Genetic analysis of pungency deficiency in Japanese chili pepper 'Shishito' (Capsicum annuum) revealed its unique heredity and brought the discovery of two genetic loci involved with the reduction of pungency.

The sensation of pungency generated by capsaicinoids is a characteristic trait of chili peppers (Capsicum spp.), and the presence or absence of pungency is central in determining its usage as a spice or a vegetable. In the present study, we aimed to clarify the heredity and genetic factors involved in the deficiency of pungency (quite low pungency) that is uniquely observed in the Japanese chili pepper 'Shishito' (Capsicum annuum). First, the F2 population ('Shishito' × pungent variety 'Takanotsume') was used for segregation analysis, and pungency level was investigated using capsaicinoid quantification with high-performance liquid chromatography. Also, restriction site associated DNA sequencing of the F2 population was performed, and genetic map construction and quantitative trait locus (QTL) mapping were implemented. The results indicated that the F2 population showed varying capsaicinoid content and two major QTLs were detected, Shql3 and Shql7, which explained 39.8 and 19.7% of the genetic variance, respectively. According to these results, the quite low pungency of 'Shishito' was a quantitative trait that involved at least the two loci. Further, this trait was completely separate from general non-pungent traits controlled by individual recessive genes, as described in previous studies. The present study is the first report to investigate the genetic mechanism of pungency deficiency in Japanese chili peppers, and our results provide new insights into the genetic regulation of pungency in chili pepper.

New findings on the fungal species Tricholoma matsutake from Ukraine, and revision of its taxonomy and biogeography based on multilocus phylogenetic analyses.

Matsutake mushrooms are among the best-known edible wild mushroom taxa worldwide. The representative Tricholoma matsutake is from East Asia and the northern and central regions of Europe. Here, we report the existence of T. matsutake under fir trees in Eastern Europe (i.e., Ukraine), as confirmed by phylogenetic analysis of nine loci on the nuclear and mitochondrial genomes. All specimens from Japan, Bhutan, China, North Korea, South Korea, Sweden, Finland, and Ukraine formed a T. matsutake clade according to the phylogeny of the internal transcribed spacer region. The European population of T. matsutake was clustered based on the ß2 tubulin gene, with a moderate bootstrap value. In contrast, based on analyses of three loci, i.e., rpb2, tef1, and the ß2 tubulin gene, T. matsutake specimens sampled from Bhutan and China belonged to a clade independent of the other specimens of this species, implying a genetically isolated population. As biologically available type specimens of T. matsutake have not been designated since its description as a new species from Japan in 1925, we established an epitype of this fungus, sampled in a Pinus densiflora forest in Nagano, Japan.

Effects of adding a commercial capsaicin fertilizer on the feeding behavior of sika deer (Cervus nippon).

We investigated the inhibitory effect of capsaicin fertilizer on feeding in deer. We tested four captive adult female deer. In Experiment 1, in addition to the treatment (intact) containing only a solid feed (HC), we mixed the fertilizer not containing capsaicin (F) or the capsaicin fertilizer (CF) in the solid feed. In addition, the solid feed was put on a wire net that capsaicin fertilizer was placed 5 cm below (SCF). We investigated their feeding behavior response. In Experiment 2, we changed the amount of substance (fertilizer and capsaicin fertilizer) mixed in the HC. We mixed different amounts (0, 50, 100, and 200 g) of the treatments other than the intact with HC and presented them to the deer, and investigated their feeding behavior response. In Experiment 1, intake in the F and CF decreased (p < .05). In Experiment 2, HC intake was significantly lower in the 100 and 200 g CF (p < .05). However, HC intake relatively increased by the last day in the CF 200 g too. The capsaicin fertilizer decreased the feeding behavior of deer by directly touching the mucous membranes of the deer nose and lips. However, the effects were decreased over time.

The pungent-variable sweet chili pepper 'Shishito' (Capsicum annuum) provides insights regarding the relationship between pungency, the number of seeds, and gene expression involving capsaicinoid biosynthesis.

Pungent traits caused by capsaicinoids are characteristic of chili peppers (Capsicum spp.), and the pungent-variable sweet chili pepper 'Shishito' (Capsicum annuum) is unique in being known for the pungency in fruits with few seeds. In the present study, we tried to clarify the relationship between the number of seeds and pungency in 'Shishito'. First, we investigated the pungency of 'Shishito' by simple sensory evaluations and quantifications of capsaicinoids by high-performance liquid chromatography. As a result, few-seeded fruits had a larger fluctuation of capsaicinoid content than many-seeded ones. This indicates that the number of seeds, in particular a decrease of the seeds, has some sort of connection with the pungency of 'Shishito'. Then, we analyzed the relationship between pungency and gene expression involving capsaicinoid biosynthesis at the individual fruit level. We vertically separated the placental septum in which capsaicinoids are synthesized and performed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for 18 genes involved in capsaicinoid biosynthesis. We also used the placental septum for capsaicinoid quantification so that the gene expression levels and capsaicinoid contents in the same fruits were obtained, and their correlations were analyzed using 20 biological replicates. Among the 18 genes, expression levels of 11 genes (WRKY9, CaMYB31, AT3, BCAT, BCKDH, KAS I, KAS III, ACL, CaKR1, FAT, and pAMT) had a significant positive correlation with the capsaicinoid concentration, and they were considered to upregulate capsaicinoid biosynthesis. These results provide new insights regarding the environmental variation of the pungency traits in chili peppers and the relationship between pungency, the number of seeds, and gene expression involved in capsaicinoid biosynthesis.

A low-pungency S3212 genotype of Capsicum frutescens caused by a mutation in the putative aminotransferase (p-AMT) gene.

The purpose of this study was to identify the genetic mechanism underlying capsinoid biosynthesis in S3212, a low-pungency genotype of Capsicum frutescens. Screening of C. frutescens accessions for capsaicinoid and capsiate contents by high-performance liquid chromatography revealed that low-pungency S3212 contained high levels of capsiate but no capsaicin. Comparison of DNA coding sequences of pungent (T1 and Bird Eye) and low-pungency (S3212) genotypes uncovered a significant 12-bp deletion mutation in exon 7 of the p-AMT gene of S3212. In addition, p-AMT gene transcript levels in placental tissue were positively correlated with the degree of pungency. S3212, the low-pungency genotype, exhibited no significant p-AMT transcript levels, whereas T1, one of the pungent genotypes, displayed high transcript levels of this gene. We therefore conclude that the deletion mutation in the p-AMT gene is related to the loss of pungency in placental tissue and has given rise to the low-pungency S3212 C. frutescens genotype. C. frutescens S3212 represents a good natural source of capsinoids. Finally, our basic characterization of the uncovered p-AMT gene mutation should contribute to future studies of capsinoid biosynthesis in Capsicum.

Multiple-herbicide resistance in Echinochloa crus-galli var. formosensis, an allohexaploid weed species, in dry-seeded rice.

Biotypes of Echinochloa crus-galli var. formosensis with resistance to cyhalofop-butyl, an acetyl-CoA carboxylase (ACCase) inhibitor, have been found in dry-seeded rice fields in Okayama, Japan. We collected two lines with suspected resistance (Ecf27 and Ecf108) from dry-seeded rice fields and investigated their sensitivity to cyhalofop-butyl and other herbicides. Both lines exhibited approximately 7-fold higher resistance to cyhalofop-butyl than a susceptible line. Ecf108 was susceptible to penoxsulam, an acetolactate synthase (ALS) inhibitor. On the other hand, Ecf27 showed resistance to penoxsulam and two other ALS inhibitors: propyrisulfuron and pyriminobac-methyl. The alternative herbicides butachlor, thiobencarb, and bispyribac-sodium effectively controlled both lines. To examine the molecular mechanisms of resistance, we amplified and sequenced the target-site encoding genes in Ecf27, Ecf108, and susceptible lines. Partial sequences of six ACCase genes and full-length sequences of three ALS genes were examined. One of the ACCase gene sequences encodes a truncated aberrant protein due to a frameshift mutation in both lines. Comparisons of the genes among Ecf27, Ecf108, and the susceptible lines revealed that none of the ACCases and ALSs in Ecf27 and Ecf108 have amino acid substitutions that are known to confer herbicide resistance, although a single amino acid substitution was found in each of three ACCases in Ecf108. Our study reveals the existence of a multiple-herbicide resistant biotype of E. crus-galli var. formosensis at Okayama, Japan that shows resistance to cyhalofop-butyl and several ALS inhibitors. We also found a biotype that is resistant only to cyhalofop-butyl among the tested herbicides. The resistance mechanisms are likely to be non-target-site based, at least in the multiple-herbicide resistant biotype.

A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker for the identification of Amaranthus cruentus species.

A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker was developed to identify the Amaranthus cruentus species by comparing sequences of the starch branching enzyme (SBE) locus among the three cultivated grain amaranths. We determined the partial SBE genomic sequence in 72 accessions collected from diverse locations around the world by direct sequence analysis. Then, we aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR-RFLP analysis. The result indicated that MseI would recognize the sequence 5'-T/TAA-3' in intron 11 from A. cruentus SBE. A restriction analysis of the amplified 278-bp portion of the SBE gene using the MseI restriction enzyme resulted in species-specific RFLP patterns among A. cruentus, Amaranthus caudatus and Amaranthus hypochondriacus. Two different bands, 174-bp and 104-bp, were generated in A. cruentus, while A. caudatus and A. hypochondriacus remained undigested (278-bp). Thus, we propose that the PCR-RFLP analysis of the amaranth SBE gene provides a sensitive, rapid, simple and useful technique for identifying the A. cruentus species among the cultivated grain amaranths.