 11 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 R 11 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 R 11 . 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 R 11 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 R 11 in sunflower breeding

A real-time non-contact monitoring method of subsea pipelines

To monitoring the subsea pipeline in real-time, a special potentiometric sensor array and a potential prediction model are presented in this paper. Firstly, to measure the potential of seawater, a special potentiometric sensor array with Ag/AgCl all-solid-state reference electrodes is first developed in this paper. Secondly, according to the obtained distribution law of electric field intensities a prediction model of the pipeline potentials is developed. Finally, the potentiometric sensor array is applied in sink experiment and the prediction model is validated by the sink measurements. The maximum error for pipeline potential prediction model is 1.1 mV. The proposed non-contact monitoring method for subsea pipeline can predict the potential of sea pipeline in real-time, thus providing important information for further subsea pipeline maintenance