Kenichi Matsushima

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.

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.

Origin and evolution of the waxy phenotype in Amaranthus hypochondriacus: evidence from the genetic diversity in the Waxy locus

The existence of polymorphism in the Waxy locus in a large gene pool of 53 strains with various waxy phenotypes from samples of Amaranthus hypochondriacus collected from different regions was investigated in an origin-and-evolution study. First, we screened all strains for a mutation point (G–A polymorphism in exon 6) by using PCR–RFLP and/or direct sequence analysis. The results showed that the nonsense mutation in the coding region (exon 6) of the Waxy gene was responsible for the change in perisperm starch, leading to a waxy phenotype in all strains. Second, phylogenetic analysis, which was based on the Waxy variation, indicated diverse waxy types occurring separately and independently in certain domesticated regions in Mexico. Finally, we designated nine molecular types by comparing obvious structural variations in the coding region of the Waxy gene. Among the molecular types, A. hypochondriacus contained Type III in three subtypes with the waxy phenotype, with evolutionary routes that could originate from Type II in accordance with G–A polymorphism. In addition, these types had the same mutation points by which the Waxy gene was converted into the waxy phenotype. Therefore, the present results showed that the nonsense mutation is a unique event in the evolution of waxy phenotypes in this crop. This study will provide useful information for understanding the evolutionary process of the waxy phenotype.

Characterization of a new granule-bound starch synthase gene found in amaranth grains (Amaranthus cruentus L.)

To clarify the mechanism underlying amylose synthesis in the amaranth pericarp, we attempted to identify new GBSS isoforms. A new GBSS-encoding gene (i.e., CrGBSSIb) was isolated from amaranth leaf tissue. The CrGBSSIb gene consists of 4699-bp, including a 1938-bp open reading frame encoding 606 amino acids. A comparison of the cDNA and genomic sequences suggested that CrGBSSIb contains 12 introns and 13 exons. Interestingly, a phylogenetic analysis revealed that the amaranth GBSSIb gene evolved independently from the other GBSSI isoforms within this crop (i.e., intraspecies) and differed from the other plant GBSSII genes. The expression patterns of two GBSS isoforms revealed that CrGBSSIb and CrGBSSI are expressed during the early and late phases of seed development, respectively. Additionally, a high CrGBSSIb transcript level was detected in leaf tissue. This result indicates that CrGBSSI and CrGBSSIb, which affect amylose synthesis in amaranth grains, are active in the perisperm and pericarp, respectively. Therefore, CrGBSSIb encodes an enzyme associated with amylose synthesis. The enzyme may be primarily responsible for amylose metabolism in pericarp tissue.