Identification of <Emphasis Type="Italic">Pm58</Emphasis> from <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

Key message

A novel powdery mildew-resistance gene, designated Pm58, was introgressed directly from Aegilops tauschii to hexaploid wheat, mapped to chromosome 2DS, and confirmed to be effective under field conditions. Selectable KASP? markers were developed for MAS.

Abstract

Powdery mildew caused by Blumeria graminis (DC.) f. sp. tritici (Bgt) remains a significant threat to wheat (Triticum aestivum L.) production. The rapid breakdown of race-specific resistance to Bgt reinforces the need to identify novel sources of resistance. The d-genome species, Aegilops tauschii, is an excellent source of disease resistance that is transferrable to T. aestivum. The powdery mildew-resistant Ae. tauschii accession TA1662 (2n?=?2x?=?DD) was crossed directly with the susceptible hard white wheat line KS05HW14 (2n?=?6x?=?AABBDD) followed by backcrossing to develop a population of 96 BC₂F₄ introgression lines (ILs). Genotyping-by-sequencing was used to develop a genome-wide genetic map that was anchored to the Ae. tauschii reference genome. A detached-leaf Bgt assay was used to screen BC₂F_4:6 ILs, and resistance was found to segregate as a single locus (χ?=?2.0, P value?=?0.157). The resistance gene, referred to as Pm58, mapped to chromosome 2DS. Pm58 was evaluated under field conditions in replicated trials in 2015 and 2016. In both years, a single QTL spanning the Pm58 locus was identified that reduced powdery mildew severity and explained 21% of field variation (P value?<?0.01). KASP? assays were developed from closely linked GBS-SNP markers, a refined genetic map was developed, and four markers that cosegregate with Pm58 were identified. This novel source of powdery mildew-resistance and closely linked genetic markers will support efforts to develop wheat varieties with powdery mildew resistance.

     

<Emphasis Type="Italic">Sr33</Emphasis> and <Emphasis Type="Italic">Sr35</Emphasis> gene homolog identification in genomes of cereals related to <Emphasis Type="Italic">Aegilops tauschii</Emphasis> and <Emphasis Type="Italic">Triticum monococcum</Emphasis>

Using bioinformatics analysis, the homologs of genes Sr33 and Sr35 were identified in the genomes of Triticum aestivum, Hordeum vulgare, and Triticum urartu. It is known that these genes confer resistance to highly virulent wheat stem rust races (Ug99). To identify amino acid sites important for this resistance, the found homologs were compared with the Sr33 and Sr35 protein sequences. It was found that sequences S5DMA6 and E9P785 are the closest homologs of protein RGAle, a Sr33 gene product, and sequences M7YFA9 (CNL-C) and F2E9R2 are homologs of protein CNL9, a Sr35 gene product. It is assumed that the homologs of genes Sr33 and Sr35, which were obtained from the wild relatives of wheat and barley, can confer resistance to various forms of stem rust and can be used in the future breeding programs aimed at improvement of national wheat varieties.     

Fine genetic mapping of greenbug aphid-resistance gene <Emphasis Type="Italic">Gb3</Emphasis> in <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

The greenbug, Schizaphis graminum (Rondani), is an important aphid pest of small grain crops especially wheat (Triticum aestivum L., 2n = 6x = 42, genomes AABBDD) in many parts of the world. The greenbug-resistance gene Gb3 originated from Aegilops tauschii Coss. (2n = 2x = 14, genome D^tD^t) has shown consistent and durable resistance against prevailing greenbug biotypes in wheat fields. We previously mapped Gb3 in a recombination-rich, telomeric bin of wheat chromosome arm 7DL. In this study, high-resolution genetic mapping was carried out using an F_2:3 segregating population derived from two Ae. tauschii accessions, the resistant PI 268210 (original donor of Gb3 in the hexaploid wheat germplasm line ‘Largo’) and susceptible AL8/78. Molecular markers were developed by exploring bin-mapped wheat RFLPs, SSRs, ESTs and the Ae. tauschii physical map (BAC contigs). Wheat EST and Ae. tauschii BAC end sequences located in the deletion bin 7DL3-0.82–1.00 were used to design STS (sequence tagged site) or CAPS (Cleaved Amplified Polymorphic Sequence) markers. Forty-five PCR-based markers were developed and mapped to the chromosomal region spanning the Gb3 locus. The greenbug-resistance gene Gb3 now was delimited in an interval of 1.1 cM by two molecular markers (HI067J6-R and HI009B3-R). This localized high-resolution genetic map with markers closely linked to Gb3 lays a solid foundation for map based cloning of Gb3 and marker-assisted selection of this gene in wheat breeding.     

Genome-wide identification,classification, and expression analysis of the phytocyanin gene family in <Emphasis Type="Italic">Phalaenopsis equestris</Emphasis>

Phytocyanins (PCs) are ancient blue copper-binding proteins in plants that bind to single type I copper atoms and function as electron transporters. PCs play an important role in plant development and stress resistance. Many PCs are considered to be chimeric arabinogalactan proteins (AGPs). Previously, 38, 62, and 84 PC genes were identified in Arabidopsis thaliana, Oryza sativa, and Brassica rapa, respectively. In this study, we identified 30 putative PC genes in the orchid Phalaenopsis equestris through comprehensive bioinformatics analysis. Based on phylogeny and motif constitution, the P. equestris phytocyanins (PePCs) were divided into five subclasses: 10 early nodulin-like proteins, 10 uclacyanin-like proteins, five stellacyanin-like proteins, four plantacyanin-like proteins, and one unknown protein. Structural and glycosylation predictions suggested that 16 PePCs were glycosylphosphatidylinositol-anchored proteins localized to the plasma membrane, 22 PePCs contain N-glycosylation sites, and 14 are chimeric AGPs. Phylogenetic analysis indicated that each subfamily was derived from a common ancestor before the divergence of monocot and dicot lineages and that the expansion of the PC subfamilies occurred after the divergence of orchids and Arabidopsis. The number of exons in PC genes was conserved. Expression analysis in four tissues revealed that nine PC genes were highly expressed in flowers, stems, and roots, suggesting that these genes play important roles in growth and development in P. equestris. The results of this study lay the foundation for further analysis of the functions of this gene family in plants.     

Genetic diversity of <Emphasis Type="Italic">avenin-like b</Emphasis> genes in <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss

Avenin-like storage proteins influence the rheological properties and processing quality in common wheat, and the discovery of new alleles will benefit wheat quality improvement. In this study, 13 avenin-like b alleles (TaALPb7D-A–M) were discovered in 108 Aegilops tauschii Coss. accessions. Ten alleles were reported for the first time, while the remaining three alleles were the same as alleles in other species. A total of 15 nucleotide changes were detected in the 13 alleles, resulting in only 11 amino acid changes because of synonymous mutations. Alleles TaALPb7D-E, TaALPb7D-G, and TaALPb7D-J encoded the same protein. These polymorphic sites existed in the N-terminus, Repetitive region (Left), Repetitive region (Right) and C-terminus domains, with no polymorphisms in the signal peptide sequence nor in those encoding the 18 conserved cysteine residues. Phylogenetic analysis divided the TaALPb7Ds into four clades. The Ae. tauschii alleles were distributed in all four clades, while the alleles derived from common wheat, TaALPb7D-G and TaALPb7D-C, belonged to clade III and IV, respectively. Alleles TaALPb7D-G and TaALPb7D-C were the most widely distributed, being present in nine and six countries, respectively. Iran and Turkey exhibited the highest genetic diversity with respect to TaALPb7D alleles, accessions from these countries carrying seven and six alleles, respectively, which implied that these countries were the centers of origin of the avenin-like b gene. The new alleles discovered and the phylogenetic analysis of avenin-like b genes will provide breeding materials and a theoretical basis for wheat quality improvement.     

Genome-wide identification,characterization, and expression analysis of <Emphasis Type="Italic">SnRK2</Emphasis> family in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis>

The sucrose non-fermenting 1-related protein kinase 2 (SnRK2) gene family belongs to a group of plant-specific serine/threonine kinase family involved in abscisic acid (ABA) signaling and biotic and abiotic stress response. Although genome-wide analyses of the SnRK2 gene family have been conducted in some species, little is known about the SnRK2 gene family in rubber tree (Hevea brasiliensis). In this study, we identified 10 SnRK2s designated as HbSnRK2.1 to HbSnRK2.10 in the rubber tree genome. The subsequently constructed phylogenetic tree demonstrated that HbSnRK2s have three subfamilies that correlate well with those of Arabidopsis sp. and rice subfamilies. All SnRK2 genes contained nine exons and eight introns. Although the C-terminus was divergent, eight conserved motifs were found. Motifs 1–6 were common to all HbSnRK2s. Expression analysis results showed that 7 of the 10 HbSnRK2s were highly expressed in latex. HbSnRK2.7 was predominantly expressed and simultaneously regulated by abscisic acid, jasmonic acid, and ethylene treatment in laticifers. HbSnRK identification and characterization provided further understanding on the role of ABA signal in the rubber tree.     

Low molecular weight glutenin subunit gene composition at <Emphasis Type="Italic">Glu</Emphasis>-<Emphasis Type="Italic">D3</Emphasis> loci of <Emphasis Type="Italic">Aegilops tauschii</Emphasis> and common wheat and a further view of wheat evolution

Key message

A comprehensive comparison of LMW-GS genes between Ae. tauschii and its progeny common wheat.

Abstract

Low molecular weight glutenin subunits (LMW-GSs) are determinant of wheat flour processing quality. However, the LMW-GS gene composition in Aegilops tauschii, the wheat D genome progenitor, has not been comprehensively elucidated and the impact of allohexaploidization on the Glu-D3 locus remains elusive. In this work, using the LMW-GS gene molecular marker system and the full-length gene-cloning method, LMW-GS genes at the Glu-D3 loci of 218 Ae. tauschii and 173 common wheat (Triticum aestivum L.) were characterized. Each Ae. tauschii contained 11 LMW-GS genes, and the whole collection was divided into 25 haplotypes (AeH01–AeH25). The Glu-D3 locus in common wheat lacked the LMW-GS genes D3-417, D3-507 and D3-552, but shared eight genes of identical open reading frame (ORF) sequences when compared to that of Ae. tauschii. Therefore, the allohexaploidization induces deletions, but exerts no influence on LMW-GS gene coding sequences at the Glu-D3 locus. 92.17% Ae. tauschii had 7-9 LMW-GSs, more than the six subunits in common wheat. The haplotypes AeH16, AeH20 and AeH23 of Ae. tauschii ssp. strangulate distributed in southeastern Caspian Iran were the main putative D genome donor of common wheat. These results facilitate the utilization of the Ae. tauschii glutenin gene resources and the understanding of wheat evolution.

     

Identification and genetic characterization of an <Emphasis Type="Italic">Aegilops tauschii</Emphasis> ortholog of the wheat leaf rust disease resistance gene <Emphasis Type="Italic">Lr1</Emphasis>

Aegilops tauschii (goat grass) is the progenitor of the D genome in hexaploid bread wheat. We have screened more than 200 Ae. tauschii accessions for resistance against leaf rust (Puccinia triticina) isolates, which are avirulent on the leaf rust resistance gene Lr1. Approximately 3.5% of the Ae. tauschii accessions displayed the same low infection type as the tester line Thatcher Lr1. The accession Tr.t. 213, which showed resistance after artificial infection with Lr1 isolates both in Mexico and in Switzerland, was chosen for further analysis. Genetic analysis showed that the resistance in this accession is controlled by a single dominant gene, which mapped at the same chromosomal position as Lr1 in wheat. It was delimited in a 1.3-cM region between the restriction fragment length polymorphism (RFLP) markers ABC718 and PSR567 on chromosome 5DL of Ae. tauschii. The gene was more tightly linked to PSR567 (0.47 cM) than to ABC718 (0.79 cM). These results indicate that the resistance gene in Ae. tauschii accession Tr.t. 213 is an ortholog of the leaf rust resistance gene Lr1 of bread wheat, suggesting that Lr1 originally evolved in diploid goat grass and was introgressed into the wheat D genome during or after domestication of hexaploid wheat. Compared to hexaploid wheat, higher marker polymorphism and recombination frequencies were observed in the region of the Lr1 ortholog in Ae. tauschii. The identification of Lr1^Ae, the orthologous gene of wheat Lr1, in Ae. tauschii will allow map-based cloning of Lr1 from this genetically simpler, diploid genome.Hong-Qing Ling and Jiwen Qiu have contributed equally to this work     

Characterization of low-molecular-weight glutenin genes in <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

This paper reports the characterization of the low-molecular-weight (LMW) glutenin gene family of Aegilops tauschii (syn. Triticum tauschii), the D-genome donor of hexaploid wheat. By analysis of bacterial artificial chromosome (BAC) clones positive for hybridization with an LMW glutenin probe, seven unique LMW glutenin genes were identified. These genes were sequenced, including their untranslated 3 and 5 flanking regions. The deduced amino acid sequences of the genes revealed four putative active genes and three pseudogenes. All these genes had a very high level of similarity to LMW glutenins characterized in hexaploid wheat. The predicted molecular weights of the mature proteins were between 32.2 kDa and 39.6 kDa, and the predicted isoelectric points of the proteins were between 7.53 and 8.06. All the deduced proteins were of the LMW-m type. The organization of the seven LMW glutenin genes appears to be interspersed over at least several hundred kilo base pairs, as indicated by the presence of only one gene or pseudogene per BAC clone. Southern blot analysis of genomic DNA of Ae. tauschii and the BAC clones containing the seven LMW glutenin genes indicated that the BAC clones contained all LMW glutenin-hybridizing bands present in the genome. Two-dimensional gel electrophoresis of an LMW glutenin extract from Ae. tauschii was conducted and showed the presence of at least 11 distinct proteins. Further analysis indicated that some of the observed proteins were modified gliadins. These results suggest that the actual number of typical LMW glutenins may in fact be much lower than previously thought, with a number of modified gliadins also being present in the polymeric fraction.     

Chromosomal location of <Emphasis Type="Italic">Pm35</Emphasis>, a novel <Emphasis Type="Italic">Aegilops tauschii</Emphasis> derived powdery mildew resistance gene introgressed into common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

A single gene controlling powdery mildew resistance was identified in the North Carolina germplasm line NC96BGTD3 (NCD3) using genetic analysis of F₂ derived lines from a NCD3 X Saluda cross. Microsatellite markers linked to this Pm gene were identified and their most likely order was Xcfd7, 10.3 cM, Xgdm43, 8.6 cM, Xcfd26, 11.9 cM, Pm gene. These markers and the Pm gene were assigned to chromosome 5DL by means of Chinese Spring Nullitetrasomic (Nulli5D-tetra5A) and ditelosomic (Dt5DL) lines. A detached leaf test showed a distinctive disease reaction to six pathogen isolates among the NCD3 Pm gene, Pm2 (5DS) and Pm34 (5DL). An allelism test showed independence between Pm34 and the NCD3 Pm gene. Together, the tests provided strong evidence for the presence of a novel Pm gene in NCD3, and this gene was designated Pm35.     

Genome-wide identification and expression analysis of the molecular chaperone binding protein <Emphasis Type="Italic">BiP</Emphasis> genes in <Emphasis Type="Italic">Citrus</Emphasis>

Here, we report for the first time the genome-wide identification and expression analysis of the molecular chaperone BiP genes in Citrus. Six genes encoding the conserved protein domain family GPR78/BiP/KAR2 were identified in the genome of Citrus sinensis and C. clementina. Two of them, named here as CsBiP1 and CsBiP2, were classified as true BiPs based on their deduced amino acid sequences. Alignment of the deduced amino acid sequences of CsBiP1 and CsBiP2 with BiP homologs from soybean and Arabidopsis showed that they contain all the conserved functional motifs of BiPs. Analysis of the promoter region of CsBiPs revealed the existence of cis-acting regulatory sequences involved in abiotic, heat-shock, and endoplasmic reticulum (ER) stress responses. Publicly available RNA-seq data indicated that CsBiP1 is abundantly expressed in leaf, flower, fruit, and callus, whereas CsBiP2 expression is rarely detected in any tissues under normal conditions. Comparative quantitative real-time PCR (qPCR) analysis of expression of these genes between C. sinensis grafted on the drought-tolerant “Rangpur” lime (C. limonia) and -sensitive “Flying Dragon” trifoliate orange (Poncirus trifoliata) rootstocks showed that CsBiP1 was upregulated by drought stress on the former but downregulated on the latter, whereas the CsBiP2 mRNA levels were downregulated on drought-stressed “Flying Dragon,” but remained constant on “Rangpur.” CsBiP2 upregulation was only observed in C. sinensis seedlings subjected to osmotic and cold treatments. Taken together, these results indicate the existence of two highly conserved BiP genes in Citrus that are differentially regulated in the different tissues and in response to abiotic stresses.     

Identification,characterization, and expression of the <Emphasis Type="Italic">SWEET</Emphasis> gene family in <Emphasis Type="Italic">Phalaenopsis equestris</Emphasis> and <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

Sugars are important molecules that function not only as primary metabolites, but also as nutrients and signal molecules in plants. The sugar transport protein genes family SWEET has been recently identified. The availability of the Dendrobium officinale and Phalaenopsis equestris genome sequences offered the opportunity to study the SWEET gene family in this two orchid species. We identified 22 and 16 putative SWEET genes, respectively, in the genomes of D. officinale and P. equestris using comprehensive bioinformatics analysis. Based on phylogenetic comparisons with SWEET proteins from Arabidopsis and rice, the DoSWEET and PeSWEET proteins could be divided into four clades; among these, clade II specifically lacked PeSWEETs and clade IV specifically lacked DoSWEETs, and there were orthologs present between D. officinale and P. equestris. Protein sequence alignments suggest that there is a predicted serine phosphorylation site in each of the highly conserved MtN3/saliva domain regions. Gene expression analysis in four tissues showed that three PeSWEET genes were most highly expressed in the flower, leaf, stem, and root, suggesting that these genes might play important roles in growth and development in P. equestris. Analysis of gene expression in different floral organs showed that five PeSWEET genes were highly expressed in the column (gynostemium), implying their possible involvement in reproductive development in this species. The expression patterns of seven PeSWEETs in response to different abiotic stresses showed that three genes were upregulated significantly in response to high temperature and two genes were differently expressed at low temperature. The results of this study lay the foundation for further functional analysis of SWEET genes in orchids.     

Production of intergeneric hybrid between dwarfing polish wheat (<Emphasis Type="Italic">Triticum polonicum</Emphasis> L.) and <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Cosson. with reference to wheat origin

Dwarfing polish wheat is a dwarfing accession of Triticum polonicum L. from Xinjiang of China. In the present study, the artificial hybridization between dwarfing polish wheat and two accessions of Aegilops tauschii Cosson. (AS60 and AS65) was carried out, and the F₁ hybrids were obtained successfully without using embryo rescue techniques for the first time. The crossabilities of hybrids T. polonicum × Ae. tauschii (AS60) and T. polonicum × Ae. tauschii (AS65) were 1.67% and 0.60% respectively. Only the hybrids of T. polonicum × Ae. tauschii (AS60) germinated well, and 24 F₁ hybrid plants were obtained. All the F₁ hybrid plants grew vigorously, and the morphological traits were similar to bread wheat. The F₁ plants had some obvious traits inherited from T. polonicum and Ae. tauschii and were completely sterile. Chromosome pairing in the hybrid was characterized by a large number of univalents, with an average of 20.56 and 0.22 bivalents per PMC, and no ring bivalents and multivalents were observed. Furthermore, the potential value of the F1 hybrids between T. polonicum and Ae. tauschii for studying wheat origin and breeding are discussed. The article is published in the original.     

Polyhydroxybutyrate in <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Bradyrhizobium</Emphasis>: quantification and <Emphasis Type="Italic">phbC</Emphasis> gene expression

Polyhydroxyalkanoates (PHAs) are hydroxyalkanoate polymers that are produced and accumulate by many kinds of bacteria. These polymers act as an energy store for bacteria. Polyhydroxybutyrate (PHB) is the most studied polymer in the PHA family. These polymers have awakened interest in the environmental and industrial research areas because they are biodegradable and have thermoplastic qualities, like polypropylene. In this work, we analyzed the PHB production in Bradyrhizobium sp., Rhizobium leguminosarum bv. phaseoli, and Rhizobium huautlense cultured with two different carbon sources. We did biochemical quantification of PHB production during the three phases of growth. Moreover, these samples were used for RNA extraction and phbC gene expression analysis via real-time PCR. The bacteria showed different manner of growth, PHB accumulation and phbC gene expression when different quantity and quality of carbon sources were used. These results showed that under different growth media conditions, the growth and metabolism of different species of bacteria were influenced. These differences reflect the increase or decrease in PHB accumulation.     

Phylogenetic and evolutionary relationships and developmental expression patterns of the zebrafish <Emphasis Type="Italic">twist</Emphasis> gene family

Four members of the twist gene family (twist1a, 1b, 2, and 3) are found in the zebrafish, and they are thought to have arisen through three rounds of gene duplication, two of which occurred prior to the tetrapod-fish split. Phylogenetic analysis groups most of the vertebrate Twist1 peptides into clade I, except for the Twist1b proteins of the acanthopterygian fish (medaka, pufferfish, stickleback), which clustered within clade III. Paralogies and orthologies among the zebrafish, medaka, and human twist genes were determined using comparative synteny analysis of the chromosomal regions flanking these genes. Comparative nucleotide substitution analyses also revealed a faster rate of nucleotide mutation/substitution in the acanthopterygian twist1b compared to the zebrafish twist1b, thus accounting for their anomalous phylogenetic clustering. We also observed minimal expression overlap among the four twist genes, suggesting that despite their significant peptide similarity, their regulatory controls have diverged considerably, with minimal functional redundancy between them. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome-wide identification and evolution of <Emphasis Type="Italic">TC1</Emphasis>/<Emphasis Type="Italic">Mariner</Emphasis> in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>) genome

TC1/Mariner transposons belong to class II transposable elements (TEs) that use DNA-mediated “cut and paste” mechanism to transpose, and they have been identified in almost all organisms. Although silkworm (Bombyx mori) has a large amount of TC1/Mariner elements, the genome wide information of this superfamily in the silkworm is unknown. In this study, we have identified 2670 TC1/Mariner (Bmmar) elements in the silkworm genome. All the TEs were classified into 22 families by means of fgclust, a tool of repetitive sequence classification, seven of which was first reported in this study. Phylogenetic and structure analyses based on the catalytic domain (DDxD/E) of transposase sequences indicated that all members of TC1/Mariner were grouped into five subgroups: Mariner, Tc1, maT, DD40D and DD41D/E. Of these five subgroups, maT rather than Mariner possessed most members of TC1/Mariner (51.23%) in the silkworm genome. In particular, phylogenetic analysis and structure analysis revealed that Bmmar15 (DD40D) formed a new basal subgroup of TC1/Mariner element in insects, which was referred to as bmori. Furthermore, we concluded that DD40D appeared to intermediate between mariner and Tc1. Finally, we estimated the insertion time for each copy of TC1/Mariner in the silkworm and found that most of members were dramatically amplified during a period from 0 to 1 mya. Moreover, the detailed functional data analysis showed that Bmmar1, Bmmar6 and Bmmar9 had EST evidence and intact transposases. These implied that TC1/Mariner might have potential transpositional activity. In conclusion, this study provides some new insights into the landscape, origin and evolution of TC1/Mariner in the insect genomes.