The integrated LIM-peptidase domain of the CSA1-CHS3/DAR4 paired immune receptor detects changes in DA1 peptidase inhibitors in Arabidopsis.

White blister rust, caused by the oomycete Albugo candida, is a widespread disease of Brassica crops. The Brassica relative Arabidopsis thaliana uses the paired immune receptor complex CSA1-CHS3/DAR4 to resist Albugo infection. The CHS3/DAR4 sensor NLR, which functions together with its partner, the helper NLR CSA1, carries an integrated domain (ID) with homology to DA1 peptidases. Using domain swaps with several DA1 homologs, we show that the LIM-peptidase domain of the family member CHS3/DAR4 functions as an integrated decoy for the family member DAR3, which interacts with and inhibits the peptidase activities of the three closely related peptidases DA1, DAR1, and DAR2. Albugo infection rapidly lowers DAR3 levels and activates DA1 peptidase activity, thereby promoting endoreduplication of host tissues to support pathogen growth. We propose that the paired immune receptor CSA1-CHS3/DAR4 detects the actions of a putative Albugo effector that reduces DAR3 levels, resulting in defense activation.

Variation in the expression of a transmembrane protein influences cell growth in Arabidopsis thaliana petals by altering auxin responses.

BACKGROUND: The same species of plant can exhibit very diverse sizes and shapes of organs that are genetically determined. Characterising genetic variation underlying this morphological diversity is an important objective in evolutionary studies and it also helps identify the functions of genes influencing plant growth and development. Extensive screens of mutagenised Arabidopsis populations have identified multiple genes and mechanisms affecting organ size and shape, but relatively few studies have exploited the rich diversity of natural populations to identify genes involved in growth control. RESULTS: We screened a relatively well characterised collection of Arabidopsis thaliana accessions for variation in petal size. Association analyses identified sequence and gene expression variation on chromosome 4 that made a substantial contribution to differences in petal area. Variation in the expression of a previously uncharacterised gene At4g16850 (named as KSK) had a substantial role on variation in organ size by influencing cell size. Over-expression of KSK led to larger petals with larger cells and promoted the formation of stamenoid features. The expression of auxin-responsive genes known to limit cell growth was reduced in response to KSK over-expression. ANT expression was also reduced in KSK over-expression lines, consistent with altered floral identities. Auxin responses were reduced in KSK over-expressing cells, consistent with changes in auxin-responsive gene expression. KSK may therefore influence auxin responses during petal development. CONCLUSIONS: Understanding how genetic variation influences plant growth is important for both evolutionary and mechanistic studies. We used natural populations of Arabidopsis thaliana to identify sequence variation in a promoter region of Arabidopsis accessions that mediated differences in the expression of a previously uncharacterised membrane protein. This variation contributed to altered auxin responses and cell size during petal growth.

Reduced chromatin accessibility underlies gene expression differences in homologous chromosome arms of diploid Aegilops tauschii and hexaploid wheat.

BACKGROUND: Polyploidy is centrally important in the evolution and domestication of plants because it leads to major genomic changes, such as altered patterns of gene expression, which are thought to underlie the emergence of new traits. Despite the common occurrence of these globally altered patterns of gene expression in polyploids, the mechanisms involved are not well understood. RESULTS: Using a precisely defined framework of highly conserved syntenic genes on hexaploid wheat chromosome 3DL and its progenitor 3 L chromosome arm of diploid Aegilops tauschii, we show that 70% of these gene pairs exhibited proportionately reduced gene expression, in which expression in the hexaploid context of the 3DL genes was ∼40% of the levels observed in diploid Ae tauschii. Several genes showed elevated expression during the later stages of grain development in wheat compared with Ae tauschii. Gene sequence and methylation differences probably accounted for only a few cases of differences in gene expression. In contrast, chromosome-wide patterns of reduced chromatin accessibility of genes in the hexaploid chromosome arm compared with its diploid progenitor were correlated with both reduced gene expression and the imposition of new patterns of gene expression. CONCLUSIONS: Our pilot-scale analyses show that chromatin compaction may orchestrate reduced gene expression levels in the hexaploid chromosome arm of wheat compared to its diploid progenitor chromosome arm.

Independent assessment and improvement of wheat genome sequence assemblies using Fosill jumping libraries.

Background: The accurate sequencing and assembly of very large, often polyploid, genomes remains a challenging task, limiting long-range sequence information and phased sequence variation for applications such as plant breeding. The 15-Gb hexaploid bread wheat (Triticum aestivum) genome has been particularly challenging to sequence, and several different approaches have recently generated long-range assemblies. Mapping and understanding the types of assembly errors are important for optimising future sequencing and assembly approaches and for comparative genomics. Results: Here we use a Fosill 38-kb jumping library to assess medium and longer-range order of different publicly available wheat genome assemblies. Modifications to the Fosill protocol generated longer Illumina sequences and enabled comprehensive genome coverage. Analyses of two independent Bacterial Artificial Chromosome (BAC)-based chromosome-scale assemblies, two independent Illumina whole genome shotgun assemblies, and a hybrid Single Molecule Real Time (SMRT-PacBio) and short read (Illumina) assembly were carried out. We revealed a surprising scale and variety of discrepancies using Fosill mate-pair mapping and validated several of each class. In addition, Fosill mate-pairs were used to scaffold a whole genome Illumina assembly, leading to a 3-fold increase in N50 values. Conclusions: Our analyses, using an independent means to validate different wheat genome assemblies, show that whole genome shotgun assemblies based solely on Illumina sequences are significantly more accurate by all measures compared to BAC-based chromosome-scale assemblies and hybrid SMRT-Illumina approaches. Although current whole genome assemblies are reasonably accurate and useful, additional improvements will be needed to generate complete assemblies of wheat genomes using open-source, computationally efficient, and cost-effective methods.

Genome sequence of the progenitor of the wheat D genome Aegilops tauschii.

Aegilops tauschii is the diploid progenitor of the D genome of hexaploid wheat (Triticum aestivum, genomes AABBDD) and an important genetic resource for wheat. The large size and highly repetitive nature of the Ae. tauschii genome has until now precluded the development of a reference-quality genome sequence. Here we use an array of advanced technologies, including ordered-clone genome sequencing, whole-genome shotgun sequencing, and BioNano optical genome mapping, to generate a reference-quality genome sequence for Ae. tauschii ssp. strangulata accession AL8/78, which is closely related to the wheat D genome. We show that compared to other sequenced plant genomes, including a much larger conifer genome, the Ae. tauschii genome contains unprecedented amounts of very similar repeated sequences. Our genome comparisons reveal that the Ae. tauschii genome has a greater number of dispersed duplicated genes than other sequenced genomes and its chromosomes have been structurally evolving an order of magnitude faster than those of other grass genomes. The decay of colinearity with other grass genomes correlates with recombination rates along chromosomes. We propose that the vast amounts of very similar repeated sequences cause frequent errors in recombination and lead to gene duplications and structural chromosome changes that drive fast genome evolution.

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations.

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis.

The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana The peptidase is activated by two RING E3 ligases, Big Brother (BB) and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PROTEOLYSIS 1 (PRT1) of the N-end rule pathway. DA1 peptidase activity also cleaves the deubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TEOSINTE BRANCED 1/CYCLOIDEA/PCF 15 (TCP15) and TCP22, which promote cell proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins.

Sequence variations in OsAGPase significantly associated with amylose content and viscosity properties in rice (Oryza sativa L.).

Sequence-based variations in starch synthesis-related genes (SSRGs) exert a basic influence on the determination of eating quality in rice (Oryza sativa L.). This study aimed to investigate the relationship between the sequence variations from parts of 10 SSRGs and the amylose content (AC) plus rapid viscosity analysis (RVA) profiles in a heuristic rice core set by association mapping (AM). In total, 86 sequence variations were found in 10 sequenced amplicons, including 79 single-nucleotide polymorphisms (SNPs), six insertion-deletions (InDels) and one polymorphic simple sequence repeat (SSR). Among them, 61 variations were exon-based, of which 41 should lead to amino acid changes. Four subpopulations were revealed by population structure analysis based on 170 genome-wide SSR genotypes. The final AM showed a sum of four significant associations between three phenotypic indices and three sequence variations. An ADP-glucose pyrophosphorylase small unit 1 (OsAGPS1) SNP (A-G) was significantly associated with increased AC (P<0·001, R(2)=15·6%), while a 12-bp deletion of AGPase large unit 4 (OsAGPL4) was significantly related to the decreased breakdown viscosity (BDV) (P<0·001, R(2)=16·6%) in both general linear model (GLM) and mixed linear model (MLM). This study provides a new perspective of allele mining for breeding strategies based on marker-assisted selection.

SNP marker integration and QTL analysis of 12 agronomic and morphological traits in F8 RILs of pepper (Capsicum annuum L.).

Red pepper, Capsicum annuum L., has been attracting geneticists' and breeders' attention as one of the important agronomic crops. This study was to integrate 41 SNP markers newly developed from comparative transcriptomes into a previous linkage map, and map 12 agronomic and morphological traits into the integrated map. A total of 39 markers found precise position and were assigned to 13 linkage groups (LGs) as well as the unassigned LGe, leading to total 458 molecular markers present in this genetic map. Linkage mapping was supported by the physical mapping to tomato and potato genomes using BLAST retrieving, revealing at least two-thirds of the markers mapped to the corresponding LGs. A sum of 23 quantitative trait loci from 11 traits was detected using the composite interval mapping algorithm. A consistent interval between a035_1 and a170_1 on LG5 was detected as a main-effect locus among the resistance QTLs to Phytophthora capsici at high-, intermediate- and low-level tests, and interactions between the QTLs for high-level resistance test were found. Considering the epistatic effect, those QTLs could explain up to 98.25% of the phenotype variations of resistance. Moreover, 17 QTLs for another eight traits were found to locate on LG3, 4, and 12 mostly with varying phenotypic contribution. Furthermore, the locus for corolla color was mapped to LG10 as a marker. The integrated map and the QTLs identified would be helpful for current genetics research and crop breeding, especially in the Solanaceae family.

Transcriptome profiling and molecular marker discovery in red pepper, Capsicum annuum L. TF68.

Transcriptome from high throughput sequencing-by-synthesis is a good resource of molecular markers. In this study, we present utility of massively parallel sequencing by synthesis for profiling the transcriptome of red pepper (Capsicum annuum L. TF68) using 454 GS-FLX pyrosequencing. Through the generation of approximately 30.63 megabases (Mb) of expressed sequence tag (EST) data with the average length of 375 base pairs (bp), 9,818 contigs and 23,712 singletons were obtained by raw reads assembly. Using BLAST alignment against NCBI non-redundant and a UniProt protein database, 30% of the tentative consensus sequences were assigned to specific function annotation, while 24% returned alignments of unknown function, leaving up to 46% with no alignment. Functional classification using FunCat revealed that sequences with putative known function were distributed cross 18 categories. All unigenes have an approximately equal distribution on chromosomes by aligning with tomato (Solanum lycopersicum) pseudomolecules. Furthermore, 1,536 high quality single nucleotide discrepancies were discovered using the Bukang mature fruit cDNA collection (dbEST ID: 23667) as a reference. Moreover, 758 simple sequence repeat (SSR) motif loci were mined from 614 contigs, from which 572 primer sets were designed. The SSR motifs corresponded to di- and tri- nucleotide motifs (27.03 and 61.92%, respectively). These molecular markers may be of great value for application in linkage mapping and association mapping research.

Molecular cloning and characterization of hatching enzyme-like gene in the silkworm, Bombyx mori.

Hatching is the important process for the life of the metazoan, in which hatching enzyme (HE) plays a key role. In this paper, we cloned the full-length sequence of hatching enzyme-like cDNA from bluish-silkworm-eggs of Bombyx mori (BmHEL) by the method of in silico cloning, SMART cDNA synthesis and RACE-PCR technique. The BmHEL is 974 bp in length, and contains an ORF of 885 bp, encoding 294 amino acids residues. The deduced amino acid sequence of BmHEL has 30.3-47.1% identities to that of HE identified in the other species. Two similar signature sequences of HE gene family harbor in the BmHEL. The BmHEL gene structure is 6-exon-5-intron, and a promoter region with high scores has been predicted, which harbors some basal elements and some embryo-development related transcription factor binding sites. In the silkworm eggs at different developmental stages during incubation, the BmHEL transcripts can be detected and keep at a low level during the early stages, increase dramatically since 7th day of incubation, and reach to the maximum on 9th day. Change of BmHEL transcripts is in accordance with the process of embryo development and hatching, indicated that it plays an important role in these processes. Moreover, BmHEL transcript can be detected in the midgut and testis at larval stage, suggested that BmHEL may have other biological functions. To the best of our knowledge, this is the first report on HE gene in the Lepidoptera insects and will be helpful to provide a molecular basis for understanding the complicated mechanism underlying silkworm hatching.