Phenotypic traits of the radish ls mutant
Our research group previously established a radish mutagenesis system and constructed a radish mutant pool (Li et al., 2019); after manyyears of self-fertilization, they were used to screen the radish ls mutant with stable silique and ovule phenotypes (Fig.1). There were significant differences between the ls mutant and WT radish in silique length and number of seeds per silique (Fig.2A–F). Specifically, the average length of silique in the ls mutant was 20.50 cm, compared to 9.10 cm in the WT radish (Fig.2G), and the average number of seeds per silique in the radish ls mutant was 9.5, whereas the average number of ovules of WT radish was 4.5 cm (Fig.2H). The statistical results showed that the siliques in the radish ls mutant were 2.3 times longer than those of WT radish, and there was a greater number of seeds per silique in the ls mutant than WT, indicating that the ls mutant not only has a longer silique than WT radishes but also a significantly higher number of ovules per silique. The radish ls mutant, with its significantly increased number of seeds per silique, has great research value and is an important experimental material for studying radish ovule formation and improving radish seed yield.
Detection of silique-related molecular markers and gene expression in the radish ls mutant
The results of screening of 42 simple-sequence repeat (SSR) molecular markers related to crop traits have been published (see Materials and Methods), and showed that the amplification products of the SSR molecular marker primers HZ001, SRC9-022, and OI12F11 were different between WT and ls mutant radishes. There were also abundant polymorphisms in the ls mutant and WT radish molecular markers (Fig. 3A). The bands of WT radish amplified by molecular markers HZ001 and SRC9-022 primers were faint (Fig. 3A). By contrast, amplification of the radish ls mutant by the molecular marker OI12F11 did not produce bands (Fig. 3A). The LS1 gene was derived from the BnaA09g39480D sequence of the B. napus gene (Shen et al., 2019) through sequence alignment by Blast (LR778317), and the LS2 gene was designed from the radish gene (XM_018634088) sequence obtained by homologous alignment in the National Center for Biotechnology Information database. Semi-quantitative polymerase chain reaction (PCR) results showed that the expression of LS1 and LS2 in the silique of the radish ls mutant was lower than that in WT radish. In addition, there were clear differences in the expression of LS1 and LS2 between mutant and WT radish cotyledons.
Verification and qualification of Arabidopsis-transformed plants overexpressing LS2 (RsNAC66)
Transformed Arabidopsis lines overexpressing LS2 (RsNAC66) were screened using amplification fragments of the kanamycin gene by PCR (Fig. 4A). The semi-quantitative PCR results showed that the expression of LS2 (RsNAC66) was significantly enhanced in the transgenic plants (Fig. 4B, C). Relative expression of the LS2 (RsNAC66) gene in the transgenic lines (lines 3, 5, 7, 9) was upregulated to different degrees compared to WT, and increased 6,054-, 2,427-, 2-, and 99-fold in the siliques of lines 3, 5, 7, and 9, respectively (Fig. 4C). These results showed that the expression of LS2 (RsNAC66) was significantly increased in the overexpressed lines.
Statistical analyses of the phenotypic characteristics of A. thaliana-transformed plants overexpressing LS2 (RsNAC66)
Statistical analyses showed that the average plant height of A. thaliana transgenic lines 3 and 7 was higher than that of WT, and the plant height of transformed line 7 was significantly different from that of WT (P < 0.01; Fig. 5A). There was no significant difference in plant height between transformed lines 3, 5, and 9 and WT (P > 0.05). A. thaliana transgenic line 5 showed abnormal bolting (Fig. 5B), and early bolting and flowering were observed during week 6 of A. thaliana growth. The average silique length and number of ovule per silique of transgenic A. thaliana were measured and analyzed (Fig. 5C–F). Among them, the silique length of transgenic lines 5 and 9 was longer than that of WT (P < 0.05). There were more ovules per silique in lines 3, 7, and 9 than in WT (P < 0.01).
Detection and analyses of the expression of silique development-related genes
AP2, OPT4, and Atsus2 are silique development regulatory genes (Kunst et al., 1989; Bowman et al., 1989; Chai et al., 2011; Huang et al., 2013; Niuetal., 2002; Ohto et al., 2005). The heterologous expression of LS2 (RsNAC66) affects the development of Arabidopsis siliques. We further examined the correlation between the two genes in siliques. As shown in Fig. 11, the relative expression of AP2 in the transgenic lines was upregulated in siliques, as well as 6-, 17-, 1- and 2-fold in the four tested transgenic lines (3, 5, 7, and 9), respectively (Fig. 5G). OPT4 and Atsus2 were both expressed at high levels in the four tested transgenic lines (3, 5, 7, and 9). The high expression and function of LS2 (RsNAC66) in siliques suggest that the AP2 gene coordinated with LS2 (RsNAC66) is involved in the regulation of silique development.