Identification of the responsible chromosomes of the larval and cocoon phenotypes in S. ricini
In order to examine the feasibility of research aiming at identifying trait-related genes in S. ricini , we tried to carry out forward genetic analysis using S. ricini and S. c. pryeri . Some morphological traits are different between S. ricini and S. c. pryeri (Figs. 1 and 4A), thus such traits can be good targets for forward genetic analysis.
As shown in Figs. 4B and 4C, the phenotypes originally derived fromS. c. pryeri were isolated in backcross generation 1 (BC1) individuals which were obtained by the crossing (S. ricini x S. c. pryeri ) x S. ricini . Here, we tried to identify the responsible chromosomes for 4 phenotypes, namely, ‘Blue,’ ‘Yellow,’ ‘Spot’ and ‘Red cocoon.’ ‘Blue’ and ‘Yellow’ refer to blue and yellow larval integument, respectively. ‘Spot’ refers to black spots on larval integument. ‘Red cocoon’ phenotype literally illustrates the colour of cocoons which some BC1 individuals produce.
Since meiotic recombination does not occur in lepidopteran females, all chromosomes of the BC1 individuals should be S. ricini -S. c. pryeri heterozygotic or S. ricini -S. ricini homozygotic, and not chimeric. Considering that the above-mentioned four phenotypes derived from S. c. pryeri are dominant, responsible chromosomes should be heterozygotic in all BC1 individuals.
Genomic PCR with chromosome-specific markers, which can molecularly distinguish S. ricini and S. c. pryeri , revealed that chromosome 8, 13, 3 and 12 were uniformly heterozygotic in all examined ‘Blue,’ ‘Yellow,’ ‘Spot’ and ‘Red cocoon’ individuals, meaning that those chromosomes were responsible for ‘Blue,’ ‘Yellow,’ ‘Spot’ and ‘Red cocoon’ traits, respectively (Fig. 5). This is the first report which demonstrated that forward genetic analysis is achievable in S. ricini (and S. c. pryeri ). Although the responsible genes of the four phenotypes have not been identified yet, it will not be long before those were identified.
Larvae of many lepidopteran species are sometimes called ‘green caterpillar’ because of their greenish integument. Greenish colour found in lepidopteran larvae is mixture of yellow and blue pigments, namely carotenoids and bilins. Regarding ‘Blue,’ the substance which forms blue colour was elucidated to be biliverdin IXγ (Saitoh, 2011). In the larval integument of S. c. pryeri , Biliverdin IXγ binds to Biliverdin-Binding protein II (BBP-II). However, BBP-II protein amount in the larval integument of S. c. pryeri and S. ricinidoes not significantly differ (Saitoh, 2011), indicating that BBP-II encoding gene is not a responsible gene for ‘Blue’ phenotype.
In contrast to ‘Blue’, the exact substances which form ‘Yellow’ colour has yet to be identified. There are a few mutant strains of B. mori which show yellow integument colour phenotype, and ‘lemon’ and ‘lemon lethal’ represent one example (Meng et al., 2009). The yellow colour of ‘lemon’ and ‘lemon lethal’ has already been elucidated to be xanthopterin, not carotenoid. Furthermore, ‘lemon’ and ‘lemon lethal’ are recessive phenotypes, indicating these two mutant phenotypes ofB. mori has nothing to do with ‘Yellow’ of S. ricinibecause ‘Yellow’ is a dominant phenotype. Identifying the responsible genes for ‘Blue’ and ‘Yellow’ phenotype will allow us to reveal the genetic basis of formation of larval ‘green’ colour.
Spot pattern of larvae of S. c. pryeri and ‘Spot’ individuals is uniformly formed in every segment of larval body. As shown in Fig. S6, lateral sides of a single larval body segment have three spots on each side. In addition, the dorsal side of a segment have five spots. In total, eleven spots can be found in a single segment. Although there are a number of silkworm strains which show various patterns of black spots on larval integument, none of those strains have uniform spot patterns similar to ‘Spot’ individuals.
‘Red cocoon’ phenotype exhibits brightly shining red colour. Interestingly, ‘Red cocoon’ phenotype has never been observed in F1 individuals. Colour of cocoons of S. c. pryeriand F1 individuals is grey, and thus, this ‘Grey cocoon’ is the epistatic trait to ‘Red cocoon’ phenotype (Fig. 4C). ‘Red cocoon’ is one of the economic traits of S. ricini . Some eco-races ofS. ricini in India are known to spin red cocoons, similar to our ‘Red cocoon’ individuals. However, it is difficult to produce red silk from red cocoons because red pigments are mainly present in sericin layer and are easily swept away during the degumming step (Fig. S7). Therefore, Eri-silk made from red cocoons is not as red as they appeared to be before the degumming step. We are now attempting to identify the red pigment and reveal the genetic basis of red colour formation in cocoons, which will provide us with much information about improvement on the degumming condition.