Role of microsatellites in genetic analysis of Bombyx mori silkworm: a review

Construction of DNA profiles

Microsatellites have proved a useful tool for generating band patterns that enable discrimination of silkworm lines. Kim et al.^8,33,34 found that 25–28% of the amplified alleles are specific to the lines, due to which a small number of microsatellites, one to three, allow identifying approximately 20% of the analyzed materials without resorting to the genotyping of other loci. Hou et al.³⁵ and Chandrakanth et al.³⁶ likewise indicate that the analyzed genotypes are homozygous for a substantial part of the microsatellites used. Since each locus is multiallelic, those that amplify as a single band in each line are powerful tools for identification, as demonstrated by Li et al.³⁷ when discriminating between two closely related lines identical in their morphological characteristics: Dazao and P50.

DNA fingerprinting also represents a tool for the identification of insect cell lines. Between 3 and 8 microsatellites have therefore been used to generate profiles of cell lines susceptible to the nuclear polyhedrosis virus of B. mori (BmNPV), developed to study replication and expression mechanisms of the virus^38–40. McIntosh et al.⁴¹ obtained stable DNA profiles even after performing 200 subcultures by using coding regions (aldolase, prolactin receptor, interleukin-1β) as molecular markers. Microsatellites are less stable, however, due to their high mutation rate—between 10^-4 and 10^-6 per locus per generation⁴². Thus, for future identification of cell lines, it is advisable to evaluate and select SSR loci that present relatively low mutation rates.

Analysis of genetic diversity

Sericulture depends on the strategic use of silkworm germplasm to develop hybrids with high yields of silk that resist or tolerate disease and adverse climatic conditions⁴³, based on knowledge of the extent and distribution of the genetic diversity available in both the domesticated silkworm B. mori and its wild relative Bombyx mandarina Moore (1872). In this context, between 500 and 700 microsatellites were developed in B. mori, of which 5 to 27 markers have been used for analyzing genetic diversity (Table 1).

Miao et al.¹⁵ and Zhan et al.²⁰ discovered that the genome of B. mori lines with contrasting characteristics is similar, in terms of the low percentage of polymorphic SSR loci, ranging between 17% and 24% compared, for example, with 85% for the European bee, Apis mellifera⁴⁴, and 55% in laboratory rats⁴⁵. Together, these data attribute the origin of B. mori to a single domestication event from a reduced population of B. mandarina¹⁵. Xia et al.⁴⁶, however, on conducting a complete genome analysis on domesticated lines and wild individuals report that B. mori harbors 83% of the genetic variability of wild populations, indicating that the origin was possibly not limited to a reduced population or a single domestication event⁴³.

The low percentage of polymorphic SSR loci found among domesticated lines of B. mori should not be interpreted as evidence of the reduction of genetic diversity with respect to wild populations. The low figure could instead be the result of size homoplasy, a process of change by which convergent mutations cause microsatellites, belonging to different lineages, to have the same length in base pairs⁴⁷.

Size homoplasy is favored when mechanisms are present that neutralize elongation of the microsatellites and limit the number of repeats, since possible alleles are reduced and mutations are more likely to converge in the same length. These conditions appear to be present in the B. mori genome, in which most microsatellites are of reduced size^16,17,20. In 2016, De Barba et al.⁴⁸ proposed a new method for genotyping microsatellites using high-throughput sequencing; this would allow direct access to the microsatellite sequences in B. mori and evaluate whether the low percentage of polymorphic loci is due to the presence of size homoplasy.

Although B. mori has not experienced a drastic reduction in genetic diversity compared to B. mandarina, the wide genetic distances between domesticated and wild populations⁴⁹ indicate that these species have a marked genetic differentiation and distant relationships. This is likely due to the absence of genetic flow, a result of the inability of B. mori to fly and to survive without human intervention⁵⁰. Thus, B. mandarina represents a potential unique source of genetic material for sericulture⁴³.

Cluster analyzes to determine the relationships between B. mori lines provide contradictory results. Reddy et al.¹⁴, Qian et al.⁵¹, Thiyagu and Kamble⁵² and Chandrakanth et al.³⁶ report that grouping the materials based on the microsatellites analyzed corresponded to type of voltinism, geographical origin, silk productivity, color or shape of the cocoon. However, the groups formed in studies that analyzed a larger sample of germplasm—69 lines on average, compared to 18 in the works cited above—exhibit mixtures of genotypes with variability in these characteristics^8,33–35,37.

Several different scenarios may explain why B. mori genotypes, with apparently diverse traits, are grouped together, the main one being the hybridization that has historically been used, in pure lines, to perform the introgression of genes that increase silk yield or survival rate in various environmental conditions^55–58. This would alter their phenotypic characteristics but, due to backcrossing, they would maintain a similar—or even the same—genotype, depending on the microsatellites analyzed.

Various pure lines may also share an origin in the same ancestral population of B. mandarina, but would have been selected to express different phenotypes, maintaining a high similarity at the genotypic level^7,37,59. This might also explain why pure lines with similar characteristics are not always grouped, given that they would have a distant relationship, but would have been selected to express similar traits^35,37.

Traditional genetic improvement in B. mori implies the use of phenotypic characteristics or geographical origin to differentiate and select parents with contrasting characteristics with which to perform crosses. However, due to the scenarios already mentioned, these characteristics do not always make it possible to accurately determine genetic relations between materials⁶⁰. Microsatellites thus represent an important tool for making accurate estimates of genetic diversity and relationships in order to develop genetically improved materials (hybrids), bearing in mind that, comparatively, performance is better than that of other markers such as RAPDs, RFPLs and ISSRs⁶.

Microsatellites not only represent a marker with a good performance in analysis of genetic diversity in B. mori. They constitute a versatile tool that can be adjusted to meet the research requirements and the resources available. For example, they can potentially be genotyped with high-throughput sequencing for greater accuracy⁴⁸ and even be identified and analyzed simultaneously with SNPs to strengthen inferences about diversity, structure and genetic relationships⁶¹. However, they can also be identified by means of polyacrylamide gels or capillary sequencing equipment when fluorescently labeled⁵. As such, they provide accessible and profitable information for managing germplasm banks and promoting regional initiatives in developing countries that do not have the resources to access cutting-edge technology, yet view sericulture as an opportunity to generate employment and improve the conditions of the rural population¹.

Linkage maps

Determination of the relative positions of microsatellite markers in the chromosomes of the B. mori genome began with the low-density linkage map developed by Prasad et al.¹⁶. The medium density one constructed by Miao et al.¹⁵ followed, with an average distance between markers of 6.3 cM and 29 linkage groups. Zhan et al.²⁰ subsequently increased the density of this map using new lines and mapping populations of B. mori, decreasing the average distance between markers to 4.8 cM (Table 2).

The density achieved with the linkage map of SSR markers is below the results expected by the authors due to the high homology (low percentage of polymorphic loci) between the loci of the B. mori lines used to generate the mapping populations^15,20. Nevertheless, the resolution is sufficient to carry out the preliminary gene screening (Table 3) and identification of QTLs.

Exclusive linkage maps for the Z chromosome were developed by Nagaraja et al.⁶² and Miao et al.⁶³ with the purpose of contributing to identification of genes related to control of the duration of larval stages, diapause, moltinism, body size and color, etc., and to analyze the role played by characteristics linked to sex in evolutionary processes in Lepidoptera, and differentiation of geographic races⁶⁴.

Identification of genes and QTLs

Identification of SSR markers linked to genes was carried out in order to understand the molecular basis of characteristics of agronomic and scientific interest. The SSR loci identified by Miao et al.¹⁵ and Zhan et al.²⁰ enabled the identification of genes related to characteristics such as thermotolerance, resistance to the Z strain of the virus of densonosis in B. mori, tolerance to fluorinated compounds, and absence of wing scales; studies that were used to develop improved lines with marker-assisted selection^9,65,66. Genes related to pigmentation patterns were also identified, in cocoons and larvae, and in development processes such as the formation of extremities, thoracic segments, cell adhesion and regulation of molting (Table 3).

Microsatellites have also allowed tracking of QTLs in B. mori related to weight of cocoon, cortex, pupa and filament^10,20, which are mainly located on chromosome 1 where they are strongly linked to SSR loci (LOD>11.0), contribute significantly to phenotypic variation (30 %) and have a simultaneous effect on the above characteristics. The location of the QTLs in chromosome 1 was delimited to a region of 290 kb, in which 12 candidate genes were identified that will allow study of the molecular mechanisms underlying these characters of agronomic interest in B. mori¹⁰. Additionally, Gao et al.⁶⁷ discovered that Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) resistance is a polygenic characteristic.

Linkage maps with microsatellites have allowed us to advance in the knowledge of characteristics of economic interest in B. mori, as well as in the Lepidoptera genetic architecture. However, Xu et al.⁶⁸ and Li et al.⁶⁹ indicate that the most used models have not been adjusted according to the particular genetic characteristics of the silkworm, because they ignore the effects of gender and the presence of aquiasmatic meiosis, a process that implies the absence of chromosomal cross-linking in the germinal line of the females. As a result, these authors have proposed models that allow correcting the potential biases and lack of precision of the most used methods for mapping QTLs in B. mori. Xu et al.⁶⁸ propose a statistical model to analyze QTLs in F2 populations, while Li et al.⁶⁹ focus their method on the use of backcrossing and the rational selection of mapping populations to first identify the chromosomes with QTLs and subsequently their position.