Genebanks hold comprehensive collections of cultivars, landraces and crop wild relatives of all major food crops, but their detailed characterization has so far been limited to sparse core sets. The analysis of genome-wide genotyping-by-sequencing data for almost all barley accessions of the German ex situ genebank provides insights into the global population structure of domesticated barley and points out redundancies and coverage gaps in one of the world's major genebanks. Our large sample size and dense marker data afford great power for genome-wide association scans. We detect known and novel loci underlying morphological traits differentiating barley genepools, find evidence for convergent selection for barbless awns in barley and rice and show that a major-effect resistance locus conferring resistance to bymovirus infection has been favored by traditional farmers. This study outlines future directions for genomics-assisted genebank management and the utilization of germplasm collections for linking natural variation to human selection during crop evolution.

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), PtdIns(3)P, PtdIns(3,4,5)P3 and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to regulate GTPase activity in vitro [6] [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that PtdIns(4,5)P2 is required for both early and late events in endocytic coated vesicle formation.

Mapping-by-sequencing has emerged as a powerful technique for genetic mapping in several plant and animal species. As this resequencing-based method requires a reference genome, its application to complex plant genomes with incomplete and fragmented sequence resources remains challenging. We perform exome sequencing of phenotypic bulks of a mapping population of barley segregating for a mutant phenotype that increases the rate of leaf initiation. Read depth analysis identifies a candidate gene, which is confirmed by the analysis of independent mutant alleles. Our method illustrates how the genomic resources of barley together with exome resequencing can underpin mapping-by-sequencing.

The annexins are a multigene family of Ca(2+)-dependent phospholipid-binding proteins which contain novel types of Ca2+ sites. Using site-directed mutagenesis, we generated mutant proteins that show defects in the Ca(2+)-binding sites in a particular member of this family, the src tyrosine kinase substrate annexin II. Analysis of the relative Ca(2+)-binding affinities of annexin II mutants in a combined Ca2+/phospholipid-binding assay revealed two distinct types of Ca(2+)-binding sites. Three so-called type II sites are found in annexin repeats 2, 3 and 4 respectively. Two so-called type III sites are located in the first repeat and involve the glutamic acid residues at positions 52 and 95. Both types of sites were recently identified by X-ray crystallography in annexins V and I [Huber, Schneider, Mayr, Römisch and Paques (1990) FEBS Lett. 275, 15-21; Weng, Luecke, Song, Kang, Kim and Huber (1993) Protein Sci. 2, 448-458], indicating that similar principles govern Ca2+ binding to annexins in crystals and in solution. The two types of Ca(2+)-binding sites differ not only in their architecture but also in their affinity for the bivalent cation. The Ca2+ concentration needed for half-maximal phosphatidylserine binding is 5-10 microM for an annexin II derivative with intact type II but defective type III sites (TM annexin II) whereas a mutant protein containing defective type II but unaltered type III sites (CM annexin II) requires 200-300 microM Ca2+ for the same activity. Annexin II mutants with defects in the type II and/or type III sites also show different subcellular distributions. When expressed transiently in HeLa cells, TM annexin II acquires the typical location in the cortical cytoskeleton observed for the wild-type molecule. In contrast, CM annexin II remains essentially cytosolic, as does a mutant protein containing defects in both type II and type III Ca(2+)-binding sites (TCM annexin II). This indicates that the intracellular association of annexin II with the submembraneous cytoskeleton depends only on the occupation of type II Ca(2+)-binding sites.

Annexin II is a Ca(2+)-regulated membrane- and cytoskeleton-binding protein implicated in membrane transport events along the Ca(2+)-regulated secretory and the early endocytic pathway. Biochemical properties of this annexin and its intracellular distribution are regulated by complex formation with p11 (S100A10), a member of the S100 protein family. The annexin II-p11 interaction is mediated through the unique N-terminal domain of annexin II and is inhibited by protein kinase C phosphorylation of a serine residue in annexin II. To map this regulatory serine phosphorylation site we developed a baculovirus-mediated expression system for wild-type annexin II and for a series of annexin II mutants which contained substitutions in one or more serine residues present in the N-terminal domain. The different mutant derivatives were purified and shown to display the same biochemical properties as recombinant wild-type annexin II and the authentic protein purified from porcine intestine. However, significant differences in phosphate incorporation were observed when the individual serine mutants were subjected to phosphorylation by protein kinase C. A comparison of the phosphorylation patterns obtained identified Ser-II as the protein kinase C site responsible for regulating the annexin II-p11 interaction. Ser-II lies within the sequence mediating p11 binding, i.e. amino-acid residues 1 to 14 of annexin II, and phosphorylation at this site most likely leads to a direct spatial interference with p11 binding.

Site-directed mutagenesis was employed to map and characterize Ca2 + -binding sites in annexin 11, a member of the annexin family of Ca2+-and phospholipid-binding proteins which serves as a major cellular substrate for the tyrosine kinase encoded by the src oncogene. Several single amino acid substitutions were introduced in the human annexin I1 and the various mutant proteins were scored for their affinity towards Ca2+ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J . Biol. Chem. 266, 14732-147391 that a Ca2+-binding site is present in the third of the four repeat segments whch comprise the 33-kDa protein core of annexin 11. In addition to Gly206 and Thr207, which are localized in the highly conserved endonexin fold of the third repeat, Glu246 is involved in the formation of this site. Thus the architecture of this Ca2+-binding site in solution is very similar, if not identical, to that of Ca2+ sites identified recently in annexin V crystals [Huber, R., Schneider, M., Mayr, I., Romisch, J. and Paques, E.-P. (1990) FEBS Lett. 275,[15][16][17][18][19][20][21]. In addition to the site in repeat 3, we have mapped sites of presumably similar architecture in repeats 2 and 4 of annexin 11. Again, an acidic amino acid which is located 40 residues C-terminal to the conserved glycine at position 4 of the endonexin fold is indispensable for highaffinity Ca2+ binding: Asp161 in the second and Asp321 in the fourth repeat. In contrast, repeat 1 does not contain an acidic amino acid at a corresponding position and also shows deviations from the other repeats in the sequence surrounding the conserved glycine. These results on annexin I1 together with the crystallographic information on annexin V reveal that annexins can differ in the position of the Ca2+ sites. Ca2+-binding sites of similar structure are present in repeats 2, 3, and 4 of annexin I1 while in annexin V they occur in repeats 1, 2, and 4. We also synthesized an annexin I1 derivative with mutations in all three Ca2+ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca2+, indicating the presence of (an) additional Ca2 + site(s) of presumably different architecture.The annexins form a multigene family of Ca2+-dependent membrane-binding and phospholipid-binding proteins. Although the precise biological function of the annexins is not known, their biochemical properties imply that the proteins are involved in membrane phenomena, e.g. membrane fusion events during exocytosis, membrane-cytoskeleton linkage, membrane channel formation (for review see Klee, 1988;Moss et al., 1991). In addition to common biochemical properties the annexins share a characteristic structural element, the annexin repeat. This segment of 70-80 amino acids is repeated four (32 -39-kDa annexins) or eight times (68-kDa annexin) along the polypeptide chain of the individual annexin. The annexin repeats show intramolecular as well as intermolecular sequence similarities which are par...

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Inflorescence architecture in small-grain cereals has a direct effect on yield and is an important selection target in breeding for yield improvement. We analyzed the recessive mutation laxatum-a (lax-a) in barley (Hordeum vulgare), which causes pleiotropic changes in spike development, resulting in (1) extended rachis internodes conferring a more relaxed inflorescence, (2) broadened base of the lemma awns, (3) thinner grains that are largely exposed due to reduced marginal growth of the palea and lemma, and (4) and homeotic conversion of lodicules into two stamenoid structures. Map-based cloning enforced by mapping-by-sequencing of the mutant lax-a locus enabled the identification of a homolog of BLADE-ON-PETIOLE1 (BOP1) and BOP2 as the causal gene. Interestingly, the recently identified barley uniculme4 gene also is a BOP1/2 homolog and has been shown to regulate tillering and leaf sheath development. While the Arabidopsis (Arabidopsis thaliana) BOP1 and BOP2 genes act redundantly, the barley genes contribute independent effects in specifying the developmental growth of vegetative and reproductive organs, respectively. Analysis of natural genetic diversity revealed strikingly different haplotype diversity for the two paralogous barley genes, likely affected by the respective genomic environments, since no indication for an active selection process was detected.The inflorescence is the most prominent part of smallgrain cereal plants, producing the carbohydrate-rich grains that are harvested for food, feed, and fiber. However, our understanding of the genetic factors that regulate inflorescence architecture remains limited. What is clear is that the appearance and shape of the inflorescence has been under constant visual selection since early domestication and is still ongoing in modern plant breeding due to the impact of inflorescence architecture on crop yield. For instance, in barley (Hordeum vulgare), strong selection has been exerted on spontaneously occurring alleles of nonbrittle rachis1 (btr1) and btr2 that prevent dehiscence of the rachis at maturity (Pourkheirandish et al., 2015), six-rowed spike1 (vrs1) that determines whether the inflorescence exhibits two or six rows of grain (Komatsuda et al., 2007), and nudum (nud) that controls whether the grain is hulled or hull-less (Taketa et al., 2008). Ultimately, knowing all of the genes that control cereal inflorescence architecture will provide targets for understanding and exploiting natural or induced genetic diversity toward improving both yield potential and end-use characteristics.The barley inflorescence or spike forms an unbranched main rachis carrying triplets of sessile single-floreted