Kimiko Yamamoto

Construction of a single nucleotide polymorphism linkage map for the silkworm, Bombyx mori, based on bacterial artificial chromosome end sequences.

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female × an F1 (p50T × C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest.

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens.

Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F2 progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.

Application of Scanning Probe Microscopy to Genetic Analysis

We are developing an integrated technique involving of nanometer-size dissection of chromosome fragments by atomic force microscopy (AFM) and direct detection of the location of genome library clones by scanning near-field optical/atomic force microscopy (SNOM/AFM). The locations of nucleus organizer regions (NORs) on barley chromosomes and a bacterial artificial chromosome (BAC) clone were successfully detected by SNOM/AFM. Nanometer-scale dissection of silkworm pachytene chromosomes was also performed by AFM, and we succeeded in three successive dissection events of the chromosome region approximately 250 nm apart from each other. If this type of integrated method can be established in the near future, we will easily obtain the nucleotide sequences with positional information on chromosomes, which lead to a time- and cost-saving genome analysis technique.

The scanning probe microscope as a novel genomic analysis tool

We have developed a novel and efficient genomic analysis tool that combines scanning probe microscopy (SPM) and image processing with molecular biology techniques to accelerate genomic research. To examine the correlation between chromosome volume and DNA content, we scanned human metaphase chromosome sets with an atomic force microscope to examine the chromosome volume distribution. We found that the chromosome volume distribution agreed with DNA length distribution (obtained from a public database), and that the short arm to long arm volume ratio showed good agreement with the genomic position of the centromere. We were also able to predict the genomic position of an arbitrary gene marker with high accuracy by combining a scanning near-field optical/atomic force microscope and image processing techniques using fluorescence in situ hybridization. Thus, a novel SPM-based system developed here will be an effective tool to rapidly and accurately map DNA markers and construct physical map, which contributes to the advancement of genomic science.

Deficiency of a pyrroline-5-carboxylate reductase produces the yellowish green cocoon ‘Ryokuken’ of the silkworm, Bombyx mori

The silkworm cocoon colour has attracted researchers involved in genetics, physiology and ecology for a long time. ‘Ryokuken’ cocoons are yellowish green in colour due to unusual flavonoids, prolinylflavonols, while ‘Sasamayu’ cocoons are light green and contain only simple flavonol glucosides. We found a novel gene associated with the cocoon colour change resulting from a change in flavonoid composition and named it Lg (light green cocoon). In the middle silk glands of the +Lg/+Lg larvae, 1-pyrroline-5-carboxylic acid (P5C) was found to accumulate due to a decrease in the activity of pyrroline-5-carboxylate reductase (P5CR), an enzyme reducing P5C to proline. Sequence analysis of BmP5CR1, the candidate gene for Lg, revealed a 1.9u2009kb insertion and a 4u2009bp deletion within the 1st intron, a 97u2009bp deletion within the 4th intron, and au2009>u2009300u2009bp insertion within the 3′-UTR, in addition to two amino acid changes on exons 3 and 4 in +Lg/+Lg compared to Lg/Lg. Decreased expression of BmP5CR1 was observed in all of the investigated tissues, including the middle silk glands in +Lg/+Lg, which was probably caused by structural changes in the intronic regions of BmP5CR1. Furthermore, a BmP5CR1 knockout strain exhibited a yellowish green cocoon with the formation of prolinylflavonols. These results indicate that the yellowish green cocoon is produced by a BmP5CR1 deficiency. To our knowledge, this is the first report showing that the defect of an enzyme associated with intermediate metabolism promotes the conjugation of phytochemicals derived from foods with endogenously accumulating metabolites in animal tissues.

Toll ligand Spätzle3 controls melanization in the stripe pattern formation in caterpillars

Significance A stripe pattern is widely observed among animals and often used for warning or camouflage in caterpillars. However, its genetic background is largely unknown. This study showed that the Toll ligand Spätzle3 (Spz-3) is responsible for the silkworm Zebra locus, which causes black stripes on the anterior margin of each segment. Exhaustive knockdown experiments of spz- and Toll-related genes clarified that spz-3 and Toll-8 are involved in the melanin pigmentation of Zebra and another mutant. The Spz-3/Toll-8 signaling pathway is suggested to induce Zebra stripe-specific expression of the pigmentation gene yellow. This study sheds light on not only the unique aspect of Spz/Toll functions but also, stripe pattern pigmentation in caterpillars through co-option of a Toll signaling pathway. A stripe pattern is an aposematic or camouflage coloration often observed among various caterpillars. However, how this ecologically important pattern is formed is largely unknown. The silkworm dominant mutant Zebra (Ze) has a black stripe in the anterior margin of each dorsal segment. Here, fine linkage mapping of 3,135 larvae revealed a 63-kbp region responsible for the Ze locus, which contained three candidate genes, including the Toll ligand gene spätzle3 (spz-3). Both electroporation-mediated ectopic expression and RNAi analyses showed that, among candidate genes, only processed spz-3 induced melanin pigmentation and that Toll-8 was the candidate receptor gene of spz-3. This Toll ligand/receptor set is also involved in melanization of other mutant Striped (pS), which has broader stripes. Additional knockdown of 5 other spz family and 10 Toll-related genes caused no drastic change in the pigmentation of either mutant, suggesting that only spz-3/Toll-8 is mainly involved in the melanization process rather than pattern formation. The downstream pigmentation gene yellow was specifically up-regulated in the striped region of the Ze mutant, but spz-3 showed no such region-specific expression. Toll signaling pathways are known to be involved in innate immunity, dorsoventral axis formation, and neurotrophic functions. This study provides direct evidence that a Toll signaling pathway is co-opted to control the melanization process and adaptive striped pattern formation in caterpillars.