*2.7. Alternative Splicing Transcripts and Novel Genes*

Alternative splicing (AS) creates more versatile regulating RNAs and translatable mRNAs, thus a more enriched diversity in transcriptome and possible code proteins [19]. In this experiment, a total of 68,385 AS events, derived from 12,044 annotated genes, had been revealed (Figure 6A,B). Among them, the retained intron was the most common event type (34.4%–34.9%), followed by the alternative 3- splicing sites (31.3%–31.7%), alternative 5 splicing sites (17.6%–17.8%), skipped exon (12.7%–12.8%), a mix of two or more different types of AS (complex, 2.6%) and mutually exclusive exons (0.8%). The number of AS events between the varieties and among N rates did not show any statistically significant difference. However, the AS events relative to clean reads in all category type was consistently 1–4% higher in NPB than that in YD6, which probably reflected the varietal differences.

Totally 352 novel genes were found in the RNA-seq data. Among them, 74 and 174 genes were unique to NPB and YD6, respectively; 104 genes were common in the two varieties (Figure 6C). Novel genes responsive to LN were 58 and 89 specific to NPB and YD6 respectively; 55 were common in the two varieties (Figure 6D). These corresponding novel transcripts numbers were 48, 102 and 60 in MN, 48, 93 and 52 in HN, respectively (Figure 6E,F). These results suggest that deep sequencing may reveal more tissue-specific transcripts. Comparison of the novel gene number among N rates within a variety also revealed similar trends, though the numbers were always bigger in YD6 than that in NPB (Figure 6G,H). The novel genes revealed were always more in the *indica* variety YD6 than the *japonica* variety NPB, probably partially attributable to that the transcript annotation in the *japonica* subspecies was more fully covered in the reference genome.

**Figure 6.** Profiles of alternative splice (AS) and novel genes. (**A,B**) Events and types of AS; (**C–H**) Novel genes between varieties in general (**C**); at LN (**D**); at MN (**E**); at HN (**F**); Novel genes in response to N rate in NPB (**G**); Novel genes in response to N rate in YD6 (**H**).
