ANALYSIS OF DNA METHYLATION PATTERN ASSOCIATED WITH FLORAL BUD DISTORTION IN SOYBEAN (Gylcine max L.).
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Date
2017-08-28
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Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola, Maharashtra.
Abstract
The present investigation entitled, “Analysis of DNA methylation pattern associated with floral bud distortion in soybean (Glycine max L.)” was carried out during 2016-17 at Biotechnology Centre, Department of Agricultural Botany, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola to investigate methylation pattern associated with floral bud distortion in soybean.
DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In the present attempt, cytosine methylation profile was determined in elite soybean (Glycine max L.) cultivar JS-335. Methylation-sensitive amplified polymorphism was used to generate genome-wide methylation profiles and compared the patterns of cytosine methylation in floral bud distorted and asymptomatic plant.
It has been learned that soybean plant is affected by the disorder known as floral bud distortion (FBD), no pod formation take place at R6 stage which results in huge yield loss in symptomatic plant(s). The methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP) used to investigate the epigenetic changes associated with FBD in soybean. The samples of genomic DNA were double-digested with either HpaII or MspI and with EcoRI. To analyze epigenetic changes, 25 MSAP primer combinations detected a few DNA methylation patterns in the plant tissue of symptomatic and asymptomatic plant. A total of 378 and 392 clear and reproducible bands were amplified from symptomatic and asymptomatic samples, respectively. In the symptomatic plant, out of 378 MSAP sites, 273 (72.22%) were found un-methylated, 31 (8.20%) were hemi-methylated, 35 (9.25%) were fully methylated; and 39 (10.31%) hyper-methylated sites were amplified. Whereas, in asymptomatic plant, out of 392 MSAP sites, 315 (80.35%) found un-methylated, 21(5.35%) hemi-methylated, 29 (7.39%) fully methylated and 25 (60.37) hyper-methylated sites were amplified. An increased level of methylation was recorded in symptomatic plant (27.28%) than that of asymptomatic (19.13%). In fact, there was evidence of increase in fully methylated ratio to 19.57% in symptomatic plant and that in case of asymptomatic revealed decrease of 12.75%. These results indicated that there were greater differences of the genomic DNA methylation extent and also variation in the pattern of methylation between the symptomatic and asymptomatic plant. It revealed that the methylation status varied substantially during the development stage of flower.
The polymorphic DNA methylation fragments that showed polymorphic bands were excised from Polyacrylamide gel and cloned in vector pTZ57R/T. However, total 10 polymorphic bands were excised varying from 150 to 250 bp, out of which only 2 bands were successfully purified from the PAGE. Further these methylated fragments were cloned in vector pTZ57R/T (2886 bp).
These findings successfully generated extent and pattern of cytosine methylation in floral bud distorted and asymptomatic plants. In the present investigation, although limited numbers of methylation sensitive polymorphic fragments were obtained this helped to calculate the extent and pattern of methylation associated with floral bud distortion in soybean.
Description
The present investigation entitled, “Analysis of DNA methylation pattern associated with floral bud distortion in soybean (Glycine max L.)” was carried out during 2016-17 at Biotechnology Centre, Department of Agricultural Botany, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola to investigate methylation pattern associated with floral bud distortion in soybean.
Soybean was infected with the disorder known as floral bud distortion. Attempts to understand the symptoms and causal agent were limited because of unawareness about its peculiar symptoms and cause. Therefore present investigation intends to understand the epigenetic changes that have been taken place in soybean. As the epigenetic changes could be due to environmental effect leading to DNA methylation, histone modification, RNAi mechanism of gene silencing.
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Citation
KAD, MISS. SNEHAL KRISHNA. (2017). Analysis of DNA methylation pattern associated with floral bud distortion in soybean (Gylcine max L.). Biotechnology Centre. Department of Agricultural Botany. Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola. M. Sc. 2017. Print. xi, 78p. (Unpublished).