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. 2024 Sep 30;56(1):68.
doi: 10.1186/s12711-024-00937-z.

QTL analysis to identify genes involved in the trade-off between silk protein synthesis and larva-pupa transition in silkworms

Rui Gao  1 , Chunlin Li  1 , Ang Zhou  1 , Xiachao Wang  1 , Kupeng Lu  1 , Weidong Zuo  1 , Hai Hu  1 , Minjin Han  1 , Xiaoling Tong  1 , Fangyin Dai  2
Affiliations

QTL analysis to identify genes involved in the trade-off between silk protein synthesis and larva-pupa transition in silkworms

Rui Gao et al. Genet Sel Evol. .

Abstract

Background: Insect-based food and feed are increasingly attracting attention. As a domesticated insect, the silkworm (Bombyx mori) has a highly nutritious pupa that can be easily raised in large quantities through large-scale farming, making it a highly promising source of food. The ratio of pupa to cocoon (RPC) refers to the proportion of the weight of the cocoon that is attributed to pupae, and is of significant value for edible utilization, as a higher RPC means a higher ratio of conversion of mulberry leaves to pupa. In silkworm production, there is a trade-off between RPC and cocoon shell ratiao(CSR), which refers the ratio of silk protein to the entire cocoon, during metamorphosis process. Understanding the genetic basis of this balance is crucial for breeding edible strains with a high RPC and further advancing its use as feed.

Results: Using QTL-seq, we identified a quantitative trait locus (QTL) for the balance between RPC and CSR that is located on chromosome 11 and covers a 9,773,115-bp region. This locus is an artificial selection hot spot that contains ten non-overlapping genomic regions under selection that were involved in the domestication and genetic breeding processes. These regions include 17 genes, nine of which are highly expressed in the silk gland, which is a vital component in the trade-off between RPC and CSR. These genes are annotate with function related with epigenetic modifications and the regulation of DNA replication et al. We identified one and two single nucleotide polymorphisms (SNPs) in the exons of teh KWMTBOMO06541 and KWMTBOMO06485 genes that result in amino acid changes in the protein domains. These SNPs have been strongly selected for during the domestication process. The KWMTBOMO06485 gene encodes the Bombyx mori (Bm) tRNA methyltransferase (BmDnmt2) and its knockout results in a significant change in the trade-off between CSR and RPC in both sexes.

Conclusions: Taken together, our results contribute to a better understanding of the genetic basis of RPC and CSR. The identified QTL and genes that affect RPC can be used for marker-assisted and genomic selection of silkworm strains with a high RPC. This will further enhance the production efficiency of silkworms and of closely-related insects for edible and feed purposes.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Trait performance for traits related to silk yield and pupa weight ratio in the two parental lines and the pooling sequencing experimental design of the bulk segregant analysis (BSA). a Photographs of the cocoon and pupa of the silkworm strains, IS-Dazao and 872B. b Cocoon weight (CW), pupa weight (PW), the ratio of pupa to cocoon (RPC) and cocoon shell ratio (CSR) of females and males from the IS-Dazao and 872B strains. Sample sizes n for IS-Dazao females and males are 80 and 90, respectively, and for 872B females and males of are 78 and 88, respectively. c SNP density map on each chromosome of the merged DNA pools of the CSR_L. and CSR_H. Y-axis: chromosome number; X-axis: physical distance of chromosomes; window size is 100 kb
Fig. 2
Fig. 2
QTL mapping analysis of the CSR/RPC. a The Δ(SNP-index) and G' values of the SNPs in the merged CSR_L and CSR_H pools were calculated in the whole genome via a sliding window method with a window size of 1e6 bp. The numbers along the top of the figure represent silkworm chromosome numbers. X-axis: physical distance of each chromosome (Mb); the lines for CI_95 and CI_99 in Δ(SNP-index) means the two-sided confidence intervals of 0.95 and 0.99, respectively; the line in the G' value plot means FDR = 0.01. b Δ(SNP-index) statistics for each SNPs within the QTL region, and points indicate SNPs with a Δ(SNP-index) ≥ 0.45. X-axis: Physical position within the mapping region of silkworm Chr11
Fig. 3
Fig. 3
Selection signals within the mapped regions during domestication and genetic breeding processes. a, b Analysis of the selection pressure of the QTL region during domestication (a) and breeding (b) of silkworms via a sliding window method with a window size of 5 kb and a step-size of 500 bp. Threshold line in (a): πLocal ≤ lowest 5% (0.0014), FST Wild-Local ≥ top 1% (0.63) and ROD Wild-Local ≥ top 5% (0.7). Threshold line in (b): πCHN-I ≤ lowest 5% (0.0008), FST Local-CHN-I ≥ top5% (0.35) and ROD Local-CHN-I ≥ top 5% (0.75). Green shadow box: region selected by domestication. Blue shadow box: region subjected to selection during CHN-I improvement. The SNPs are extracted from the re-sequenced data of silkworm strains in Additional file 2 Table S1, and sample sizes n of Wild, Local and CHN-I silkworms are 51, 205 and 105, respectively. c Genomic start and end positions of the window bins (bp) in the artificially-selected region. Up: domestication-selected regions. Down: improvement-selected region. d Heat-map of the tissue expression of genes in regions under artificial selection. Red boxes indicate the genes with a higher expression level in the silk gland (SG). ASG: anterior silk gland; MSG_A: anterior segment of middle silk gland; MSG_M: middle segment of middle silk gland; MSG_P: posterior segment of middle silk gland; PSG: posterior silk gland; FB: fat body; MG: midgut; MT: Markov tube; OV: ovary; TT: testis; av: mean FPKM values of three biological replicates. The data consisted of the FPKM values derived from the transcriptome of tissues dissected from the 3rd day of the 5th instar larvae of silkworm strain P50, as provided in KAIKObase version 4.0.0
Fig. 4
Fig. 4
Sequence variation analysis of the candidate genes. a1 Selection signal in the genomic region around the BmDnmt2 gene during domestication of silkworm, with a sliding window size of 5 kb and a step-size of 500 bp. Threshold line: FST Wild-Local ≥ top 1% (0.62) and ROD Wild-Local ≥ top 5% (0.7). a2 The gray box indicates the predicted genes within the region. The blue box indicates the BmDnmt2 gene, the combination of black boxes and lines is the gene structure diagram of BmDnmt2, and the bottom is the allele heat-map of SNPs in Wild, Local, and Improved populations in the genome region where BmDnmt2 is located. The SNPs are extracted from the re-sequenced data of silkworm strains in Additional file 2 Table S1, Sample sizes n of Wild, Local and Improved silkworms are 51, 205 and 194, respectively. Blue: Reference allele, Red: Alternate allele, Yellow: heterozygous genotype, and Grey: missing genotype. Arrows indicate the SNPs that are located in the exons of the gene. From left-to-right are the + 881 and + 502 bases of the coding region, respectively. a3 Allele frequency of SNPs in + 881 and + 502 bases of the coding region in different silkworm populations, and the corresponding amino acids encoded by each allele. Sample sizes of Wild, Local and Improved silkworms are the same with these in a2. The graphical description of the allele frequency is at the bottom. AF_Alt: allele frequency of the alternate allele of the SNPs in Wild, Local and Improved silkworms. AF_Ref: allele frequency of reference allele of the SNPs in the same groups. A Pearson chi-square test was used to compare the difference in allele frequencies between Wild and Local silkworm groups. ***The Chi-square value corresponded to a P < 0.001. b Selection signal in the genomic region around the BmMsr gene; legend details are the same as for Fig. 3a; the blue box indicates the BmMsr gene and the arrow indicates that the SNP is located at + 252 base of the gene coding region
Fig. 5
Fig. 5
Effect of BmDnmt2 on RPC and silk yield traits. a Detection of mutant alleles in the BmDnmt2-KO genomic sequence. The gene structure of BmDnmt2 is shown at the top. The two sgRNA sites (sgR1 and sgR2) of BmDnmt2 are marked in red, and the 178 bp deletion of BmDnmt2-KO between the two sites is shown in the middle. The sequencing results are shown below. At the bottom is the result of the PCR amplification of the deletion sequence in the wild-type and BmDnmt2-KO line. b Detection of mutant alleles in the BmDnmt2-KO coding sequence. There were 74 bp between the two sites in the wild-type, an 86 bp deletion in BmDnmt2-KO between the two sites in the coding sequence and a stop codon (TGA) was introduced. The results of base sequencing and gel electrophoresis are shown below. c Schematic diagram of structural changes in the BmDnmt2-KO protein. The brown box is the DNA methylase domain (PF00145.19). d Phenotypic investigation of RPC, CSW, CW, and PW in females (left) and males (right) of wild-type (WT) and BmDnmt2-KO (KO), respectively. Sample size is shown in parentheses following WT and KO, respectively. Center line, median; white dots, mean; box limits, upper and lower quartiles; whiskers, 1.5 × the interquartile range; black dots, outliers (**P < 0.01, ***P < 0.005, two-sided Wilcoxon test)
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