Genetic diversity and structure revealed by genomic microsatellite markers in Centella asiatica (L.) Urb., a plant with medicinal potential

 Abstract

Background: Around the world, medicinal plants are utilised for various purposes. Centella asiatica is one of the important medicinal plants widely used in many medicinal systems. Nevertheless, analysis of the genetic diversity would pave the way for its most suitable utilisation.

Methods and results: The present study analyses the genetic diversity and structure of eighty C. asiatica accessions collected from the southern states of India, using ten genomic microsatellite markers. The mean Nei’s gene diversity (0.46) indicates considerable genetic diversity. Analysis of molecular variance (82.48%) exhibited significant genetic variance between samples within the population. The cluster analysis brought out the structure of the analysed populations as three subpopulations based on the genetic differentiation.

Conclusions: The study showed significant intra-population variation, predominant inbreeding and population differentiation in C. asiatica. The findings will help better understanding of the genetic structure and gene pool of the plant.

Full article link: https://rdcu.be/cAvFI

Sakthipriya M., S. S. Vishnu, S. Sujith, P. Rajeesh Kumar, and K. K. Sabu. 2018. Analysis of genetic diversity of Centella asiatica using SSR markers. International Journal of Applied Sciences and Biotechnology Vol 6(2): 103-109. http://dx.doi.org/10.3126/ijasbt.v6i2.19583

Abstract
Genetic diversity represents the heritable variation within and between populations of organisms. A better understanding of genetic diversity and its distribution is essential for its conservation and use. In this study the genetic diversity analysis was performed in 30 accessions of Centella asiatica (L.) Urb. using 10 SSR markers. C. asiatica is a tropical medicinal plant from Apiaceae family native to Southeast Asian countries. It is among the top herbs in the category of anti-aging and CNS drugs used worldwide. The whole plant and aerial parts are used widely in traditional and alternative medicines due to its ample pharmacological activities. The molecular screening through microsatellite markers showed low polymorphism (0.019) between the samples analyzed. Further studies, including the effect of environmental factors, genetic composition or possibility of inbreeding, are required to analyze the probable reason for the low variability exists in the species.

Int. J. Appl. Sci. Biotechnol. Vol 6(2): 103-109

Keywords: Centella asiatica; genetic diversity; SSR; microsatellites; medicinal plant

Transcriptome mining for EST-INDELs and development of EST-SSR markers in turmeric

Sabu K. K., M. D. Shehenaz and J. Amrutha. 2018. Transcriptome mining for EST-INDELs and development of EST-SSR markers in turmeric (Curcuma longa L.). International Journal of Agriculture, Environment and Biotechnology 11(3): 487-492. http://ndpublisher.in/countpdfdownload.php?id=3040&pdf=IJAEBv11n3i.pdf

ABSTRACT
Curcuma longa L. commonly known as turmeric is used as a culinary spice in India and many Asian countries. Turmeric has anti-fungal, anti-bacterial, anti-malarial and anti-cancer properties to mention a few. We have analyzed the transcriptome of C. longa retrieved from NCBI SRA database (SRR495223) for the development and validation of EST-SSR markers and identification of EST-InDels to be used for C. longa and related genera. A total of 337 primers were developed and 20 primers having the rating of 100% with the help of NetPrimer were selected and used for PCR validation in C. longa, Amomum subulatum Roxb. (large cardamom) and Elettaria cardamomum (L.) Maton (small cardamom). It was found that around 50% primers generated PCR products in both types of cardamom and 85% in C. longa. The developed primers worked with curcuma, large cardamom and small cardamom plants at varying levels. Besides, the transcriptome analysis detected high amount of deletion and 18 addition of bases which could be screened through the development of CAPS markers using the tool SNP2CAPS and it was found that 93 restriction enzyme can be used for screening these InDels.

Highlights

• Transcriptome sequence of Curcuma longa was used for the development of EST-SSR markers.
• The EST-SSR primers were validated in Curcuma longa and its related taxa such as Amomum subulatum and Elettaria cardamomum
• EST-InDels were also successfully identified in the transcriptome sequences of Curcuma longa.

Keywords: Curcuma longa, EST-SSR, EST-InDel, PCR, ClustalW, WebSat, NetPrimer, SNP2CAPS

Rapidly evolving sex-specific sequences in Calamus travancoricus Bedd. ex. Becc. and Calamus nagbettai R.R.Fernald & Dey

Gene sequences mediating sexual reproduction are more divergent within and between closely related species. Microsatellite or simple sequence repeat (SSR) markers are valuable molecular tools for analysis of genetic variability, phylogeny, and also for identifying sex at seedling stage in dioecious plant species. Calamus travancoricus Bedd. ex. Becc. and Calamus nagbettai R.R.Fernald & Dey are economically important rattan species. The dioecious nature of the Calamus spp. limits its breeding and cultivation. The sex of rattans can only be identified after attaining reproductive maturity which ranges from 5 to 15 years. A study was carried out in this background and 9 putative sex-specific PCR products were identified as sex markers for C. travancoricus and C. nagbettai and sequenced by Sanger method. The sequence homology search revealed occurrence of identical sequences in many plant species across different families indicating the conserved nature of the sequences. However, these sequences were not present in opposite sex in the studied species, indicating divergent evolution favoring sex determination. Annotation of these sequences revealed that most of these are mediating sexual reproduction by and large. An adequate sex ratio is to be maintained for these dioecious palms in natural habitat for producing offsprings having equal gene complements for continual evolution and sustainable utilization. Developing scientific management strategies and improved utilization of canes could help to generate employment locally and thus contribute to the socioeconomics sustainably.

https://link.springer.com/article/10.1007%2Fs11295-017-1220-2

Development and validation of EST-SSR and identification of EST-SNP markers for snake gourd (Trichosanthes cucumerina var. cucumerina L.)

Snake gourd is a natural antibiotic and the plant is commonly used in folk medicines throughout India. The present study was carried out using transcriptomic sequence of Trichosanthes kirilowii Maxim. for the development and validation of EST-SSR markers and EST sequences of T. dioica Roxb. for identification of EST-SNPs in T. cucumerina var. cucumerina L. A total of 382 primers were developed and out of which 58 were found to be suitable for genetic analysis. For experimental validation, 20 primers were randomly selected and amplified in T. cucumerina var. cucumerina and two related genera, viz. Coccinia grandis and Momordica charantia. It was found that 90% of the primers generated PCR products in T. cucumerina var. cucumerina and considerable cross generic amplification in M. charantia and C. grandis. Further, identification of EST-SNPs in T. cucumerina var. cucumerina was also carried out. The SNPs detected were converted to CAPS markers using the tool SNP2CAPS. The newly developed SSR and SNP markers offer novel additions to the genomic resources of this important medicinal herb for population studies, phylogenetics analyses and estimation of genetic diversity.

Citation: Silpa S, Shinsy M Y, Sabu K K. 2016. Development and validation of EST-SSR and identification of EST-SNP markers for snake gourd (Trichosanthes cucumerina var. cucumerina L.). Abrahamia 2: 51-56

Development and Cross-genera Transferability of Ginger EST-SSR Markers for Cardamom

Background: Cardamom (Elettaria cardamomum Maton) is an important commodity spice that comes under the ginger family (Zingiberaceae). Several genetic markers are widely being used to analyze plant genomes. However, genetic mapping of cardamom has never been attempted owing to the lack of sufficient number of high quality genetic markers and other pertinent genome information.

Objective: The goals of the present study were to design SSR markers from the EST sequences of ginger (Zingiber officinale) and to validate the same in cardamom for demonstrating cross generic transferability. Method: 38,116 expressed sequence tags of ginger downloaded from the NCBI dbEST database were used to develop and validate co-dominant, multi-allelic SSR markers.

Results: A total of 1214 SSRs including mono, di, tri, tetra and hexa repeats were identified in the study. The validation through SSR-PCR followed by agarose gel electrophoresis was carried out for the developed markers. Genetic analysis of the SSR markers showed polymorphism and it clearly differentiated wild genotypes from cultivars and wild escapes from the plantations. Large cardamom, Amomum subulatum Roxb. was used as an outgroup and the newly developed EST-SSR markers were amplified well in this species.

Conclusion: The newly developed EST-SSRs could be useful as reproducible markers for cardamom genetic studies.

See https://benthamscience.com/journals/current-bioinformatics/article/147712/

ISSR and SSR markers reveal sex-specific DNA sequences in three Calamus species from India

Calamus species yields the raw materials for the cane industry. However, the extraction of the cane from the forests is being carried out indiscriminately without considering the sustenance of the species. Plants of both sexes should co-exist for reproductive success. The sex of Calamus plants can be identified only after flowering and hence proper planning of managed forestry practices cannot be accomplished. A study was carried out in this background and male specific ISSR markers for C. tenuis and C. flagellumand SSR markers for C. thwaitesii were identified. The diagnostic potential of these markers can be exploited to sex the Calamus species at the seedling stage for proper breeding and agroforestry management.

This article has just been published:

Title ISSR and SSR markers reveal sex-specific DNA sequences in three Calamus species from India Journal Agroforestry Systems, (), 1-5 DOI 10.1007/s10457-016-9952-9

This article is available as 'Online First':  http://link.springer.com/article/10.1007/s10457-016-9952-9

Development of EST-SSR Markers to Assess Genetic Diversity in Elettaria Cardamomum Maton

Elettaria cardamomum Maton is one of the most ancient and valuable spice crops. Cardamom is cultivated following intensive pesticide usage where alleles present in the wild cardamom genotypes could positively contribute towards genetic improvement of the cultivars. However, the genetic map or whole-genome sequence of E. cardamomum is not available and very limited information on simple sequence repeat (SSR) markers are publicly available. We have tested whether SSRs from Curcuma longa can be used to analyze genetic diversity E. cardamomum.

Int J Appl Sci Biotechnol, Vol 3(2): 188-192

N Anjali, Sowmya S Dharan, F Nadiya, K K Sabu

DOI: http://dx.doi.org/10.3126/ijasbt.v3i2.12380  

Keywords: EST-SSR; microsatellite; genetic diversity; Elettaria cardamomum; cardamom; marker development

Full Text: PDF

UKM Scientists Developed High Yielding Superior Red Rice

After seven years of research UKM’s scientists with the cooperation of MARDI’s research officers have successfully produced a variety of rice which not only can increase padi yield but also has properties with low gylcaemic index suitable for diabetics.

Its Plant Genetics and Biotechnology expert, from the Faculty of Science and Technology, Prof Dr. R. Wickneswari Ratnam assisted by some 14 other scientists from UKM, Malaysia Agricultural Research and Development Institute (MARDI), Universiti Putra Malaysia (UPM) , Malaysia Nuclear Agency (MNA) and UM had been doing research on this since 2002 and have succeeded in producing the new padi variant G33 named UKMRC9 which can increase local red rice production.

Prof Wickneswari described it as a superior red rice developed through conventional breeding involving controlled cross-breeding between cultivar MR219 and wild rice Oryza rufipogon. It involved the transfer of genes of the wild type to the common paddy produced by MARDI now extensively cultivated in the country.

She was assisted by Dr K K Sabu, Dr. Lim Li Sze, Dr. Atiqur Bhuiyan, Dr. Abdul Rahim Harun, En. Parviz Fasahat, Puan Ngu Mee Siing, En. Abdullah M Zain, Puan Site Zuraini Abdul Rahman, Dr. Narimah Md Kairudin, Dr. Tilakavati Karupiah, Prof. Aminah Abdullah, Dr. Kharidah Muhammad, Chua Khun Aik and Tan Choon Heen in planning and carrying out the seven years of research. Read full article.

Genetic diversity among cardamom germplasm accessions assessed by SSR and ISSR analysis

Abstract: Cardamom (Elettaria cardamom Maton; Zingiberacea), the ‘Queen of spices’ is one of the most important and highly priced spices. The important areas of cultivation in Kerala are Nelliampathy, Wynad and Idukki. The origin of cardamom is believed to be in the Western Ghats, where presumably rich and untapped genetic diversity of the cardamom still resides. Biodiversity present in the wild relatives of crop plants in Kerala have not yet been addressed properly (Nayar, 2011). The present study aims to collect and evaluate genetic diversity among wild cardamom populations in comparison to the cultivated counterparts.

Citation

Ashitha J P, Anija T A, Anjali, N and K K Sabu. 2013. Genetic diversity among cardamom germplasm accessions assessed by SSR and ISSR analysis. Proc of the 25th Kerala Science Congress held at Technopark at Thiruvananthapuram during 29th January to 1st February 2013, pp. 263-265 (03-27).

Link to Google Scholar citation

Identification and Validation of Quantitative Trait Loci for Agronomic Traits in Advanced Backcross Breeding Lines Derived from Oryza rufipogon × Oryza sativa Cultivar MR219

Ratnam Wickneswari, M. A. R. Bhuiyan, Sabu Kalluvettankuzhy K., Li Sze Lim, Michael J. Thomson, Md. Kairudin Narimah and Md. Zain Abdullah

Abstract 
A backcross breeding strategy was used to identify quantitative trait loci (QTLs) associated with 14 traits in a BC2F2 population derived from a cross between MR219, an indica rice cultivar and an accession of Oryza rufipogon (IRGC 105491). A total of 261 lines were genotyped with 96 microsatellite markers and evaluated for plant morphology, yield components and growth period. The genetic linkage map generated for this population with an average interval size of 16.2 cM, spanning 1,553.4 cM (Kosambi) of the rice genome. Thirty-eight QTLs were identified with composite interval mapping (CIM), whereas simple interval mapping (SIM) resulted in 47 QTLs (LOD >3.0). The O. rufipogon allele was favourable for 59% of QTLs detected through CIM. Of 261 BC2F2 families, 26 advanced backcross breeding lines (BC2F5) were used for QTL validation. These lines were selected on the basis of the yield traits potentiality in BC2F3 and BC2F4 generations. The field trial was conducted at three different locations in Malaysia using randomized complete block design with three replications. Trait based marker analysis was done for QTL determination. Twenty-five QTLs were detected in BC2F5 generation whereas 29 QTLs were detected in BC2F2 generation of the same population. Two QTLs (qPL-1 and qSPL-7) were not considered for validation due to their low R 2 values and two QTLs (qPSS-3-2 and qGW-3-2) were not detected in the BC2F5 population. Fifteen QTLs showed the beneficial effect to enhance the trait value of the breeding lines. QTL validation aided to select the promising lines for further utilization. [More details]

Keywords
Backcross breeding – Composite interval mapping – Oryza rufipogon – Oryza sativa – QTL identification – QTL validation – Trait-based marker analysis

Genetic studies in rattan palms (Calamus sp.) using SSR markers

Natural variation is essential for conservation and utilization of plant genetic resources. Human interference on forests may cause depletion of natural genetic variation in the gene pool and even to genetic erosion of certain species. Rattans are one such species currently under crisis due to over-exploitation. They represent high value non-wood forest produce primarily used for making furniture, handicraft items and an important raw material in the cottage industry which contributes significantly in rural economy. There are about 1 lakh people in Kerala living out of this valuable resource. However, number of small scale industrial (SSI) units depending on these forest produce are decreasing drastically.  Besides, over exploitation of the rattans has exerted severe pressure on existing populations and sometimes resulted in loss of genetic diversity.

There are about 600 rattan species in 13 genera globally and 51 species in 4 genera in India, of which 75% are endemic and 40.5% threatened. The Western Ghats region of India is rich in species diversity of rattans with one genus, viz. Calamus having 27 species of which 21 are identified.

Presently associated (as Principal Investigator) to a research project on ‘Genetic studies on selected rattan palms (Calamus sp.) using microsatellite markers’.

Objectives of the project are to collect natural populations of the selected rattan palms, viz. C. brandisii, C. gamblei, C. nagabettai, C. thwaitesii, C. travancoricus and C. hookerianus and analyze genetic diversity, population structure and differentiation using SSR markers.

The project is being funded by Kerala Biotechnology Commission/KSCSTE.

Genetic Structure of Oryza rufipogon Griff. Natural Populations

Genetic Structure of Oryza rufipogon Griff. Natural Populations in Malaysia: Implications for Conservation and Genetic Introgression of Cultivated Rice

Abstract
Thirty polymorphic Oryza sativa microsatellite loci (SSRs) were used to study population genetic structure of O. rufipogon Griff. natural populations in Malaysia. A total of 445 alleles were detected with an average of 14.8 alleles per locus in 176 individuals of O. rufipogon sampled from the states of Penang, Kedah, Kelantan and Terengganu where the natural populations are still found. The Kelantan population in the northeast of Peninsular Malaysia had the highest level of genetic diversity as measured by the mean number of alleles per locus, Aa=7.67, average number of effective alleles, Ae=5.50, percentage of polymorphic loci, P=100%, observed heterozygosity, Ho=0.631 and expected heterozygosity, He=0.798. In contrast, the Terengganu population in the east showed the lowest level of genetic diversity measured by the same criteria (Aa=4.23, Ae=2.10, P= 100%, Ho=0.549 and He=0.449). Model–based clustering analysis using the STRUCTURE 2.2 program placed all the individuals into 12 clusters that corresponded to the geographic sampling locations. Neighbour-joining tree was constructed based on Nei’s genetic distance to further assess the genetic structure of the O. rufipogon individuals, showed good agreement (93.8%) with the model-based cluster analysis. However, the neighbour-joining tree identified subpopulations that STRUCTURE could not identify. The classification of individuals from the same populations under the same cluster supported the population differentiation. These two analyses seemed to indicate expansion of populations from the northeast of Peninsular Malaysia (Tumpat, Pasir Mas and Kota Bahru, Kelantan) not only to the immediate south of the region i.e. Terengganu but also into the northwest (i.e. Penang and Kedah) with the former being more recent. Oryza rufipogon accession IRGC105491 and O. sativa ssp. indica cultivar MR219, which were included in this study for comparisons with the local wild rice accessions, indicated that introgression of cultivated rice could change genetic composition and affect the population genetic structure of wild rice. This possibility should be carefully considered in plans to conserve this wild rice.

Published in:
Tropical Plant Biol. (2010) 3:227–239

Postdoc research: Improvement of rice yield through molecular breeding

ABSTRACT
This research was conducted to identify possible trait enhancing quantitative trait loci (QTLs) from common wild rice, Oryza rufipogon. Two hundred and sixty-six BC2F2 families were developed from an interspecific cross between O. rufipogon accession no. IRGC 105491 and O. sativa cv. ‘MR219’during 2002-2003. Field evaluation of the BC2F2 families for 18 yield and yield related traits was carried out in 2004 at Malaysian Agricultural Research and Development Institute, Seberang Perai. Ninety three SSR (simple sequence repeat) markers were used to create a genetic linkage map spanning the twelve rice chromosomes. Ninety one QTLs were detected by means of marker regression with a significance level of p < 0.01. Among the putative QTLs, 21 were significant and 70 were suggestive. QTLs having favourable effects were noticed for days to heading (decrease heading period up to 6%), grain filling (increased filled grains plant-1 up to 12%), grain yield (increased grain yield plant-1 up to 10%), resistance to bacterial leaf blight (increased resistance by 13%), and tolerance to brown planthopper infection (increased tolerance by 40%). Quantitative data analysis suggests that about 10% of the total of 266 BC2F2 families demonstrated nearly 27% higher yield potential in comparison to the parental cultivar ‘MR219’. This increase is mostly due to higher tillering ability which is characteristic of O. rufipogon. These families also showed a considerable increase in tolerance against bacterial leaf blight and brown plant hopper infestation. The selected BC2F2 families can offer an efficient source of genetic diversity for future breeding programmes. The study concludes that even though wild rice is inferior to most of the agronomic traits, they could positively contribute to the overall yield enhancement in cultivated rice.