Development of the differential detection reverse transcription loop-mediated isothermal amplification method for Barley yellow mosaic virus or Japanese soil-borne wheat mosaic virus from barley.

A differential detection reverse transcription loop-mediated isothermal amplification (DD-RT-LAMP) method was developed to detect either Barley yellow mosaic virus (BaYMV) or Japanese soil-borne wheat mosaic virus (JSBWMV) simultaneously. Both primer sets, which recognized either BaYMV or JSBWMV genomic RNA, amplified DNA more efficiently at 65°C using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak annealing temperatures of BaYMV and JSBWMV amplification products using specific primer sets were 86.9°C-87.7°C and 84.5°C-85.0°C, respectively, and were clearly distinguishable during an annealing step following the isothermal amplification, monitored using a fluorescence detection device. In the field samples of barley (Hordeum vulgare L.) tested, BaYMV or JSBWMV were detected by DD-RT-LAMP, and the detection results of DD-RT-LAMP were correspondent with the results of reverse transcription-PCR.

Association mapping and validation of QTLs for flour yield in the soft winter wheat variety Kitahonami.

The winter wheat variety Kitahonami shows a superior flour yield in comparison to other Japanese soft wheat varieties. To map the quantitative trait loci (QTL) associated with this trait, association mapping was performed using a panel of lines from Kitahonami's pedigree, along with leading Japanese varieties and advanced breeding lines. Using a mixed linear model corrected for kernel types and familial relatedness, 62 marker-trait associations for flour yield were identified and classified into 21 QTLs. In eighteen of these, Kitahonami alleles showed positive effects. Pedigree analysis demonstrated that a continuous pyramiding of QTLs had occurred throughout the breeding history of Kitahonami. Linkage analyses using three sets of doubled haploid populations from crosses in which Kitahonami was used as a parent were performed, leading to the validation of five of the eight QTLs tested. Among these, QTLs on chromosomes 3B and 7A showed highly significant and consistent effects across the three populations. This study shows that pedigree-based association mapping using breeding materials can be a useful method for QTL identification at the early stages of breeding programs.

Differential detection of Wheat yellow mosaic virus, Japanese soil-borne wheat mosaic virus and Chinese wheat mosaic virus by reverse transcription loop-mediated isothermal amplification reaction.

A differential detection method for three wheat viruses: Wheat yellow mosaic virus (WYMV), Japanese soil-borne mosaic virus (JSBWMV) and Chinese wheat mosaic virus (CWMV) using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction was developed. All three primer sets, which were designed from the genome sequences of WYMV, JSBWMV and CWMV respectively, worked most efficiently at 65 °C and could detect each virus RNA within 10 min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak denaturing temperatures of WYMV, JSBWMV and CWMV primer sets were 87.6 °C, 84.8 °C and 86.4 °C, respectively and were clearly distinguished by the isothermal DNA amplification and fluorescence detection device. The RT-LAMP assay including all three primer sets was found to be 100 times more sensitive than RT-PCR for WYMV and JSBWMV and as sensitive as RT-PCR for CWMV. The RT-LAMP method was validated for the simultaneous detection of these viruses in wheat and barley leaves.