DNA based diagnostic for the quantification of sugarcane root DNA in the field

J S Pierre,D M Hartley,A L Rae,D Giblot-Ducray,J M Perroux,A C Mckay

doi:10.1038/s41598-018-34844-3

Abstract

Plant root systems play many key roles including nutrient and water uptake, interface with soil microorganisms and resistance to lodging. As for other crops, large and systematic studies of sugarcane root systems have always been hampered by the opaque and solid nature of the soil. In recent years, methods for efficient extraction of DNA from soil and for species-specific DNA amplification have been developed. Such tools could have potential to greatly improve root phenotyping and health diagnostic capability in sugarcane. In this paper, we present a fast, specific and efficient method for the quantification of sugarcane live root cells in soil samples. Previous studies were typically based on mass and length, so we established a calibration to convert root DNA quantity to live root mass. This diagnostic was validated on field samples and used to investigate the fate of the root system after harvest prior to regrowth of the ratoon crop. Two weeks after harvest, the sugarcane roots from the previous crop were still viable. This raises the question of the role that the root system of the harvested crop plays in the performance of the next crop and demonstrates how this test can be used to answer research questions.

Highlights

Plant root systems play a key role in determining nutrient and water uptake and are the interface between soil microorganisms and plants
Results show that the method is robust, specific to sugarcane and can detect small amounts of live roots in soil samples, which will enable high throughput field phenotyping of sugarcane root systems
Since the assay relies on the amplification of the Internal Transcribed Spacer (ITS) sequences in real time PCR, the ITS copy number directly influences the quantification results

Summary

Result

For all varieties and depths tested, the correlations between root dry weight and target DNA copy number all had a coefficient of determination (r2) above 0.95 except one (Q242 50–100 cm depth; r2 = 0.92) Based on these correlations, the assay efficiency was calculated to be on average 97%. The DNA assay was used to assess soil core samples collected from a field trial that was designed to test the effect of different types of nitrogen input on sugarcane yield. The decline in root mass was positively and significantly correlated with the depth of the soil core sample (p < 0.001) whereas time did not have a significant effect (p = 0.442) on the quantity of viable sugarcane root in the soil over the two week period. Root weight density values were calculated using the Q208-50 cm calibration factor and an average soil bulk density of 1.5 g cm−3

Discussion

Findings

Materials and Methods