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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (3): 385-393.doi: 10.3724/SP.J.1006.2021.04123


Genetic analysis and molecular characterization of multilocular trait in the srb mutant of Brassica rapa L.

YANG Yang(), LI Huai-Lin(), HU Li-Min, FAN Chu-Chuan*(), ZHOU Yong-Ming   

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2020-06-05 Accepted:2020-09-13 Online:2021-03-12 Published:2020-10-09
  • Contact: FAN Chu-Chuan E-mail:yangyangyy91@163.com;1228023730@qq.com;fanchuchuan@mail.hzau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31671279);National Natural Science Foundation of China(31971976);National Natural Science Foundation of China(31371240)


Multilocular silique is considered as a trait associated with high yield in rapeseed, and we studied the genetic regulation of the multilocular trait in B. rapa var. srb. This mutant showed a stable multilocular phenotype, ranging from 94.7% to 100% multilocular siliques per plant and 3.5 carpels per silique. Genetic analyses showed that the multilocular trait was monogenically governed by a recessive nuclear gene. Comparative sequencing analysis revealed that there was a novel C-to-G single-nucleotide mutation in the core CLE motif of BrCLV3, leading to histidine mutation at position 12 in conserved domain to aspartic acid, which was named Brclv3Asp12. The analysis of segregated population by SNP marker showed that the C/G single-nucleotide variation in Brclv3Asp12 was co-segregated with the multilocular phenotype. Transgenic complementation studies and in vitro peptide assays further confirmed that the Brclv3Asp12 allelic mutation in srb could lead to reduced activity of the CLV3 peptide, resulting in the formation of multilocular phenotype. Therefore, the study preliminarily clarified the mechanism involved in multilocular silique formation in srb mutant.

Key words: B. rapa var. srb, multilocular silique, BrCLV3, allelism test, comparative sequencing, functional analysis

Table 1

Primer sequences used in this study"

Primer name
Primer sequence (5°-3°)

Fig. 1

Phenotypic observation of floral and silique morphology of mutilocular B. rapa a-b, c-f, g-j respesent the number of floral organs in WT, ml4 and srb, respectively; k-l, m-p, q-v respesent siliques with different carpel numbers in WT, ml4 and srb, respectively; cross-sections of gynoecia of WT (two carpels, w), ml4 (3-4 carpels, x and y) and srb (2-4 carpels, w to y) at stage 9-11; lc: locule; O: ovule; M: medial region; L: lateral region. Bar = 2 mm (k, m, o, q, s, u), 0.5 mm (l, o, p, r, t, v), and 100 μm (w-y)."

Table 2

Performance of multilocular silique trait in parents and F1 hybrids"

Range (%)
Mean±SD (%)
ml4 mutant 79.6-100.0 97.90±5.10
srb mutant 96.0-100.0 96.40±0.03
WT 0
ml4*srb F1 100.0
srb*ml4 F1 100.0
WT*srb F1 0
srb*WT F1 0

Fig. 2

BrCLV3 gene structure and amino acid sequences alignment a: BrCLV3 gene structure and natural variations between the alleles from WT and srb; black boxes represents the coding regions; there are two single nucleotide substitutions and one ATAT insertion/deletion in BrCLV3 gene regions between WT and srb. b: alignment of the amino acid sequences of CLV3 from B. rapa (srb, WT, ml4, Chiifu-401, and Arabidopsis); the putative signal sequence cleavage site is indicated by arrow, the conserved domain (CLE motif) at its C-terminus is underlined, and the amino acid changes in srb and ml4 are highlighted by the red highlighted font."

Fig. 3

Co-segregation analysis of genotype and multilocular phenotype in srb*WT F2 population by Brclv3Asp12 allele-specific SNP marker"

Fig. 4

Phenotypes of 35S::Brclv3Asp12 transgenic plants in the clv3-2 background The phenotypes of bilocular siliques (a, b) and normal inflorescence (q, r) in ler, multilocular siliques with 5-6 locular (i-l) and enlarged inflorescence (m, n) in clv3-2, and 35S::Brclv3Asp12 transgenic plants with partly rescue phenotypes, including 2-6 carpels (c-l) and normal inflorescence (o, p). Gene expression (s) and phenotype analysis of carpel numbers per plant (t) in five independent 35S::Brclv3Asp12 transgenic lines, which showed partially rescued phenotypes; the data and error bars represent the mean ± SD (n ≥ 15 plants for each line). Bars superscripted by lowercase letters indicate significant differences at the 0.05 probability level. Bar = 0.5 mm."

Fig. 5

Effects of different peptides on the SAMs in Arabidopsis 1: ler (a) and clv3-2 (b, c, d) plants were grown on 1/2 MS liquid media (a, b) containing 1 μmol L-1 Brclv3Asp12 peptide (c) and 1 μmol L-1 BrCLV3 peptide (d), and 9-day-old Arabidopsis seedlings were observed; the area of the SAM was measured on a median plane by calculating the area above the straight line from the upper edges of two opposite leaf primordia (arrowheads); Bar = 50 μm. 2: area of the SAM after different peptide treatments for nine days (e), the data and error bars represent the mean ± SD (n ≥ 20 plants for each line). Bars superscripted by lowercase letters indicate significant differences at the 0.01 probability level."

Fig. 6

Effects of different peptides on the root and RAM in Arabidopsis Arabidopsis seedlings of 8-day-old were grown on agar media without (a, h) or with 1 nmol L-1 (b, c, i, j, o, r), 10 nmol L-1 (d, e, k, l, p, s), 100 nmol L-1 (f, g, m, n, q, t) of the BrCLV3Asp12 (b, d, f, i, k, m) or Brclv3 (c, e, g, j, l, n) peptides, respectively. The data and error bars represent the mean ± SD (n ≥ 26 plants for each line). ** indicates significant difference at the 0.01 probability level. Bar = 1 cm (a-g). Bar=100 μm (h-n). The black arrows indicate the border between the root meristematic and elongation zones."

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