Echinochloa crus-galli (L.) P. Beauv. (barnyardgrass) is one of the most noxious weeds infesting Italian rice fields. It is characterized by high genetic intraspecific variability and has developed resistance to several classes of herbicides. The aim of our study was to assess, for the first time in Italy, the genetic diversity in E. crus-galli from differently managed rice fields in the Lombardy region (Northern Italy) using eight specific SSR markers. To this purpose, an amplification protocol was optimized, testing different DNA concentrations, PCR mixtures, and temperatures. A total of 48 alleles were identified in 144 samples. SSR fingerprint analysis using R 3.6.3 software (poppr, polysat, and StAMPP) allowed us to handle SSRs as codominant and polyploid data. The results suggested that genetic richness and diversity were high. The analysis of molecular variance (AMOVA) indicated that genetic variation exists mainly between agronomic managements (47.23%) and among populations (37.01%). Hierarchical clustering and PCoA were in concordance with the identification of four distinct genetic groups. Our results confirm that SSR markers represent a valuable and affordable tool for the assessment of E. crus-galli genetic diversity and would grant useful information to plan more targeted, effective, and sustainable control strategies against barnyardgrass. The improved methodology applied here allowed us to assess the genetic variability of an allo-hexaploid species without information loss and biased results.

Several mechanisms involved in weed herbicide resistance are unknown, particularly those acting at the epigenetic level, such as the capacity of small-non-coding RNAs (sncRNAs) to target messenger RNAs of genes involved in herbicide detoxification. The transcription of these sncRNAs is stimulated by epigenetic factors, thereby affecting gene expression. This study was carried out in order to evaluate, for the first time in Echinochloa crus-galli (L.) P. Beauv. (barnyardgrass), the capacity of miRNAs to regulate the expression of genes associated with bispyribac-sodium detoxification. The expression profiles of eight miRNAs with a high degree of complementarity (≥80%) with mRNAs of genes involved in herbicide detoxification (CYP450, GST and eIF4B) were determined by qRT-PCR before and after herbicide spraying. Five of the miRNAs studied (gra-miR7487c, gma-miR396f, gra-miR8759, osa-miR395f, ath-miR847) showed an increased expression after herbicide application in both susceptible and resistant biotypes. All the miRNAs, except gra-miR8759, were more highly expressed in the herbicide-resistant biotypes. In specimens with increased expression of miRNAs, we observed reduced expression of the target genes. The remaining three miRNAs (ata-miR166c-5p, ath-miR396b-5p and osa-miR5538) showed no over-expression after herbicide treatment, and no difference in expression was recorded between susceptible and resistant biotypes. Our results represent a first overview of the capacity of miRNAs to regulate the expression of genes involved in bispyribac-sodium detoxification in the genus Echinochloa. Further research is required to identify novel miRNAs and target genes to develop more focused and sustainable strategies of weed control.