Genetic diversity and relationships in mulberry (genus Morus) as revealed by RAPD and ISSR marker assays

BACKGROUND: The genus Morus, known as mulberry, is a dioecious and cross-pollinating plant that is the sole food for the domesticated silkworm, Bombyx mori. Traditional methods using morphological traits for classification are largely unsuccessful in establishing the diversity and relationships among different mulberry species because of environmental influence on traits of interest. As a more robust alternative, PCR based marker assays including RAPD and ISSR were employed to study the genetic diversity and interrelationships among twelve domesticated and three wild mulberry species. RESULTS: RAPD analysis using 19 random primers generated 128 discrete markers ranging from 500–3000 bp in size. One-hundred-nineteen of these were polymorphic (92%), with an average of 6.26 markers per primer. Among these were a few putative species-specific amplification products which could be useful for germplasm classification and introgression studies. The ISSR analysis employed six anchored primers, 4 of which generated 93 polymorphic markers with an average of 23.25 markers per primer. Cluster analysis of RAPD and ISSR data using the WINBOOT package to calculate the Dice coefficient resulted into two clusters, one comprising polyploid wild species and the other with domesticated (mostly diploid) species. CONCLUSION: These results suggest that RAPD and ISSR markers are useful for mulberry genetic diversity analysis and germplasm characterization, and that putative species-specific markers may be obtained which can be converted to SCARs after further studies

Genetic diversity and population structure of Indian golden silkmoth (Antheraea assama)

Background The Indian golden saturniid silkmoth (Antheraea assama), popularly known as muga silkmoth, is a semi-domesticated silk producing insect confined to a narrow habitat range of the northeastern region of India. Owing to the prevailing socio-political problems, the muga silkworm habitats in the northeastern region have not been accessible hampering the phylogeography studies of this rare silkmoth. Recently, we have been successful in our attempt to collect muga cocoon samples, although to a limited extent, from their natural habitats. Out of 87 microsatellite markers developed previously for A. assama, 13 informative markers were employed to genotype 97 individuals from six populations and analyzed their population structure and genetic variation. Methodology/Principal Findings We observed highly significant genetic diversity in one of the populations (WWS-1, a population derived from West Garo Hills region of Meghalaya state). Further analysis with and without WWS-1 population revealed that dramatic genetic differentiation (global FST = 0.301) was due to high genetic diversity contributed by WWS-1 population. Analysis of the remaining five populations (excluding WWS-1) showed a marked reduction in the number of alleles at all the employed loci. Structure analysis showed the presence of only two clusters: one formed by WWS-1 population and the other included the remaining five populations, inferring that there is no significant genetic diversity within and between these five populations, and suggesting that these five populations are probably derived from a single population. Patterns of recent population bottlenecks were not evident in any of the six populations studied. Conclusions/Significance A. assama inhabiting the WWS-1 region revealed very high genetic diversity, and was genetically divergent from the five populations studied. The efforts should be continued to identify and study such populations from this region as well as other muga silkworm habitats. The information generated will be very useful in conservation of dwindling muga culture in Northeast India

Monitoring recombinant baculovirus replication in Spodoptera litura (Lepidoptera: Noctuidae) larvae by using firefly luciferase gene as a reporter

An engineered virus, vAcluc, carrying the firefly luciferase (luc) gene as a reporter was injected into the hemocoel and different regions of the gut of Spodoptera litura F. to study the route of infection and virus dissemination. Luciferase activity was detected at 72 h postinfection in all the tissues examined (i.e., gut, gonads, cuticle, fat body, and the hemolymph). However, at 144 h post-infection expression was restricted primarily to the fat body tissue. Although injection of virus into the hemocoel was the most effective way of inoculation, injection into the gut also resulted in luc expression, albeit at a lower level, suggesting that nonoccluded viruses can also be infected orally

Differential gene expression during early embryonic development in diapause and non-diapause eggs of multivoltine silkworm <i style="">Bombyx mori</i>

1143-1151Quantification of the differential expression of metabolic enzyme and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm B. mori was carried out by semi-quantitative RT-PCR. Data analysis revealed that, the phosphofructokinase (PFK) expression started at a higher level in the early stage (6 h after oviposition) in non-diapause eggs, while in diapause induced eggs, it started at a lower level. However, the PFK gene expression in diapause eggs was comparatively higher than in non-diapause eggs. PFK facilitates use of carbohydrate reserves. The lower level of PFK gene expression in the early stage of diapause induced eggs but comparatively higher level of expression than in non-diapause eggs is due to enzyme inactivation via protein phosphorylation during early embryogenesis followed by de-phosphorylation in later stage. The sorbitol dehydrogenase-2 (SDH-2) gene was down regulated in diapause induced eggs up to 24 h and its expression levels in diapause induced eggs coincided with that of PFK gene at 48h in non-diapause eggs. During carbohydrate metabolism, there is an initial temporary accumulation of sorbitol which acts as protectant. The down regulation of SDH-2 gene during the first 24 hours in diapause induced eggs was due to the requirement of sorbitol as protectant. However, since the diapause process culminates by 48 h, the SDH-2 gene expression increased and coincided with that of PFK gene expression. The trehalase (Tre) gene expression was at a lower level in diapause induced eggs compared to non-diapausing eggs. The induction of Tre activity is to regulate uptake and use of sugar by the tissues. The non-diapause eggs revealed maximum expression of GPase gene with major fluctuations as well as an overall higher expression compared to diapause induced eggs. The diapause process requires less energy source which reflects lower activity of the gene. Heat shock protein (Hsp)genes (Hsp20.4, 40, 70, and 90) revealed differential levels of expression in both the eggs at all stages of embryonic development. The present study thus provides an overview of the differential expression levels of metabolic enzyme and Hsp genes in non-diapause and diapause induced eggs of multivoltine silkworm B. mori within 48 h after oviposition, confirming the major role of in early embryogenesis

Population structure of six <i>A. assama</i> populations prepared using STUCTURE program.

<p>Upper panel shows the structure obtained by analysis of all six populations and the lower panel shows the structure obtained by excluding WWS-1 population.</p

Locus-wise data on the number of alleles (Na), number of effective alleles (Ne), mean observed (Mean Ho) and expected (Mean He) heterozygosities, the number of migrants (Nm), fixation index (F) and F statistics obtained by analyzing five populations excluding WWS-1 population.

<p>Locus-wise data on the number of alleles (Na), number of effective alleles (Ne), mean observed (Mean Ho) and expected (Mean He) heterozygosities, the number of migrants (Nm), fixation index (F) and F statistics obtained by analyzing five populations excluding WWS-1 population.</p

Population-wise data on the mean number of alleles per locus (Na), number of effective alleles (Ne), number of private alleles, mean observed (Ho) and expected (He) heterozygosity, fixation index (F) and percentage polymorphism observed in 13 SSR loci (% P).

<p>Population-wise data on the mean number of alleles per locus (Na), number of effective alleles (Ne), number of private alleles, mean observed (Ho) and expected (He) heterozygosity, fixation index (F) and percentage polymorphism observed in 13 SSR loci (% P).</p

Locus-wise data on the number of alleles (Na), number of effective alleles (Ne), mean observed (Mean Ho) and expected (Mean He) heterozygosities, the number of migrants (Nm), fixation index (F) and F statistics.

<p>Locus-wise data on the number of alleles (Na), number of effective alleles (Ne), mean observed (Mean Ho) and expected (Mean He) heterozygosities, the number of migrants (Nm), fixation index (F) and F statistics.</p