Molecular distinction amongst varieties of Mulberry using RAPD and DAMD profiles

Background  

Mulberry trees are the most important host for rearing mulberry silkworms in sericulture. Improved varieties of mulberry tree have been developed through traditional breeding procedures. Not much work, however, has been carried out on the molecular characterization of these varieties. Random Amplified Polymorphic DNA (RAPD) and Directed Amplification of Minisatellite DNA (DAMD) methods based on Polymerase Chain Reaction are important tools to analyze genetic diversity of mulberries. These have been used to determine variation amongst nine varieties of Morus spp. maintained at Banthra Research Station of National Botanical Research Institute, Lucknow.     

Groupwise sampling: a strategy to sample core entries from RAPD marker data with application to mulberry

Key message

A strategy for effective utilization of RAPD marker data for sampling diverse entries was suggested and utilized for the development of mulberry core collection.

Abstract

Mulberry (Morus spp.) is a perennial tree cultivated mainly for its foliage in sericulture industry and also known for its edible fruits, fodder, and valued timber. In recent years, mulberry cultivation is confronted with several abiotic and biotic stresses due to inimical climatic factors and this has necessitated the genetic improvement of the crop. Core collection is an efficient way of harnessing the trait variation and novel genes available in a natural gene pool for the development of improved elite lines. In this study, we analyzed 850 mulberry accessions assembled from 23 countries with separate sets of polymorphic RAPD markers along with a limited number of ISSR, SSR, and phenotypic markers. A total of 75 accessions were duplicated in 20 clusters among five natural groups. The limitations of the RAPD marker system like problem in cross gel comparison were tackled by adopting a novel “Groupwise sampling” approach. A mulberry core collection with 100 diverse entries was selected using maximization method implemented in MSTRAT software. The mean Dice dissimilarity coefficient computed from marker data was 0.308 among core entries. Indigenous and exotic entries were not discriminated in cluster and principal component analysis supporting the spread of mulberry far from the place of origin. Accessions belonging to two wild mulberry species from Andaman Islands and Himalayan region formed separate clusters indicating the geographical, reproductive, and taxonomic distinction. The identified core collection will be available for researchers for intensive mining of desirable alleles in mulberry improvement as well as in genome resequencing program.     

Leaf cuticular wax amount and crystal morphology regulate post-harvest water loss in mulberry (Morus species)

Mulberry leaves are the sole source of food for silkworms (Bombyx mori), and moisture content of the detached leaves fed to silkworms determines silkworm growth and cocoon yield. Since leaf dehydration in commercial sericulture is a serious problem, development of new methods that minimize post-harvest water loss are greatly needed. In the present study, variability in moisture retention capacity (MRC, measured as leaf relative water content after one to 5 h of air-drying) was examined by screening 290 diverse mulberry accessions and the relationship between MRC and leaf surface (cuticular) wax amount was determined. Leaf MRC varied significantly among accessions, and was found to correlate strongly with leaf wax amount. Scanning electron microscopic analysis indicated that leaves having crystalline surface waxes of increased facet size and density were associated with high MRC accessions. Leaf MRC at 5 h after harvest was not related to other parameters such as specific leaf weight, and stomatal frequency and index. This study suggests that mulberry accessions having elevated leaf surface wax amount and crystal size and density exhibit reduced leaf post-harvest water loss, and could provide the foundation for selective breeding of improved cultivars.     

Genetic relationships of Japanese and Indian mulberry (Morus spp.) genotypes revealed by DNA fingerprinting

Mulberry (Morus spp.), a deciduous tree, originated at the foothills of the Himalayas and is used in sericulture for its leaf to feed the silkworm Bombyx mori L. Species differentiation among the genotypes of the genus Morus has never been out of debate as inter-specific hybridization events are often fertile. In the present study attempts were made to elucidate the genetic relationships among 18 mulberry genotypes collected from India and Japan using 15 Inter Simple Sequence Repeat (ISSR) and 15 Random Amplified Polymorphic DNA (RAPD) primers. The ISSR primers generated 81.13% polymorphism while the RAPDs generated 71.78% polymorphism. The polymorphic index of the primers identified UBC-812, UBC-826, UBC827, UBC-881, OPA-01, OPA-02, OPA-04 and OPH-17 as informative primers in mulberry. The genetic similarity coefficients and the dendrograms showed considerable genetic similarity among the genotypes. However, using the DNA markers, these genotypes were discriminated into two major groups in accordance with their geographic origin and species status. Distribution of the genotypes on a two-dimensional figure on the basis of the ALSCAL algorithm using Euclidean distance further confirmed the genetic divergence between these two groups. From the study it can be concluded that though morphologically Japanese and Indian mulberry genotypes show little divergence, genetic analysis using DNA markers could unravel significant genetic variation between these two groups. Similarly, while the species status of Japanese mulberry genotypes agrees with the genetic analysis, the same does not apply to Indian genotypes, in agreement with many earlier reports. The information generated in this study is of much use for taxonomical grouping and also for utilization in breeding and conservation programs.     

Mulberry improvements via plastid transformation and tissue culture engineering

The in vitro tissue culture and micropropagation studies for Morus spp., a pivotal sericulture plant, are well established. The rapid and reproducible in vitro response to plant growth regulator treatments has emerged as an essential complement of transformation studies for this plant species. A major area of study is the use of protoplast culture and fusion techniques where advantages to mulberry improvement can be applied. The advancements in genetic transformation of mulberry are reviewed, and a section on strategy for transforming plastids (chloroplasts) of mulberry is included. A role for mulberry in “molecular farming” is envisioned. The conclusions and future prospects for improvement of this economically important tree species are proposed.Key words: molecular farming, Morus spp., plastid transformation, protoplast electrofusion, sericultureThe importance of silk production is well recognized in sericulture industry that involves cultivation of host plants for silkworm rearing. India is one of the countries where sericulture is an important agro-based cottage industry involved in production of five different silk types—mori, muga, eri, tasar and oak types. This classification comes from type of host plant that act as feed for silkworm, and thus sericulture industry largely depends on the availability of host plant species. Bombyx mori (mulberry silkworm) feeds on mulberry leaves, Philosomia ricini (eri silkworm) on castor leaves, Anthraea assama (muga silkworm) on som and soalu leaves, Anthraea proylei (temperate/oak tasar silkworm) on oak leaves, and Anthraea mylitta (tropical tasar silkworm) on Terminalia leaves. A systematic and proper cultivation of novel primary and/or secondary host plants showing high yield, suitability to silkworm rearing, and resistance to different abiotic stress conditions i.e., tolerance to water stress, alkalinity and salinity are recommended for sericulture improvement.The genus Morus (commonly known as mulberry) belongs to the family Moraceae, is a group of dioecious woody trees/shrubs. Many varieties of these species are cultivated on a commercial scale in India, China, Japan and Korea for the sericulture industry.¹ In India, six species are found, namely, M. alba L., M. indica L., M. nigra L., M. atropurpurea Roxb., M. serrata Roxb. and M. laevigata Wall.² Due to higher economic return and greater employment potential, attempts are been made to increase productivity by developing high yielding mulberry varieties. At present, Mysore local, Bomaypiasbari, Kanva-2 (K2), Bilidevalaya, Kajli, Sujanpur-1 (S1), BC (2) 59, C776, RFS-175, S36 and Victory-1 are being cultivated extensively in different parts of India.     

Determination of iminosugars in mulberry leaves and silkworms using hydrophilic interaction chromatography-tandem mass spectrometry

Mulberry 1-deoxynojirimycin (DNJ, a potent α-glycosidase inhibitor) has been investigated thoroughly for its analytical methods and therapeutic potential against diabetes, whereas little attention has been given to other iminosugars such as 2-O-α-d-galactopyranosyl-DNJ (GAL-DNJ) and fagomine. For instance, concentration and composition of these iminosugars in mulberry leaves as well as sericulture products have not been fully characterized due to lack of suitable analytical methods. Here we developed a simultaneous determination method for DNJ, GAL-DNJ, and fagomine using hydrophilic interaction chromatography (HILIC) with tandem mass spectrometry (MS/MS). When mulberry leaf extracts were subjected to HILIC-MS/MS with multiple reaction monitoring (MRM), individual iminosugars could be separated and detected. The developed method is sufficiently sensitive for determining iminosugars in mulberry leaves as well as silkworms, providing new information (e.g., different amounts of iminosugars in mulberry leaf varieties; high DNJ and low GAL-DNJ in the silkworm body, especially in the blood) that is useful for producing iminosugar-rich products for nutraceutical purposes.     

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses

Molecular Biology Reports - Mulberry (Morus alba L.) is the sole food source for the mulberry silkworm, Bombyx mori and therefore important for sericulture industry. Different abiotic stress...     

Isolation of an 1-aminocyclopropane-1-carboxylate oxidase gene from mulberry (Morus alba L.) and analysis of the function of this gene in plant development and stresses response

Mulberry (Morus alba) is an important crop tree involved in sericulture and pharmaceuticals. To further understand the development and the environmental adaptability mechanism of mulberry, a cDNA of the gene MaACO1 encoding 1-aminocyclopropane-1-carboxylate oxidase was isolated from mulberry. This was used to investigate stress-responsive expression in mulberry. Developmental expression of ACC oxidase in mulberry leaves and spatial expression in mulberry flowers were also investigated. Damage and low-temperature treatment promoted the expression of MaACO1 in mulberry. In leaves, expression of the MaACO1 gene increased in cotyledons and the lowest leaves with leaf development, but showed reduced levels in emerging leaves. In flowers, the pollinated stigma showed the highest expression level, followed by the unpollinated stigma, ovary, and immature flowers. These results suggest that high MaACO1 expression may be predominantly associated with tissue aging or senescence in mulberry.     

MulSatDB: a first online database for mulberry microsatellites

Key message

Simple sequence repeat motifs were mined from the genome and EST sequences of Morus notabilis and archived in MulSatDB. Bioinformatics tools were integrated with the database for the analysis of genomic datasets.

Abstract

Mulberry is a crop of economic importance in sericulture, which shapes the lives of millions of rural people among different Eurasian and Latin American countries. Limited availability of genomic resources has constrained the molecular breeding efforts in mulberry, a poorly studied crop. Microsatellite or simple sequence repeat (SSR) has revolutionized the plant breeding and is used in linkage mapping, association studies, diversity, and parentage analysis, etc. Recent availability of mulberry whole genome assembly provided an opportunity for the development of mulberry-specific DNA markers. In this study, we mined a total of 217,312 microsatellites from whole genome and 961 microsatellites from EST sequences of Morus notabilis. Mono-repeats were predominant among both whole genome and EST sequences. The SSR containing EST sequences were functionally annotated, and SSRs mined from whole genome were mapped on chromosomes of the phylogenetically related genus—Fragaria vesca, to aid the selection of markers based on the function and location. All the mined markers were archived in the mulberry microsatellite database (MulSatDB), and the markers can be retrieved based on different criteria like marker location, repeat kind, motif type and size. Primer3plus and CMap tools are integrated with the database to design primers for PCR amplification and to visualize markers on F. vesca chromosomes, respectively. A blast tool is also integrated to collate new markers with the database. MulSatDB is the first and complete destination for mulberry researchers to browse SSR markers, design primers, and locate markers on strawberry chromosomes. MulSatDB is freely accessible at http://btismysore.in/mulsatdb.     

Standard method for detecting Bombyx mori nucleopolyhedrovirus disease-resistant silkworm varieties

Bombyx mori nucleopolyhedrovirus (BmNPV) disease is one of the most serious silkworm diseases, and it has caused great economic losses to the sericulture industry. So far, the disease has not been controlled effectively by therapeutic agents. Breeding resistant silkworm varieties breeding may be an effective way to improve resistance to BmNPV and reduce economic losses. A precise resistance-detection method will help to accelerate the breeding process. For this purpose, here we described the individual inoculation method (IIM). Details of the IIM include pathogen BmNPV preparation, mulberry leaf size, pathogen volume, rearing conditions, course of infection, and breeding conditions. Finally, a resistance comparison experiment was performed using the IIM and the traditional group inoculation method (GIM). The incidence of BmNPV infection and the within-group variance results showed that the IIM was more precise and reliable than the GIM.     

Large-Scale Intersubspecific Recombination in the Plant-Pathogenic Bacterium Xylella fastidiosa Is Associated with the Host Shift to Mulberry

Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.     

Effect of Environmental Conditions on the α-Glucosidase Inhibitory Activity of Mulberry Leaves

Mulberry leaves have been used as the sole food for silkworms in sericulture, and also as a traditional medicine for diabetes prevention. Mulberry leaf components, for example 1-deoxynojirimycin (1-DNJ), inhibit the activity of α-glucosidase and prevent increased blood glucose levels, and they are highly toxic to caterpillars other than silkworms. The α-glucosidase inhibitory activity of mulberry leaves changes with the season, but it is unknown which environmental conditions influence the α-glucosidase inhibitory activity. We investigated in this study the relationship between the α-glucosidase inhibitory activity and environmental conditions of temperature and photoperiod. The results demonstrate that low temperatures induced decreasing α-glucosidase inhibitory activity, while the induction of newly grown shoots by the scission of branches induced increasing α-glucosidase inhibitory activity. These results suggest that the α-glucosidase inhibitory activity was related to the defense mechanism of mulberry plants against insect herbivores.     

Effect of environmental conditions on the α-glucosidase inhibitory activity of mulberry leaves

Mulberry leaves have been used as the sole food for silkworms in sericulture, and also as a traditional medicine for diabetes prevention. Mulberry leaf components, for example 1-deoxynojirimycin (1-DNJ), inhibit the activity of α-glucosidase and prevent increased blood glucose levels, and they are highly toxic to caterpillars other than silkworms. The α-glucosidase inhibitory activity of mulberry leaves changes with the season, but it is unknown which environmental conditions influence the α-glucosidase inhibitory activity. We investigated in this study the relationship between the α-glucosidase inhibitory activity and environmental conditions of temperature and photoperiod. The results demonstrate that low temperatures induced decreasing α-glucosidase inhibitory activity, while the induction of newly grown shoots by the scission of branches induced increasing α-glucosidase inhibitory activity. These results suggest that the α-glucosidase inhibitory activity was related to the defense mechanism of mulberry plants against insect herbivores.     

Variations of cuticular wax in mulberry trees and their effects on gas exchange and post-harvest water loss

Though mulberry (Morus alba) tree shows great adaptations to various climate conditions, their leaf water status and photosynthesis are sensitive to climate changes. In the current study, seven widely planted mulberry cultivars in Chongqing, Southwest China, were selected to analyze leaf cuticular wax characteristics, gas exchange index, post-harvest leaf water status and their relationships, aiming to provide new theory in screening high resistant mulberry cultivars. Mulberry trees formed rounded cap-type idioblasts on the adaxial leaf surface. Film-like waxes and granule-type wax crystals covered leaf surfaces, varying in crystal density among cultivars. The stomatal aperture on the abaxial surface of cultivars with high wax amount was smaller than that of cultivars with low wax amount. The amount of total wax was negatively correlated with the net photosynthetic rate (P _N), transpiration rate (E) and stomatal conductance (g _s) and positively correlated with the moisture retention capacity. It suggested that both cuticular wax and stomatal factor might be involved in regulating water loss in mulberry leaves under field conditions. The variability in moisture retention capacity and cuticular wax characteristics might be important in evaluating and screening mulberry cultivars for increasing silk quality and silkworm productivity.