Cytogenotoxicity of raw and treated dairy manure slurry by two-stage chemical and electrocoagulation: An application of the Allium cepa bioassay.

Livestock farming is an essential agricultural practice. However, the improper management of livestock wastes and discharge of untreated or partially treated livestock manure slurry poses significant environmental problems. In this study, we aimed to compare the cytogenotoxic potential of untreated and treated dairy manure slurry treated with a two-stage chemical and electrocoagulation (EC) using the Allium cepa bioassay. The A. cepa bioassay is a well-established standard tool for assessing the cytogenotoxic effects of environmental contaminants, especially those that are occurred as complex contaminant mixtures. The dairy manure slurry was subjected to chemical treatment utilizing polyaluminum chloride (PAC) and cationic polyacrylamide (CPAM) at optimized conditions, followed by EC utilizing either aluminum (Al) or steel anodes. The treated and untreated samples were then evaluated for their potential cytogenotoxicty using the A. cepa bioassay, by measuring the nuclear abnormalities (NAs) and chromosomal aberrations (CAs), along with the mitotic indices (MIs). Our findings revealed a significant reduction in cytogenotoxic indicators in the treated liquid fraction compared to the untreated dairy manure slurry. Specifically, the frequency of total NAs showed a significant reduction from 154 ‰ to 37 ‰ when the dairy manure slurry was treated with chemical coagulation followed by EC utilizing an Al anode. Moreover, the MI exhibited a significant improvement from 7 ‰ to 123 ‰, suggesting the mitigation of toxic effects. These results collectively demonstrate the effectiveness of the two-stage chemical and EC treatment under optimal conditions in treating diary manure slurry while reducing its cytogenotoxicity for living systems. The A. cepa bioassay proved to be a sensitive and reliable method for assessing the toxicity of the treated samples. The efficient solid-liquid separation and the reduction of toxicity in the liquid fraction for biological systems achieved through this treatment process highlight its potential for sustainable management of livestock waste and the preservation of water quality. Nevertheless, further studies are required to assess the toxicity of solid fraction.

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.

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

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.

Decreased number of Gemini of coiled bodies and U12 snRNA level in amyotrophic lateral sclerosis.

Disappearance of TAR-DNA-binding protein 43 kDa (TDP-43) from the nucleus contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS), but the nuclear function of TDP-43 is not yet fully understood. TDP-43 associates with nuclear bodies including Gemini of coiled bodies (GEMs). GEMs contribute to the biogenesis of uridine-rich small nuclear RNA (U snRNA), a component of splicing machinery. The number of GEMs and a subset of U snRNAs decrease in spinal muscular atrophy, a lower motor neuron disease, suggesting that alteration of U snRNAs may also underlie the molecular pathogenesis of ALS. Here, we investigated the number of GEMs and U11/12-type small nuclear ribonucleoproteins (snRNP) by immunohistochemistry and the level of U snRNAs using real-time quantitative RT-PCR in ALS tissues. GEMs decreased in both TDP-43-depleted HeLa cells and spinal motor neurons in ALS patients. Levels of several U snRNAs decreased in TDP-43-depleted SH-SY5Y and U87-MG cells. The level of U12 snRNA was decreased in tissues affected by ALS (spinal cord, motor cortex and thalamus) but not in tissues unaffected by ALS (cerebellum, kidney and muscle). Immunohistochemical analysis revealed the decrease in U11/12-type snRNP in spinal motor neurons of ALS patients. These findings suggest that loss of TDP-43 function decreases the number of GEMs, which is followed by a disturbance of pre-mRNA splicing by the U11/U12 spliceosome in tissues affected by ALS.