Biology subjects

Ujino-Ihara, T.

Publications and source records attributed to Ujino-Ihara, T..

3 recordsLinked to original sources

bioRxiv2020

Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don

ObjectiveDue to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used for the production of a pollen-free cultivar. In order to reduce the time and cost needed for the production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica. The expected function of this gene, its two dysfunctional mutations, and genetic diversity were described recently in a related study. ResultsWe have developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. At field stations, where PCR machines are not available, LAMP markers were developed. LAMP only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be easily seen by eye. Because the causative mutations were deletions, two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers, were developed. These assays can be carried out by capillary or agarose gel electrophoresis.

genetics↗

bioRxiv2020

Identification and genetic diversity analysis of a male-sterile gene (MS1) in Japanese cedar (Cryptomeria japonica D. Don)

Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

genetics↗

bioRxiv2020

Inspecting abundantly expressed genes in male strobili in sugi (Cryptomeria japonica D. Don) via a highly accurate cDNA assembly

Sugi (Cryptomeria japonica D. Don) is an important conifer used for afforestation in Japan. The field of functional genomics is rapidly developing. The genomics of this gymnosperm species is currently being studied. Although its genomic size is 11 Gbps, it is still too large to assemble well within a short period of time. Transcriptomics is the one another approach to address this. Moreover, it is a necessary step in obtaining the complete genomic data. Here we designed a three stages assembling workflow using the de novo transcriptome assembly tools, Oases and Trinity. The three stages in transcriptomics are independent assembly, automatic and semi-automatic integration, and refinement by filtering out potential contamination. We found a set of 49,795 cDNA and an equal number of translated proteins (CJ3006NRE). According to the benchmark of BUSCO, 87.01 % were complete genes, including very high "Complete and single-copy" genes-78.47%. Compared to other full-length cDNA resources, the extent of the coverage in CJ3006NRE suggests that it may be used as the standard for further studies. When two tissue-specific libraries were compared, principal component analysis (PCA) showed that there were significant differences between male strobili and leaf and bark sets. The highest three upregulated transcription factors stood out as orthologs to angiosperms. The identified signature-like domain of the transcription factors demonstrated the accuracy of the assembly. Based on the evaluation of different resources, we demonstrate that our transcriptome assembly output is valuable and useful for further studies in functional genomics and evolutionary biology.

bioinformatics↗