The SCN bioassay for transgenic lines Y25 E12P2 and J23 E4P1 were conducted for resistance evaluation. Both lines were germinated from T1 seeds. It was accordance with initial screening assay that Y25 E12 P2 and J23 E4P1 were able to reduce the SCN egg population by 55.9% and 44.8%, respectively, compared to non-transgenic controls, cultivar JackX.
Using established RT-qPCR system for the quantitation of transgene expression combined with previous bioassay data, several T1 transgenic lines were selected for offspring seed production.
The plant tissue from SCN bioassays were shipped to UICU for RNA-seq. With the analyses of two bioassays of Y25 and Prp17 genes performed, it indicated that soybean dicer-like (DCL) was able to cleave H. glycines genes’ dsRNAs into 19 to 23 nucleotides (nt) with similar patterns as endogenous siRNA. The analyses also showed that the predominant size of siRNAs was 21-nt, the same as the dominant size of endogenous siRNA sequences of soybean reported in the literature, demonstrating that the cleaving mechanism of soybean DCL was likely the same for endogenous genes and exogenous genes. It was another evidence that host derived RNAi technique to silence nematodes worked in soybean plants.
From RNAseq data, the distribution of target siRNA in different T1 plants was following similar patterns. Compared with patterns of different generations of our transgenic soybeans, the T2 generation of Prp17 had much more unified patterns than T1 generation as well as the Y25 T1 generation. It was consistent with the phenomenon observed that the resistant ability was more fluctuated in T1 generation. Most importantly, based on our results of sequencing and bioassays, it was likely that the expression of small RNA levels were correlated to resistant ability. The resistance ability seems obtained when siRNA reached to about 200 target RPM, and in general, it strongly suggested that the higher target RPM, the higher SCN reduction in the transgenic soybean plant.