TARGET REGION AMPLIFICATION POLYMORPHISM BASED PROFILING OF SALT STRESS RESPONSIVE CANDIDATE GENES IN RICE
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Date
2021
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DRPCAU, PUSA
Abstract
The present study was undertaken to examine the molecular level genetic polymorphism in relation to salt tolerance ability among 18 genotypes of rice using 10 candidate gene specific a total of 20 TRAP markers. Experimental procedure adopted in this study was designed to ascertain the genetic variability and diversity with respect to salt tolerance amongst different rice genotypes. Using 20 TRAP primer pairs involving salt stress responsive ten candidate genes specific 10 fixed forward primers in combinations with 2 arbitrary reverse primers, reproducible amplification was successfully achieved in 18 rice genotypes. Molecular level genetic polymorphism was clearly recognized by the deviation in the physical position of the products during their resolution through electrophoresis as well as variation in the number of products generated between genotypes. Differential amplification pattern was observed amongst the genotypes and 610 polymorphic bands representing easily recognizable different types of amplified products were categorized into 291 unique products and 319 shared products. Polymorphism information content of 20 TRAP markers arrayed from 0.745 to 0.940 with an intermediate value of 0.891 across all the combinations of fixed and arbitrary primers.
Arbitrary reverse primer ME02 in combination with candidate gene specific fixed forward primers CDMK produced the least number of polymorphic variants. Similarly, another arbitrary primer ME05 produced the least number of polymorphic
variants in combination with the fixed forward primer OsHKT1; 5. However, the forward fixed primer OsHKT1; 5 produced the highest number of polymorphic variants in combination with the arbitrary reverse primer ME02. The reverse arbitrary primer ME05 yielded the highest number of polymorphic variants in combination with the forward fixed primer CCC. The primer combination SHMT1 with ME05, OsHKT2;3 with ME02, OsHKT1;3 with ME05 and OsHKT1;1 with ME02 yielded remarkably larger allelic size difference in descending order of magnitude.
Numerical variation in polymorphism information content of the markers reflected their allelic diversity and distribution frequency among the genotypes. The primer combinations OsHKT1;5 with ME02, OsHKT1;1 with ME05, OsHKT1;3 with ME05, SNAC1 with ME05 and CCC with ME05 appeared to be highly polymorphic and informative with above average values for the number of polymorphic products, number of unique products, marker index, resolving power and polymorphism information content. These primer combinations also recorded higher values for polymorphism per cent.
Experimental results clearly reflected the existence of considerable genetic diverseness at the molecular level amongst the salt tolerant and susceptible genotypes evaluated in the present study. Hierarchical classification pattern based on target region amplification polymorphism related genetic similarity among rice genotypes was in complete agreement with principal coordinate analysis dependent two-dimensional spatial distribution pattern of genetic profiles of rice genotypes. Using both the modules, the genotypes were discriminated into five multi-genotypic groups highly consistent with their salinity tolerance status. Salt responsive candidate gene based target region amplification polymorphism analysis was shown to be extremely efficient in differentiating rice genotypes based on salt tolerance status. As a result, these markers may be used to discriminate between different salt stress responsive rice genotypes and to choose genetically differentiated parental genotypes for the purpose of genetic enhancement in relation to salt tolerance capacity.