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1.
Sci Rep ; 11(1): 1496, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452328

RESUMO

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.


Assuntos
Cryptomeria/genética , Infertilidade das Plantas/genética , Alérgenos/genética , Antígenos de Plantas/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Conservação dos Recursos Naturais/métodos , Cryptomeria/metabolismo , Etiquetas de Sequências Expressas , Agricultura Florestal/métodos , Testes Genéticos/métodos , Variação Genética/genética , Japão , Fenótipo , Melhoramento Vegetal/métodos , Infertilidade das Plantas/fisiologia , Pólen/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
BMC Res Notes ; 13(1): 457, 2020 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-32993771

RESUMO

OBJECTIVE: Due 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 to produce a pollen-free cultivar. In order to reduce the time and cost needed for 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 to rapidly identify pollen-free mutants. RESULTS: We developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. LAMP markers were developed for field stations, where PCR machines are unavailable. The LAMP method only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be read by the naked eye. Because the causative mutations were deletions, we developed two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers. These assays can be visualized using capillary or agarose gel electrophoresis.


Assuntos
Cryptomeria , Infertilidade das Plantas , Pólen , Cryptomeria/genética , Melhoramento Vegetal , Pólen/genética , Reação em Cadeia da Polimerase
3.
Plant Cell Physiol ; 57(4): 754-63, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26748081

RESUMO

Magnesium (Mg) is an essential macronutrient, functioning as both a cofactor of many enzymes and as a component of Chl. Mg is abundant in plants; however, further investigation of the Mg transporters involved in Mg uptake and distribution is needed. Here, we isolated an Arabidopsis thaliana mutant sensitive to high calcium (Ca) conditions without Mg supplementation. The causal gene of the mutant encodes MRS2-4, an Mg transporter.MRS2-4 single mutants exhibited growth defects under low Mg conditions, whereas an MRS2-4 and MRS2-7 double mutant exhibited growth defects even under normal Mg concentrations. Under normal Mg conditions, the Mg concentration of the MRS2-4 mutant was lower than that of the wild type. The transcriptome profiles of mrs2-4-1 mutants under normal conditions were similar to those of wild-type plants grown under low Mg conditions. In addition, both mrs2-4 and mrs2-7 mutants were sensitive to high levels of Mg. These results indicate that both MRS2-4 and MRS2-7 are essential for Mg homeostasis, even under normal and high Mg conditions. MRS2-4-green fluorescent protein (GFP) was mainly detected in the endoplasmic reticulum. These results indicate that these two MRS2 transporter genes are essential for the ability to adapt to a wide range of environmental Mg concentrations.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Adaptação Fisiológica/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Magnésio/farmacocinética , Raízes de Plantas/metabolismo
4.
Plant J ; 71(4): 564-74, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22448681

RESUMO

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition. These T(0) plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.


Assuntos
Mutação , Oryza/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Cruzamentos Genéticos , Metilação de DNA , Técnicas de Introdução de Genes , Células Germinativas Vegetais , Germinação , Glucuronidase/genética , Recombinação Homóloga , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Pólen/genética , Regiões Promotoras Genéticas , Sementes/genética
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