A versatile computational pipeline for bacterial genome annotation improvement and comparative analysis, with Brucella as a use case

We present a bacterial genome computational analysis pipeline, called GenVar. The pipeline, based on the program GeneWise, is designed to analyze an annotated genome and automatically identify missed gene calls and sequence variants such as genes with disrupted reading frames (split genes) and those with insertions and deletions (indels). For a given genome to be analyzed, GenVar relies on a database containing closely related genomes (such as other species or strains) as well as a few additional reference genomes. GenVar also helps identify gene disruptions probably caused by sequencing errors. We exemplify GenVar's capabilities by presenting results from the analysis of four Brucella genomes. Brucella is an important human pathogen and zoonotic agent. The analysis revealed hundreds of missed gene calls, new split genes and indels, several of which are species specific and hence provide valuable clues to the understanding of the genome basis of Brucella pathogenicity and host specificity

Structural organization of an alien Thinopyrum intermedium group 7 chromosome in U.S. soft red winter wheat (Triticum aestivum L.)

Barley yellow dwarf virus (BYDV) resistance in soft red winter wheat (SRWW) cultivars has been achieved by substituting a group 7 chromosome from Thinopyrum intermedium for chromosome 7D. To localize BYDV resistance, a detailed molecular genetic analysis was done on the alien group 7 Th. intermedium chromosome to determine its structural organization. Triticeae group 7 RFLP markers and rye specific repetitive sequences used in the analysis showed that the alien chromosome in the P29 substitution line has distinguishing features. The 350–480 bp rye telomeric sequence family was present on the long arm as determined by Southern and fluorescence in situ hybridization. However, further analysis using a rye dispersed repetitive sequence indicated that this alien chromosome does not contain introgressed segments from the rye genome. The alien chromosome is homoeologous to wheat chromosomes 7A and 7D as determined by RFLP analysis. Presence of the waxy gene on chromosomes 7A, 7B, and 7D but its absence on the alien chromosome in P29 suggests some internal structural differences on the short arm between Th. intermedium and wheat group 7 chromosomes. The identification of rye telomeric sequences on the alien Thinopyrum chromosome and the homoeology to wheat chromosomes 7A and 7D provide the necessary information and tools to analyze smaller segments of the Thinopyrum chromosome and to localize BYDV resistance in SRWW cultivars.Key words: barley yellow dwarf virus, Thinopyrum intermedium, rye repetitive sequences, RFLP, homoeologous group 7

Identification and characterization of wheat-wheatgrass translocation lines and localization of barley yellow dwarf virus resistance

Stable introgression of agronomically important traits into crop plants through wide crossing often requires the generation and identification of translocation lines. However, the low efficiency of identifying lines containing translocations is a significant limitation in utilizing valuable alien chromatin-derived traits. Selection of putative wheatgrass-wheat translocation lines based on segregation ratios of progeny from γ-irradiated seed using a standard phenotypic analysis resulted in a low 4% success rate of identifying barley yellow dwarf virus (BYDV) resistant and susceptible translocation lines. However, 58% of the susceptible progeny of this irradiated seed contained a Thinopyrum intermedium chromosome-specific repetitive sequence, which indicated that γ-irradiation-induced translocations occurred at high rate. Restriction fragment length polymorphism (RFLP) analysis of susceptible lines containing alien chromatin, their resistant sister lines and other resistant lines showed that more than one third of the progeny of γ-irradiated double monosomic seeds contained wheatgrass-wheat translocations. Genomic in situ hybridization (GISH) analysis of selected lines confirmed that these were wheatgrass-wheat translocation lines. This approach of initially identifying BYDV susceptible deletion lines using an alien chromosome-specific repetitive sequence followed by RFLP analysis of their resistant sister lines efficiently identified resistant translocation lines and localized the BYDV resistance to the distal end of the introgressed Th. intermedium chromosome.Key words: gene introgression, wide crosses, chromosome, repetitive elements, RFLP, Thinopyrum intermedium

Thinopyrum 7Ai-1-derived small chromatin with Barley Yellow Dwarf Virus (BYDV) resistance gene integrated into the wheat genome with retrotransposon

Thinopyrum intermedium is a useful source of resistance genes for Barley Yellow Dwarf Virus (BYDV), one of the most damaging wheat diseases. In this study, wheat/Th. intermedium translocation lines with a BYDV resistance gene were developed using the Th. intermedium 7Ai-1 chromosome. Genomic in situ hybridization (GISH), using a Th. intermedium total genomic DNA probe, enabled detection of 7Ai-1-derived small chromatins containing a BYDV resistance gene, which were translocated onto the end of wheat chromosomes in the lines Y95011 and Y960843. Random amplified polymorphic DNA (RAPD) analyses using 120 random 10-mer primers were conducted to compare the BYDVresistant translocation lines with susceptible lines. Two primers amplified the DNA fragments specific to the resistant line that would be useful as molecular markers to identify 7Ai-1-derived BYDV resistance chromatin in the wheat genome. Additionally, the isolated Th. intermedium-specific retrotransposon-like sequence pTi28 can be used to identify Th. intermedium chromatin transferred to the wheat genome.Thinopyrum intermedium является полезным источником генов устойчивости к вирусу желтой карликовости ячменя (BYDV), одного из наиболее серьезных заболеваний пшеницы. В настоящей работе транслокационные линии пшеница/Th. intermedium с геном устойчивости BYDV получены с использованием 7Ai-1 хромосомы Th. intermedium. Геномная гибридизация in situ (GISH) с использованием тотальной ДНК Th. intermedium в качестве зонда дала возможность показать наличие небольшого фрагмента хромосомы, происходящего от хромосомы 7Ai-1 и содержащего ген устойчивости BYDV, который транслоцировался в терминальний участок одной из пшеничных хромосом в каждой из линий Y95011 и Y960843. RAPD-анализ был проведен с использованием 120 случайных 10-нуклеотидных праймеров для сравнения BYDV-устойчивых транслокационных линий с восприимчивыми линиями. Два праймера амплифицировали фрагменты ДНК, специфичные для устойчивой линии, и они могут быть использованы как молекулярные маркеры для идентификации в геноме пшеницы хроматина, транслоцированного от 7Ai-1. Кроме того, выделенная Th. intermedium-специфичная ретротранспозон-подобная последовательность pTi28 может быть использована для идентификации хроматина Th. intermedium, перенесенного в геном пшеницы