Fig 1.
Morphological comparison between the wild-type ‘Yugu1’ and the lp1 mutant.
(A) The general statures of ‘Yugu1’ and lp1 at the mature stage, bar = 10 cm. (B and C) The panicle morphology of ‘Yugu1’ (left) and that of lp1 (right), bar = 10 cm. (D, E, and G) Statistics for primary branch number, panicle length, and fertilized spikelets in ‘Yugu1’ and lp1. Welch’s two-sample t-test, n = 10 biological replications, asterisks indicate P < 0.01. (F) The primary branch morphology of ‘Yugu1’ (up) and that of lp1 (down), bar = 1 cm.
Table 1.
Morphological traits of lp1 and wild-type ‘Yugu1’.
Fig 2.
Comparisons of plant height and grain size between the wild-type ‘Yugu1’ and the lp1 mutant.
(A) Internodes and roots of ‘Yugu1’ (left) and lp1 (right) at the mature stage, bar = 10 cm. I–XV indicate the internode numbers under the panicle. (B) Comparison of internode lengths between the mutant and wild-type. Welch’s two-sample t-test, n = 3 biological replications, * indicates P < 0.05, ** indicates P < 0.01. (C) Morphological comparison of unhulled grain. The first and second rows represent the unhulled grain width of ‘Yugu1’ (up) and lp1 (down), while the third and fourth rows represent the unhulled grain lengths of ‘Yugu1’ and lp1, bar = 5 mm. (D) Morphological comparison of hulled grain. The first and second rows represent the hulled grain widths, while the third and fourth rows represent the hulled grain lengths of ‘Yugu1’ and lp1, bar = 5 mm. (E–I) Statistics of grain length, grain width, hulled grain length, hulled grain width, and 1,000-grain weight in ‘Yugu1’ and lp1. Welch’s two-sample t-test, n = 10 biological replications, asterisks indicate P < 0.01.
Fig 3.
Identification of the LP1 locus.
(A) Examples of the molecular markers used for map-based cloning. CAAS4019, In2-11, and In2-4401 are SSR and indel markers that are closely linked to the LP1 locus. M, molecular weight marker; ♀, female parents (lp1); ♂, male parents (‘SSR41’); F1, F1 generation from lp1 × ‘SSR41’ cross; P, DNA pools of recessive homozygous individual in F2 population; 1–22, different recessive homozygous individuals. (B) Fine mapping of the LP1 gene using molecular markers. (C) Co-segregation analysis of the BC1F2 population using dCAPS markers; ♀, female parents (lp1); ♂, male parents (‘Yugu1’).
Fig 4.
Conserved features of LP1 and a phylogenetic analysis.
(A) Conserved domains of the LP1 protein. LP1 has two conserved WRKY domains. (B) Phylogenetic analysis of LP1 and its homologs in Arabidopsis and rice revealed that LP1 belongs to Group I of the WRKY subfamily. The red letters stand for SiLP1 and its two closest homologous proteins in O. sativa (OsWRKY78) and Arabidopsis (AtWRKY20). Phylogenetic tree was constructed using the deduced full-length protein sequences of LP1 and other WRKYs selected from previous researches [31, 32]. MEGA5 software (www.megasoftware.net) was employed with the maximum likelihood method, JTT model and 1000 bootstrap replicates. The bootstrap value for each node is shown in the figure.
Fig 5.
Characterization of LP1 in wild-type ‘Yugu1’ and the lp1 mutant.
(A) PCR analysis of LP1 at the genomic DNA and RNA levels. Different bands were detected in the lp1 mutant at the RNA level when compared to wild-type ‘Yugu1’. (B) Comparison of the gene structures of LP1 alleles. Compared with the LP1 sequence in wild-type plants, a single G to A mutation was found in the mutant, which led to the production of three different splice variants (LP1-1, LP1-2, and LP1-3) at the transcriptomic level. (C) Comparison of WRKY domain sequences among different species. WRKY domain I (NT) indicates the first WRKY domain at the N-terminus of LP1. WRKY domain II (CT) indicates the second WRKY domain at the C-terminus of LP1. The C2H2 motif sequence in WRKY II was completely lost in all three LP1 variants in the mutant. (D) Comparison of the WRKY domain structure in ‘Yugu1’ and lp1. In silico structural modeling of the LP1 protein was performed by SWISS-MODEL (swissmodel.expasy.org). The WRKY I domain of LP1 was not affected, while the WRKY II domain structure were severely disrupted in the lp1 mutant.
Fig 6.
(A) Quantitative real-time PCR analysis of LP1 expression levels in different foxtail millet organs. Mean expression levels and standard deviations were calculated from three independent biological replications. (B) Subcellular localization of LP1 protein. The LP1-GFP fusion protein was constructed and expressed in foxtail millet mesophyll protoplasts.