Soil Microbial Diversity by DNA Analysis By: Jonjala Jackson Mentors: Dr. Elica M. Moss, Dr.Shengzuo Fang, and Prof. Ye Tian

 Poplar plantations have been established in upland areas of southwestern China in recent years  However, poor site conditions limit the growth and ecological function of poplar plantations.  Therefore, thinning practices of poplar plantations are being studied at the Chenwei Forestry Research Station.

 Thinning is the cutting down and removal of a section of trees in a forest crop  Thinning is done to provide more growing space for the remaining trees, which in turn leads to an increase in volume of individual trees  There are numerous methods of thinning which includes selective and systematic thinning

 Selective thinning involves trees being removed or retained on their individual value  Systematic thinning requires the removal of trees according to a predetermined system, such as line thinning  Line thinning, which is a combination of both systems, proves to be the most efficient management method

 This research is being conducted at Nanjing Forestry University to determine what direct effects thinning has on ◦ nutrient cycling, tree growth, forest microclimate, undergrowth plant diversity, amount and nutrient of litter, and soil microbial characteristics  The effects of thinning on soil microbial diversity will be determined through using DNA Analysis

Poplar Plantation

 To determine the effect of thinning on soil microbial diversity  To examine the effects of thinning on soil microbial activities and thus affect soil organic matter turnover

 The Chenwei Forestry Research Station is located in Sihong County near Jiangsu Province  The latitude of the experimental site is 33°32N, the longitude is 118°36E. The mean annual precipitation is mm, more than 80% of which falls during June to August. The mean annual temperature is 14.4℃.

泗洪 Nanjing city Site location Sihong county The map of Jiangsu province

 Field trips were arranged in April 2012 to collect the soil samples  The soil samples were taken from the upper 0– 5, 5-10 and cm soil layers  The collected samples were brought to the laboratory and refrigerated at 4 °C until the time of the analyses

 Soil samples were prepared for analyses by grinding the samples with a mortar and pestle and made to pass a 2 mm-sieve  The samples were then bagged in plastic bags and refrigerated at 4 °C until the time of the analyses

Break down the cell wall and membranes Centrifuge to separate the solids from the dissolved DNA Precipitate the DNA using isopropanol Centrifuge to separate the DNA from the dissolved salts and sugars Wash the DNA pellet with Ethanol and dry the pellet Dissolve DNA Overview of DNA Extraction Materials and Methods: Laboratory Work

 1% agarose gel was made with wells for sample placement. 1 g of agarose powder was placed into XTBE buffer and microwaved. 1 µl Ethium Bromide was added to solution and mixed/swirled. A comb was placed into the electrophoresis tray and the hot agarose mixture was poured into the tray. The agarose mixture cooled with comb inside (to make wells). 2 µL of Bromophenol Blue was added to 7µL of each DNA sample and placed in a well. The electrodes were attached and electrophoresis ran at 80 volts for 120 minutes. The gel was removed from the solution and placed in Bromophenol Blue staining solution for 30 minutes.

This picture depicts an image of the agarose gel used to verify results obtained from the PCR

 The initial analysis shows that after thinning, significant amounts of DNA appear to be present in samples  There are only small amounts in  Future analysis will include DGGE to determine the presence of ammonium oxidizing bacteria

 Impacts of thinning on selected soil ecological properties could be found but further research is required with respect to microbial activity and community structure  Despite these limitations, DNA-based methods such as DGGE are powerful tools that can provide insight into microbial community composition

 Alabama A&M University/Crest  Nanjing Forestry University  National Science Foundation  Dr. Elica M. Moss  Dr. Shengzuo Fang  Professor Ye Tian  Graduate Students of Dr. Fang  Rakeyta Scales

Questions?