We evaluated nine plastid (matK, rbcL, rpoC1, rpoB, rpl36-rps8, ndhJ, trnL-F, trnH-psbA, accD) and two nuclear (ITS and ITS2) barcode loci in family Zingiberaceae by analyzing 60 accessions of 20 species belonging to seven genera from India. Bidirectional sequences were recovered for every plastid locus by direct sequencing of polymerase chain reaction (PCR) amplicons in all the accessions tested. However, only 35 (58%) and 40 accessions (66%) yielded ITS and ITS2 sequences, respectively, by direct sequencing. In different bioinformatics analyses, matK and rbcL consistently resolved 15 species (75%) into monophyletic groups and five species into two paraphyletic groups. The 173 ITS sequences, including 138 cloned sequences from 23 accessions, discriminated only 12 species (60%), and the remaining species were entered into three paraphyletic groups. Phylogenetic and genealogic analyses of plastid and ITS sequences imply the possible occurrence of natural hybridizations in the evolutionary past in giving rise to species paraphyly and intragenomic ITS heterogeneity in the species tested. The results support using matK and rbcL loci for barcoding Zingiberaceae members and highlight the poor utility of ITS and the highly regarded ITS2 in barcoding this family, and also caution against proposing ITS loci for barcoding taxa based on limited sampling.

Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein—especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex “map” provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map.

30The Drosophila genome contains >13,000 protein coding genes, the majority of 31 which remain poorly investigated. Important reasons include the lack of 32 antibodies or reporter constructs to visualise these proteins. Here we present a 33 genome-wide fosmid library of ≈10,000 GFP-tagged clones, comprising tagged 34 genes and most of their regulatory information. For 880 tagged proteins we have 35 created transgenic lines and for a total of 207 lines we have assessed protein 36 expression and localisation in ovaries, embryos, pupae or adults by stainings and 37 live imaging approaches. Importantly, we can visualise many proteins at 38 endogenous expression levels and find a large fraction of them localising to 39 subcellular compartments. Using complementation tests we demonstrate that 40 two-thirds of the tagged proteins are fully functional. Moreover, our clones also 41 enable interaction proteomics from developing pupae and adult flies. Taken 42 together, this resource will enable systematic analysis of protein expression and 43 localisation in various cellular and developmental contexts. 44 Impact statement 45We provide a large-scale transgenic resource, which enables live imaging, subcellular 46 localisation and interaction proteomics of selected gene products at all stages of 47 Results 113Our goal was to generate a comprehensive resource that allows the investigation of 114 protein localisation and physical interactions for any fly protein of interest through a 115 robust, generic tagging pipeline in bacteria, which is followed by a large-scale 116 transgenesis approach (Figure 1). We based our strategy on a Drosophila 117 melanogaster FlyFos library of genomic fosmid clones, with an average size of 36 kb, 118 which covers most Drosophila genes (Ejsmont et al., 2009). Our two-step tagging 119 strategy first inserts a generic 'pre-tag' at the C-terminus of the protein, which is then 120 replaced by any tag of choice at the second tagging step, for example with a 121 superfolder-GFP (sGFP) tag to generate the sGFP TransgeneOme clone library. These 122 tagged clones are injected into fly embryos to generate transgenic fly-TransgeneOme 123 (fTRG) lines, which can be used for multiple in vivo applications. (Figure 1). 124 125 sGFP TransgeneOme -a genome-wide tagged FlyFos clone library 126We aimed to tag all protein coding genes at the C-terminus of the protein, because a 127 large number of regulatory elements reside within or overlap with the start of genes, 128 including alternative promoters, enhancer elements, nucleosome positioning 129 sequences, etc. These are more likely to be affected by a tag insertion directly after 130 the start codon. Signal sequences would also be compromised by an inserted tag after 131 the start codon. Additionally, the C-termini in the gene models are generally better 132 supported by experimental data than the N-termini due to an historical bias for 3' end 133 sequencing of ESTs. Thus, C-terminal tagging is more likely to result in a functional 134 tagged protein than N-termi...