The distinctness and diversity of Ethiopian barleys

 The relative diversity and distinctness of Ethiopian barleys has been investigated using (1) morphology/isozyme/hordein polymorphisms and (2) RFLP markers. In the former a set of 51 landraces from over the whole of Ethiopia was compared with Iranian landraces based on data from previous studies and new hordein data. The two sets of landraces were found to have a comparable diversity. The Ethiopian ones are more diverse morphologically (5 traits), are similar in numbers of alleles per protein locus (17 loci) and in genetic differentiation, but are less diverse in average heterozygosity per locus and degree of polymorphism. However, on the basis of the hordein data the two sources of germplasm are very distinct. The correlation between morphological and protein diversity is very low. In the RFLP study 28 probes evenly distributed across the genome were used to analyse 43 Ethiopian landraces (represented by one single genotype) and to compare them with modern cultivars from North America, Europe and Japan, as well as 3 landraces from Iran, 1 from Nepal, and 1 accession of H. spontaneum from Afghanistan. The major finding was that the Ethiopian germplasm appears to be significantly less diverse than the modern germplasm but that it is genotypically very distinct. The apparent contradiction between a high diversity of useful genes coming from Ethiopia and an apparently low diversity at the DNA level is discussed. Received: 22 July 1996 / Accepted: 26 July 1996     

Improvement of anther culture methods for doubled haploid production in barley breeding

There is potential to accelerate cultivar development with a doubled haploid system for breeding line production. Anther culture methodology was evaluated for U.S.A. spring barley (Hordeum vulgare L.) breeding applications. Gelrite was found to be an acceptable replacement for ficoll in the induction medium to reduce costs while maintaining embryoid and plant production levels. Beneficial effects of 28 d cold pretreatment of donor spikes for anther culture were confirmed with Pacific Northwest USA barley genotypes. A 3 d mannitol solution pretreatment of fresh anthers was shown to be less effective for green plant production compared to 28 d cold pretreatment of donor spikes. Extended donor spike cold pretreatment from 28 to 42 d did not reduce anther culture productivity. Based on this research, anther culture techniques show promise for economical and convenient application in spring barley breeding.Abbreviations DH doubled haploid - LS Linsmaier and Skoog basal medium - BAP benzylaminopurine - GLM Generalized Linear Model - SAS Statistical Analysis System     

Separating the wheat from the chaff: a prioritisation pipeline for the analysis of metabolomics datasets

Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful and widely applied method for the study of biological systems, biomarker discovery and pharmacological interventions. LC-MS measurements are, however, significantly complicated by several technical challenges, including: (1) ionisation suppression/enhancement, disturbing the correct quantification of analytes, and (2) the detection of large amounts of separate derivative ions, increasing the complexity of the spectra, but not their information content. Here we introduce an experimental and analytical strategy that leads to robust metabolome profiles in the face of these challenges. Our method is based on rigorous filtering of the measured signals based on a series of sample dilutions. Such data sets have the additional characteristic that they allow a more robust assessment of detection signal quality for each metabolite. Using our method, almost 80% of the recorded signals can be discarded as uninformative, while important information is retained. As a consequence, we obtain a broader understanding of the information content of our analyses and a better assessment of the metabolites detected in the analyzed data sets. We illustrate the applicability of this method using standard mixtures, as well as cell extracts from bacterial samples. It is evident that this method can be applied in many types of LC-MS analyses and more specifically in untargeted metabolomics.

     

Highly efficient production of phosphorylated hepatitis B core particles in yeast Pichia pastoris

Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163 g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0 mg per 1 g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.     

Occurrence and Diversity of Pome Fruit Viruses in Apple and Pear Orchards in Latvia

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus are economically important viruses infecting fruit tree species worldwide. To evaluate the occurrence of these pome fruit viruses in Latvia, a large‐scale survey was carried out in 2007. Collected samples were tested for infection by DAS ELISA and multiplex RT‐PCR. The accuracy of the detection of the viruses in multiplex RT‐PCR was confirmed by sequencing amplified PCR fragments. The results showed a wide occurrence of viruses in apple and pear commercial orchards established from non‐tested planting material. More than 89% of the tested apple trees and more than 60% of pear trees were infected with one or more pome fruit viruses. Analyses showed that the high occurrence of viruses in several apple cultivars is due to the propagation of infected clonal rootstocks and scions from infected mother trees. Sequence analyses targeting the 3′‐terminal region of the tested viruses showed various degrees of genetic diversity within respective virus isolates. This is the first report of the occurrence of ACLSV, ASGV and ASPV in apple and pear trees in Latvia and demonstrates their genetic diversity in different host genotypes.     

Genetic and physical mapping of a high recombination region on chromosome 7H(1) in barley

Approaches utilizing microlinearity between related species allow for the identification of syntenous regions and orthologous genes. Within the barley Chromosome 7H(1) is a region of high recombination flanked by molecular markers cMWG703 and MWG836. We present the constructed physical contigs linked to molecular markers across this region using bacterial artificial chromosomes (BAC) from the cultivar Morex. Barley expressed sequence tags (EST), identified by homology to rice chromosome 6 between the rice molecular markers C425A and S1434, corresponded to the barley syntenous region of Chromosome 7H(1) Bins 2–5 between molecular markers cMWG703-MWG836. Two hundred and thirteen ESTs were genetically mapped yielding 267 loci of which 101 were within the target high recombination region while 166 loci mapped elsewhere. The 101 loci were joined by 43 other genetic markers resulting in a highly saturated genetic map. In order to develop a physical map of the region, ESTs and all other molecular markers were used to identify Morex BAC clones. Seventy-four BAC contigs were formed containing 2–102 clones each with an average of 19 and a median of 13 BAC clones per contig. Comparison of the BAC contigs, generated here, with the Barley Physical Mapping Database contigs, resulted in additional overlaps and a reduction of the contig number to 56. Within cMWG703-MWG836 are 24 agriculturally important traits including the seedling spot blotch resistance locus, Rcs5. Genetic and physical analysis of this region and comparison to rice indicated an inversion distal of the Rcs5 locus. Three BAC clone contigs spanning the Rcs5 locus were identified. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural Characterization and Chemical Classification of Some Bryophytes Found in Latvia

Bryophytes are the second largest taxonomic group in the plant kingdom; yet, studies conducted to better understand their chemical composition are rare. The aim of this study was to characterize the chemical composition of bryophytes common in Northern Europe by using elemental, spectral, and non‐destructive analytical methods, such as Fourier transform IR spectrometry (FT‐IR), solid‐phase ¹³C‐NMR spectrometry, and pyrolysis‐gas chromatography/mass spectrometry (Py‐GC/MS), for the purpose of investigating their chemotaxonomic relationships on the basis of chemical‐composition data. The results of all these analyses showed that bryophytes consist mainly of carbohydrates. Judging by FT‐IR spectra, the OH groups in combination of C? O groups were the most abundant groups. The ¹³C‐NMR spectra provided information on the presence of such compounds as phenolics and lipids. It was found that the amount of phenolic compounds in bryophytes is relatively small. This finding definitely confirmed the absence of lignin in the studied bryophytes. Cluster analysis was used to better understand differences in the chemical composition of bryophyte samples and to evaluate possible usage of these methods in the chemotaxonomy of bryophytes.     

半矮秆基因brh1在大麦中的精细定位

选用从大麦、小麦和水稻中分离的RFLP标记 ,构建了大麦半矮秆基因brh1精细图谱。以快中子处理六棱大麦品种Steptoe的种子 ,从M2 代中选择出brh1突变体FN5 3。brh1是一个极易鉴别的形态学标记 ,通过对FN5 3×Morex的F2 代群体进行鉴定表明 ,brh1基因为隐性 ,前人通过BSA法将其初步定位在大麦第 1染色体 (7H)短臂上 ,靠近端粒区。这一区间还有一个控制秆锈病抗性的显性基因Rpg1。所以 ,brh1的精细定位不仅对研究其本身具有重要意义 ,同时 ,也为Rpg1的图位克隆和功能研究提供了更大的重组配子群体。定位实验全部以F2 中具有brh1特征的个体为对象完成 ,鉴定工作在苗期进行。在该精细图上 ,brh1区间长15 .2cM ,各标记间的平均距离为 0 .8cM。其中 ,大麦的cDNA克隆MWG2 0 74B与brh1共分离。 2 0 74A在靠近着丝点一侧 ,与brh1相距 0 .8cM。BCD12 9和R3139在定位群体内呈现与MWG2 0 74A共分离。CDO5 4 5位于端粒一侧 ,距离brh1为 0 .8cM。根据禾谷类作物基因组的共线性原理 ,CDO5 4 5成功定位在水稻的同源染色体即第 6染色体短臂brh1区间内。然而 ,由于在定位亲本间缺乏多态性 ,BCD12 9和MWG2 0 74的 2条主带A和B均未能定位在水稻的共线性区段内。推测MWG2 0 74的其他各带可能被定位在水稻的目标区间内 ,从而有     

QTL analysis of agronomic traits in barley based on the doubled haploid progeny of two elite North American varieties representing different germplasm groups

A better understanding of the genetics of complex traits, such as yield, may be achieved by using molecular tools. This study was conducted to estimate the number, genome location, effect and allele phase of QTLs determining agronomic traits in the two North American malting barley (Hordeum vulgare L.) quality variety standards. Using a doubled haploid population of 140 lines from the cross of two-rowed Harrington×six-rowed Morex, agronomic phenotypic data sets from nine environments, and a 107-marker linkage map, we performed QTL analyses using simple interval mapping and simplified composite interval mapping procedures. Thirty-five QTLs were associated, either across environments or in individual environments, with five grain and agronomic traits (yield, kernel plumpness, test weight, heading date, and plant height). Significant QTL×environment interaction was detected for all traits. These interactions resulted from both changes in the magnitude of response and changes in the sign of the allelic effect. QTLs for multiple traits were coincident. The vrs1 locus on chromosome 2 (2H), which determines inflorescence row type, was coincident with the largest-effect QTL determining four traits (yield, kernel plumpness, test weight, and plant height). QTL analyses were also conducted separately for each sub-population (six-rowed and two-rowed). Seven new QTLs were detected in the sub-populations. Positive transgressive segregants were found for all traits, but they were more prevalent in the six-rowed sub-population.QTL analysis should be useful for identifying candidate genes and introgressing favorable alleles between germplasm groups. Received: 18 August 2000 / Accepted: 15 December 2000