α′ Subunit of soybean β-conglycinin forms complex with rice glutelin via a disulphide bond in transgenic rice seeds*

The α′ and β subunits of soybean β-conglycinin were expressed in rice seeds in order to improve the nutritional and physiological properties of rice as a food. The α′ subunit accumulated in rice seeds at a higher level than the β subunit, but no detectable difference in mRNA transcription level between subunits was observed. Sequential extraction results indicate that the α′ subunit formed one or more disulphide bonds with glutelin. Electron microscopic analysis showed that the α′ subunit and the β subunit were transported to PB-II together with glutelin. In mature transgenic seeds, the β subunit accumulated in low electron density regions in the periphery of PB-II, whereas the α′ subunit accumulated together with glutelin in high-density regions of the periphery. The subcellular localization of mutated α′ subunits lacking one cysteine residue in the N-terminal mature region (α′ΔCys1) or five cysteine residues in the pro and N-terminal mature regions (α′ΔCys5) were also examined. Low-density regions were formed in PB-II in mature seeds of transgenic rice expressing α′ΔCys 5 and α′ΔCys1. α′ΔCys5 was localized only in the low-density regions, whereas α′ΔCys1 was found in both low- and high-density regions. These results suggest that the α′ subunit could make a complex via one or more disulphide bonds with glutelin and accumulate together in PB-II of transgenic rice seeds

ParaHaplo: A program package for haplotype-based whole-genome association study using parallel computing

<p>Abstract</p> <p>Background</p> <p>Since more than a million single-nucleotide polymorphisms (SNPs) are analyzed in any given genome-wide association study (GWAS), performing multiple comparisons can be problematic. To cope with multiple-comparison problems in GWAS, haplotype-based algorithms were developed to correct for multiple comparisons at multiple SNP loci in linkage disequilibrium. A permutation test can also control problems inherent in multiple testing; however, both the calculation of exact probability and the execution of permutation tests are time-consuming. Faster methods for calculating exact probabilities and executing permutation tests are required.</p> <p>Methods</p> <p>We developed a set of computer programs for the parallel computation of accurate P-values in haplotype-based GWAS. Our program, ParaHaplo, is intended for workstation clusters using the Intel Message Passing Interface (MPI). We compared the performance of our algorithm to that of the regular permutation test on JPT and CHB of HapMap.</p> <p>Results</p> <p>ParaHaplo can detect smaller differences between 2 populations than SNP-based GWAS. We also found that parallel-computing techniques made ParaHaplo 100-fold faster than a non-parallel version of the program.</p> <p>Conclusion</p> <p>ParaHaplo is a useful tool in conducting haplotype-based GWAS. Since the data sizes of such projects continue to increase, the use of fast computations with parallel computing--such as that used in ParaHaplo--will become increasingly important. The executable binaries and program sources of ParaHaplo are available at the following address: <url>http://sourceforge.jp/projects/parallelgwas/?_sl=1</url></p

Identification of Nine Novel Loci Associated with White Blood Cell Subtypes in a Japanese Population

White blood cells (WBCs) mediate immune systems and consist of various subtypes with distinct roles. Elucidation of the mechanism that regulates the counts of the WBC subtypes would provide useful insights into both the etiology of the immune system and disease pathogenesis. In this study, we report results of genome-wide association studies (GWAS) and a replication study for the counts of the 5 main WBC subtypes (neutrophils, lymphocytes, monocytes, basophils, and eosinophils) using 14,792 Japanese subjects enrolled in the BioBank Japan Project. We identified 12 significantly associated loci that satisfied the genome-wide significance threshold of P<5.0×10−8, of which 9 loci were novel (the CDK6 locus for the neutrophil count; the ITGA4, MLZE, STXBP6 loci, and the MHC region for the monocyte count; the SLC45A3-NUCKS1, GATA2, NAALAD2, ERG loci for the basophil count). We further evaluated associations in the identified loci using 15,600 subjects from Caucasian populations. These WBC subtype-related loci demonstrated a variety of patterns of pleiotropic associations within the WBC subtypes, or with total WBC count, platelet count, or red blood cell-related traits (n = 30,454), which suggests unique and common functional roles of these loci in the processes of hematopoiesis. This study should contribute to the understanding of the genetic backgrounds of the WBC subtypes and hematological traits

A Genome-Wide Association Study Identified AFF1 as a Susceptibility Locus for Systemic Lupus Eyrthematosus in Japanese

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes multiple organ damage. Although recent genome-wide association studies (GWAS) have contributed to discovery of SLE susceptibility genes, few studies has been performed in Asian populations. Here, we report a GWAS for SLE examining 891 SLE cases and 3,384 controls and multi-stage replication studies examining 1,387 SLE cases and 28,564 controls in Japanese subjects. Considering that expression quantitative trait loci (eQTLs) have been implicated in genetic risks for autoimmune diseases, we integrated an eQTL study into the results of the GWAS. We observed enrichments of cis-eQTL positive loci among the known SLE susceptibility loci (30.8%) compared to the genome-wide SNPs (6.9%). In addition, we identified a novel association of a variant in the AF4/FMR2 family, member 1 (AFF1) gene at 4q21 with SLE susceptibility (rs340630; P = 8.3×10−9, odds ratio = 1.21). The risk A allele of rs340630 demonstrated a cis-eQTL effect on the AFF1 transcript with enhanced expression levels (P<0.05). As AFF1 transcripts were prominently expressed in CD4+ and CD19+ peripheral blood lymphocytes, up-regulation of AFF1 may cause the abnormality in these lymphocytes, leading to disease onset

FASTPCR software for PCR, in silico PCR, and oligonucleotide assembly and analysis

This chapter introduces the software FastPCR as an integrated tools environment for PCR primer and probe design. It also predicts oligonucleotide properties based on experimental studies of PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, group-specific, unique, and overlap extension PCR for multi-fragment assembly in cloning, as well as bisulphite modification assays. It includes a programme to design oligonucleotide sets for long sequence assembly by the ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator. The program includes various bioinformatics tools for analysis of sequences with GC or AT skew, of CG content and purine-pyrimidine skew, and of linguistic sequence complexity. It also permits generation of random DNA sequence and analysis of restriction enzymes of all types. It finds or creates restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It generates consensus sequences and analyses sequence conservation. It performs efficient and complete detection of various repeat types and displays them. FastPCR allows for sequence file batch processing, which is essential for automation. The FastPCR software is available for download at http://primerdigital.com/fastpcr.html and online version at http://primerdigital.com/tools/pcr.html.Peer reviewe

Single-male paternity in coelacanths

Latimeria chalumnae, a ‘living fossil,’ is of great scientific interest, as it is closely related to the aquatic ancestors of land-living tetrapods. Latimeria show internal fertilization and bear live young, but their reproductive behaviour is poorly known. Here we present for the first time a paternity analysis of the only available material from gravid females and their offspring. We genotype two L. chalumnae females and their unborn brood for 14 microsatellite loci. We find that the embryos are closely related to each other and never show more than three different alleles per locus, providing evidence for a single father siring all of the offspring. We reconstruct the father’s genotype but cannot identify it in the population. These data suggest that coelacanths have a monogamous mating system and that individual relatedness is not important for mate choice