qPCR data of ALS gene copies expression in Echinochloa spp.

Published: 1 September 2021| Version 1 | DOI: 10.17632/gscx45tk8t.1
Contributor:
Silvia Panozzo

Description

The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors, in particular E. crus-galli and E. oryzicola. Molecular characterization led to identification three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations involved in ALS-inhibitor resistance in the different species were associated to the same gene copy (ALS1), which resulted to be significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved with herbicide resistance. This study was used to analyze the relative expression of the ALS gene copies: ALS1 carrying the mutations responsible for target-site resistance to the ALS inhibitor penoxsulam with respect to the expression of the other ALS gene copies (ALS2-3). Comparisons also were made among populations belonging to different Echinochloa species, having different resistance pattern and collected in sites far from each other. Two susceptible populations (16S and 161S) and four resistant populations (42R, 44R, 45R and 46R=102R) were included in the analyses. Among these populations, one belonged E. crus-galli species (16S) and the others belonged E. oryzicola species. They were collected in different rice areas of Italy: 16S, 161S, 42R and 44R were collected in the main Italian rice area, i.e. Lombardy and Piedmont, whereas 45R was collected in Sardinia and 46R in Emilia Romagna. Among the eight reference genes analyzed, the two with expression, on average, higher and with more primer specificity were chosen as reference genes for the gene expression study, Rubisco and 18S. Specific primers for the different ALS gene copies were designed based on an ALS gene part including two allele-specific SNPs: F1_590Tm 5’-GAGCACACACATACTTGGGGCAT-3’, F2_590Cm 5’-AGCACACACATACTTGGGGCAC-3’, R1_621 5’-GAGCATCTTCTTAATTGCTGCACGG-3’, R2_621 5’-GAGCATCTTCTTGATTGCTGCACGT-3’. cDNA diluted at 1:25 of three herbicide-treated (T) and three non-treated (NT) plants per population (in three technical replicates) were tested in the qPCR analyses to determine the relative expression of the different ALS gene copies using two reference genes. In this repository, we reported the amplification plots and dissociation curves obtained from: - the analyses of the two reference genes, Rubisco and 18S, in all populations included in the experiment; - the analyses of ALS genes with the specific primers for all populations included in the experiment.

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qPCR reactions were carried out in three technical replicates using the SYBR Green® kit in a 7300 Real-Time PCR System (Applied Biosystems) on 96-well plates PCR-96M2-HS-C® (Axygen) with a sealer MicroAmp® Optical Adhesive Film (Applied Biosystems). The reactions were carried out in a final volume of 20 μL consisting of 10 μL of cDNA (diluted 1:25), 2 μL of 10X buffer, 1.2 μL of MgCl2 solution (50 mM), 0.5 μL of dNTPs (10 μM of each nucleotide), 2 μL of SYBR Green® (Invitrogen), 0.2 μL of ROX Reference Dye (Invitrogen), 0.1 μL of Taq Platinum® (Invitrogen) and 0.4 μL of each of forward and reverse primers (10 μM). The amplification steps included an initial cycle of 95 °C for 5 min, followed by 40 cycles including a three-step amplification sequence (94 °C for 15 s, 60 °C for 10 s, 72 °C for 15 s), a fourth step where the instrument detects the fluorescence emitted at each cycle (60 °C for 35 s), and a final elongation step of 95 °C for 15 s and 60 °C for 60 s. Data were analyzed using the Sequence Detection Software (SDS) 1.4 and the Ct values means, the standard deviation, and the confidence interval per treatment were calculated. The relative expression was calculated on the mean Ct values using the ∆Ct method.