Genetic Diversity Analysis of Trichoderma Isolates Using Ssr Marker

Abstract

Trichoderma is a genus of fungi in the family Hypocreaceae found in all types of soils and is the most common culturable fungus. They occur worldwide and can be easily isolated from soil, plant organic matter, decaying wood etc. It is a well-known aggressive biocontrol agent and has a higher potential for reproduction and colonisation. In this study the genetic diversity among fourteen Trichoderma isolates (TBT- 01 to TBT- 14) was evaluated using ten SSR markers. Rhizospheric soil samples from various locations of Uttar Pradesh were collected and subjected to serial dilution and plating on a Trichoderma Selective Media. The isolates’ colonies were pure cultured on Potato Dextrose Agar for DNA isolation and molecular studies. Ten SSR primers (SSR-01, SSR- 06, SSR- 08, SSR- 10, SSR- 11, SSR-13, SSR- 14, SSR- 15, SSR- 18, and SSR- 20) were used for the PCR amplification of the DNA isolated. The study revealed that, a total of 15 alleles were obtained across the 10 primers among which 13 polymorphic and 2 monomorphic alleles were obtained. Primers SSR- 01 and SSR- 15 showed monomorphism while all other primers possessed polymorphism of varying levels. The Jaccard’s similarity coefficient calculated ranged from 0.429 to 0.929. Based on the similarity matrix the highest similarity (0.929) was observed among isolates TBT- 01 and TBT- 02, TBT- 01 and TBT- 08, TBT- 02 and TBT- 08 and isolates TBT- 13 and TBT- 14 while minimum similarity was observed among the isolates TBT- 03 and TBT-12 and also TBT- 05 and TBT- 12. The dendrogram constructed using the NTSYS software divided the isolates into a major and minor cluster. The minor cluster B comprised only one isolate TBT- 05 while the major cluster A comprised of all the other 13 isolates divided into two sub-clusters. Within the two sub-clusters A1 and A2 further sub-clustering was observed at an individual level. Isolates TBT- 01, TBT- 02 and TBT- 08 formed a single cluster, revealing their relatedness to each other while isolates TBT- 13 and TBT- 14 grouped together into one cluster indicating that they were closely related. The study revealed a significant level of genetic diversity among the isolates.