An efficient early-pooling protocol for environmental DNA metabarcoding

Environmental DNA (eDNA) metabarcoding, a method that applies high-throughput sequencing and universal primer sets to eDNA analysis, has been a promising approach for efficient, comprehensive biodiversity monitoring. However, significant money-, labor-, and time-costs are still required for performing eDNA metabarcoding. In this study, we assessed the performance of an “early-pooling” protocol (a protocol based on 1st PCR tagging) to reduce the experimental costs of library preparation for eDNA metabarcoding. Specifically, we performed three experiments to investigate the effects of 1st PCR-tagging and 2nd PCR-indexing protocols on the community composition revealed by eDNA metabarcoding, the effects of post-1st PCR exonuclease purification on tag jumping (corresponds to index hopping in 2nd PCR indexing), and the effects of the number of PCR replicates and the eDNA template volume on the number of detected OTUs. Analyses of 204 eDNA libraries from three natural aquatic ecosystems and one mock eDNA sample showed that (i) 1st PCR tagging does not cause clear biases in the outcomes of eDNA metabarcoding, (ii) post-1st PCR exonuclease purification reduces the risk of tag jumping, and (iii) increasing the eDNA template volume may increase the number of detected OTUs and reduce variations in the detected community compositions, similar to increasing the number of 1st PCR replicates. Our results show that an early-pooling protocol with post-1st PCR exonuclease purification and an increased amount of the DNA template reduces the risk of tag jumping, the costs for consumables and reagents (except for many tagged 1st PCR primers), and the handling time in library preparation, and produces similar results to a 2nd PCR-indexing protocol. Therefore, once a target metabarcoding region is selected and a set of tagged-1st PCR primers is prepared, the early-pooling protocol provides a cost, labor, and time-efficient approach for processing a large number of samples

Effect of salinity on Echinochloa crus-galli germination as affected by herbicide resistance

Salinity is one of the major abiotic stresses that may affect yield and quality of crops. Salinization, in combination with the presence of aggressive weeds, such as barnyard grass (Echinochloa spp.), can be considered one of the factors responsible for reducing yield in rice fields. The aims of the study were to evaluate the salt effect on germination and first seedling growth of six different Italian common barnyard grass (E. crus-galli) populations (three sensitive and three resistant to ALS-inhibitor herbicides) and to verify the presence of differences in salt response between populations sensitive and resistant to the ALS-inhibitor herbicides. Germination tests were conducted under nine different NaCl concentrations (from 0 mM to 400 mM). Significant differences in germination capacity were found between sensitive and resistant populations from 0 mM to 250 mM NaCl; in particular, germination capacity of the sensitive populations was higher (up to 90%) than that of the resistant ones (about 70%). The increase in salinity over 250 mM reduced progressively the germination capacity: from 300 mM onwards, no significant differences were found between sensitive and resistant populations and the germination resulted inhibited for two of them (one sensitive and one resistant). Speed of germination and root and shoot length of seedlings were also inversely related to salt concentration. Time required for achieving 50% of final germination capacity was extended from about three days at 0 mM NaCl up to about 10-12 days at 400 mM NaCl. Root length and shoot length ranged from 9.88 cm and 6.16 cm, at 0 mM NaCl, to 0.36 cm and 0.41 cm, at 400 mM NaCl. According to the results, there is no a clear evidence that response to saline conditions was related to resistance towards ALS-inhibitor herbicides, as in some cases significant differences were found between populations showing a similar herbicide sensitivity. Responses of barnyard grass to salinity are may play a role in the importance of this weed in future scenarios of salt intrusion: for example, a lower speed of germination at increasing salt levels could suggest a delayed emergence of this weed during crop establishment and first growth. To evaluate the real consequences in terms of competitions towards the crop, future studies are needed for assessing the response to salinity of the main rice varieties cultivated in the environment in which the E. crus-galli populations tested in this study were collected