Molecular Mapping and Candidate Gene Analysis for GA3 Responsive Short Internode in Watermelon (Citrullus lanatus)

Abstract

Plants with shorter internodes are suitable for high-density planting, lodging resistance and the preservation of land resources by improving yield per unit area. In this study, we identified a locus controlling the short internode trait in watermelon using Zhengzhouzigua (long internode) and Duan125 (short internode) as mapping parents. Genetic analysis indicated that F₁ plants were consistent with long internode plants, which indicates that the long internode was dominant over the short internode. The observed F₂ and BC₁ individuals fitted the expected phenotypic segregation ratios of 3:1 and 1:1, respectively. The locus was mapped on chromosome 9 using a bulked segregant analysis approach. The region was narrowed down to 8.525 kb having only one putative gene, Cla015407, flanking by CAPS90 and CAPS91 markers, which encodes gibberellin 3β-hydroxylase (GA 3β-hydroxylase). The sequence alignment of the candidate gene between both parents revealed a 13 bp deletion in the short internode parent, which resulted in a truncated protein. Before GA₃ application, significantly lower GA₃ content and shorter cell length were obtained in the short internode plants. However, the highest GA₃ content and significant increase in cell length were observed in the short internode plants after exogenous GA₃ application. In the short internode plants, the expression level of the Cla015407 was threefold lower than the long internode plants in the stem tissue. In general, our results suggested that Cla015407 might be the candidate gene responsible for the short internode phenotype in watermelon and the phenotype is responsive to exogenous GA₃ application.

Keywords: BSA-Seq; GA 3β-hydroxylase; candidate gene; cytological analysis; fine mapping.

Figure 1

Phenotypic characteristics of Zhengzhouzigua (long internode) and Duan125 (short internode) parents. (A) Long internode, and (B) Short internode plants before GA₃ application. (C) Long internode, and (D) Short internode plants after GA₃ application. (E) Internodes of the long internode plants before GA₃ application, (F) Internodes of the long internode plants after GA₃ application. (G) Internodes of the short internode plants after GA₃ application, and (H) Internodes of the short internode plants before GA₃ application.

Figure 2

Agronomic traits of long and short internode plants. (A) Number of internodes, (B) Internodes length (cm), and (C) Vine length (m) before GA₃ application. (D) Number of internodes, (E) Internodes length (cm) and (F) Vine length (m) after GA₃ application. Data were averages of three biological replications taken from three plants. LI = Long internodes, and SI= Short internodes before GA3 application. LI + GA₃ = Long internodes, and SI + GA3 = Short internodes after GA₃ application. Error bars indicates standard deviations from three repeats (n = 3). Values are means + SD (n = 3). * significant at p < 0.05; ** significant at p < 0.01 and *** significant at p < 0.001 probability levels.

Figure 3

Fine mapping and isolation of the dw gene. (A) ΔSNP index graph of BSA-seq analysis, (B) The dw gene was narrow down to an interval 8.525 kb using 430 F₂ individuals flanking by CAPS90 and CAPS91 markers, (C) The gene structure of Cla015407. (D) Sequence alignment between long and short internode parents showed a 13 bp deletion in the short internode parent.

Figure 4

The phylogenetic analysis and conserved domains of the candidate gene. (A) Phylogenetic tree for the Cla015407. The tree was constructed using MEGA 7 with Bootstrap values calculated from 1000 replicates. The Cla015407 is circled in red. (B) The conserved domain of Cla015407 gene, which was analyzed by online Pfam database.

Figure 5

Relative expression level of Cla015407 in different tissues of both long and short internode parents, Error bars indicates standard deviations from three repeats (n = 3). Values are means + SD (n = 3). LI = Long internodes, SI = Short internodes. ** Significant at p < 0.05 probability level.

Figure 6

GA₃ content in top, middle and basal internodes of long and short internode plants. (A) GA₃ content before GA₃ application, (B) GA₃ content after GA₃ application. LI = Long internode, and SI = Short internode before GA₃ application; LI + GA₃ = Long internode, and SI + GA₃ = Short internode after GA₃ application. Error bars indicates standard deviations from three repeats (n = 3). Values are means + SD (n = 3). * significant at p < 0.05 probability level.

Figure 7

Cytological observation of internode length and size of cells between long and short internode plants. (A–C) The internode cell length of long internode plants, and (G–I) The internode cell length of short internode plants from top, middle and basal internode positions, respectively before GA₃ application. (D–F) The internode cell length of long internode plants and (J–L) The internode cell length of short internode plants from top, middle, and basal internode positions, respectively after GA₃ application. Bar = 100 μm.

Figure 8

Validation of the candidate gene using an InDel marker. (A) The gene structure of Cla015407. Gray boxes represent exons and open boxes represent untranslated regions (UTRs), while lines represent introns. (B) Confirmation of the deletion in short internode through sequencing. The vertical red dotted line indicates the 13 bp deletion from 502–514 bp. (C) Co-segregation of the short internode phenotype and the 13 bp deletion of Cla015407 in F₂ population containing 135 individuals. Genotyping by PCR of the 135 individuals revealed that 33 were homozygous dominant (long internode) and 67 individuals were heterozygous, while 35 individuals were homozygous recessive (short internode).