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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (1): 27-39.doi: 10.3724/SP.J.1006.2022.04281

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Mapping and candidate gene analysis of silique number mutant in Brassica napus L.

ZHAO Gai-Hui1(), LI Shu-Yu2, ZHAN Jie-Peng1, LI Yan-Bin3, SHI Jia-Qin1,*(), WANG Xin-Fa1, WANG Han-Zhong1   

  1. 1Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, Hubei, China
    2Crop Research Institute, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, Jiangxi, China
    3Wuhan Agricultural Technology Extension Center, Wuhan 430400, Hubei, China
  • Received:2020-12-27 Accepted:2021-04-14 Online:2022-01-12 Published:2021-06-15
  • Contact: SHI Jia-Qin E-mail:3468382960@qq.com;shijiaqin@caas.cn
  • About author:First author contact:** Contributed equally to this work
  • Supported by:
    Natural Science Foundation of Jiangxi Province(20202BABL205016);Hubei Provincial Natural Science Foundation (Outstanding Youth)(2018CFA075);Breeding of New Rapeseed Varieties with High Quality, High Yield and Multiple Resistance(2018ABA087);China Agriculture Research System(CARS-13)

Abstract:

The silique number is one of the important components of yield per plant in oilseed rape (Brassica napus L.) and the exploitation and utilization of its excellent alleles are essential to increase yield. More than hundreds of silique number QTLs have been mapped in oilseed rape, but they are difficult to be fine-mapped or cloned because of their moderate and unstable effects. A oilseed rape mutant (No.7931) was detected in previous study and it had few siliques at mature stage due to the stop growth after differentiation about 10 flowers on the top of inflorescence. A F2 segregating population consisting of 3400 individuals was constructed using this mutant and another more-silique lines No.73290. Among them, we performed BSA-seq on 30 individuals with extreme more- or less-siliques and detected three associated intervals of 0-1.1 Mb, 4.7-6.2 Mb, and 11.5-12.4 Mb on the C02 chromosome. These genomic intervals contained a total of 522 annotated genes in the reference genome DarmorV8.1, among which 235 genes had functional annotation and SNP/InDel variation. At the early stage of flower bud differentiation, the shoot apical meristems of two parents were subjected to RNA-seq, and a total of 8958 differentially expressed genes (DEGs) were detected. These DEGs were significantly enriched into 20 pathways, including carbohydrate metabolism, translation, and amino acid metabolism (highly associated with flower bud differentiation) and so on, among which 99 were located in the associated intervals. By the integration of gene functional annotation as well as sequence and expression variation analysis, a total of nine candidate genes (BnaC02g00490.1D2, BnaC02g01030.1D2, BnaC02g01120.1D2, BnaC02g00270.1D2, BnaC02g02670.1D2, BnaC02g08680.1D2, BnaC02g08890.1D2, BnaC02g09480.1D2, and BnaC02g10490.1D2) were identified, which were mainly involved in the maintenance of inflorescence meristems and the regulation of flower development. The above results lay the foundation for the following fine-mapping and cloning of the silique number mutant gene in oilseed rape.

Key words: Brassica napus L., Brassica napus L., silique number mutant, silique number mutant, flower bud differentiation, flower bud differentiation, BSA-seq, BSA-seq, RNA-seq, RNA-seq

Fig. 1

Observation on the inflorescences at flowering and mature stages of the rapeseed oligocyte mutant No.7931 and polycygocyte stain No.73290 A1: No.7931 at flowering stage; 2: No.73290 at flowering stage; B1: No.7931 at mature stage; B2: No.73290 at mature stage."

Fig. 1

Observation on the inflorescences at flowering and mature stages of the rapeseed oligocyte mutant No.7931 and polycygocyte stain No.73290 A1: No.7931 at flowering stage; 2: No.73290 at flowering stage; B1: No.7931 at mature stage; B2: No.73290 at mature stage."

Table 1

Phenotypic information of No.7931 and No.73290 (mean±SD)"

亲本
Parent
主花序花器官数目
FNm
主花序角果数
SNm
株高
PH
No.7931 8.33±1.24 7.67±0.94 154.3±2.06
No.73290 199.3±1.24 93.67±2.49 150.4±1.57
t检验值t-test value 5.45E-09 6.91E-07 0.0170

Table 1

Phenotypic information of No.7931 and No.73290 (mean±SD)"

亲本
Parent
主花序花器官数目
FNm
主花序角果数
SNm
株高
PH
No.7931 8.33±1.24 7.67±0.94 154.3±2.06
No.73290 199.3±1.24 93.67±2.49 150.4±1.57
t检验值t-test value 5.45E-09 6.91E-07 0.0170

Fig. 2

Microscopic observation of silique number mutant in Brassica napus L. A, B, C, and D represent the four stages of flower primordium differentiation, calyx differentiation, petal primordium differentiation (bud axis elongation), and early budding, respectively. 1, 2 represent No.7931 and No.73290, respectively."

Fig. 2

Microscopic observation of silique number mutant in Brassica napus L. A, B, C, and D represent the four stages of flower primordium differentiation, calyx differentiation, petal primordium differentiation (bud axis elongation), and early budding, respectively. 1, 2 represent No.7931 and No.73290, respectively."

Fig. 3

Candidate interval peak map The abscissa represents the position of the Δ(SNP-index), and the ordinate represents the value of the Δ(SNP-index)."

Fig. 3

Candidate interval peak map The abscissa represents the position of the Δ(SNP-index), and the ordinate represents the value of the Δ(SNP-index)."

Fig. S1

Heat map of correlation coefficient between samples"

Fig. S1

Heat map of correlation coefficient between samples"

Table S1

Primers for qRT-PCR"

基因
Gene
正向引物
Forward primer (5°-3°)
反向引物
Reverse primer (5°-3°)
BnaA02G0314300ZS CCGACACGCTTCAGAAGGTC AGAGATCCCGCTTCGACTCC
BnaC03G0027000ZS TGAGAACACGCCGGTCAAAG CCTCGCGTTCTCTCTCTCCA
BnaC03G0100100ZS CTGATGGCCGTAGAGCATGC AGCCATCTCCTGTTGTTGCA
BnaC03G0091000ZS TCCCACTCTACCCTGCACTG GACGTTGTTCACATTCGCGC
BnaC02G0283700ZS ACAACATGGCCCTGGAAACTG CTTGGAGGCGGATGATCGTT
BnaC02G0139700ZS CGGCGGAGGTACAGACATTT TCGTCATGATGAGCCTCTCCT
BnaC02G0175400ZS GCGCCTCGTATCCATTCTCG CCTGAAGTTGTGGCGAGCTT
BnaA01G0077800ZS CCACCGTCATGTCTTCCTTCC ACGAGTTGGAAGTGTGCGTT
BnaA01G0061200ZS GGGAACGGCTTAGATGGTGC TTGCTAGTACCAGGGCTGCT
BnaC02G0013900ZS CATTGGCTCGTCATGAACATC TTCACGAGTGTTGAACTGATCC
BnaC02G0159100ZS CTGTCACTGGAAACCACCCG TCAATCGACCATGGCAAGCA
BnaA01G0009100ZS TCTGCTCTGAACGCGACCAA ACCAGCCAAAGAACCAGGGT
BnaA01G0037200ZS TCCTCAACTGTGCCGACATGT AAAGCCGTCGTCAATCTCGC
BnaC05G0266700ZS AGACTACGTGAAGCAGCCGA CTCCAGCTTCCGACCAGTCT
BraActin CTGGAATTGCTGACCGTATGAG ATCTGTTGGAAAGTGCTGAGGG

Table S1

Primers for qRT-PCR"

基因
Gene
正向引物
Forward primer (5°-3°)
反向引物
Reverse primer (5°-3°)
BnaA02G0314300ZS CCGACACGCTTCAGAAGGTC AGAGATCCCGCTTCGACTCC
BnaC03G0027000ZS TGAGAACACGCCGGTCAAAG CCTCGCGTTCTCTCTCTCCA
BnaC03G0100100ZS CTGATGGCCGTAGAGCATGC AGCCATCTCCTGTTGTTGCA
BnaC03G0091000ZS TCCCACTCTACCCTGCACTG GACGTTGTTCACATTCGCGC
BnaC02G0283700ZS ACAACATGGCCCTGGAAACTG CTTGGAGGCGGATGATCGTT
BnaC02G0139700ZS CGGCGGAGGTACAGACATTT TCGTCATGATGAGCCTCTCCT
BnaC02G0175400ZS GCGCCTCGTATCCATTCTCG CCTGAAGTTGTGGCGAGCTT
BnaA01G0077800ZS CCACCGTCATGTCTTCCTTCC ACGAGTTGGAAGTGTGCGTT
BnaA01G0061200ZS GGGAACGGCTTAGATGGTGC TTGCTAGTACCAGGGCTGCT
BnaC02G0013900ZS CATTGGCTCGTCATGAACATC TTCACGAGTGTTGAACTGATCC
BnaC02G0159100ZS CTGTCACTGGAAACCACCCG TCAATCGACCATGGCAAGCA
BnaA01G0009100ZS TCTGCTCTGAACGCGACCAA ACCAGCCAAAGAACCAGGGT
BnaA01G0037200ZS TCCTCAACTGTGCCGACATGT AAAGCCGTCGTCAATCTCGC
BnaC05G0266700ZS AGACTACGTGAAGCAGCCGA CTCCAGCTTCCGACCAGTCT
BraActin CTGGAATTGCTGACCGTATGAG ATCTGTTGGAAAGTGCTGAGGG

Fig. 4

Comparison of the relative expression abundance measured by qRT-PCR and RNA-seq"

Fig. 4

Comparison of the relative expression abundance measured by qRT-PCR and RNA-seq"

Fig. 5

GO enrichment analysis of differentially expressed genes"

Fig. 5

GO enrichment analysis of differentially expressed genes"

Fig. 6

Functional classification of differentially expressed genes"

Fig. 6

Functional classification of differentially expressed genes"

Fig. 7

KEGG analysis of differentially expressed genes"

Fig. 7

KEGG analysis of differentially expressed genes"

Table 2

Candidate gene prediction in the correlation interval"

油菜基因编号
B. napus ID
拟南芥
基因名
Name of
A. thaliana
拟南芥基因
编号
A. thaliana ID
位置Position
(Mb)
基因注释
Gene annotation
序列差异
Sequence difference
差异表达基因
Differential expressed genes
BnaC02g00490.1D2 FLC AT5g10140 0.208 K盒区和MADS转录因子家族蛋白
K-box region and MADS-box transcription factor family protein
有Yes 是No
BnaC02g01030.1D2 TPR1 AT1G80490 0.465 TPL相关的基因1 TOPLESS-related 1 无No 否No
BnaC02g01120.1D2 AGAL2 AT5G08370 0.493 α-半乳糖苷酶2 Alpha-galactosidase 2 无No 否No
BnaC02g00270.1D2 AIL6 AT5G10510 0.996 ANT基因6
AINTEGUMENTA-LIKE 6
有Yes 否No
BnaC02g02670.1D2 TFL1 AT5G03840 1.10 磷脂酰乙醇胺结合蛋白
Phosphatidylethanolamine-binding-protein
无No 是Yes
BnaC02g08680.1D2 COL1 AT5G15850 4.83 CO基因1 CONSTANS-like 1 有Yes 否No
BnaC02g08890.1D2 ELF9 AT5G16260 4.96 RNA结合(RRM/RBD/RNP基序)家族蛋白RNA binding (RRM/RBD/RNP motifs) family protein 有Yes 否No
BnaC02g09480.1D2 AIM1 AT4G29010 5.23 烯酰辅酶A水合酶/异构酶家族
Enoyl-CoA hydratase/isomerase family
有Yes 否No
BnaC02g10490.1D2 CHR17 AT5G18620 5.97 染色质重塑因子17
Chromatin remodeling factor 17
无No 是Yes

Table 2

Candidate gene prediction in the correlation interval"

油菜基因编号
B. napus ID
拟南芥
基因名
Name of
A. thaliana
拟南芥基因
编号
A. thaliana ID
位置Position
(Mb)
基因注释
Gene annotation
序列差异
Sequence difference
差异表达基因
Differential expressed genes
BnaC02g00490.1D2 FLC AT5g10140 0.208 K盒区和MADS转录因子家族蛋白
K-box region and MADS-box transcription factor family protein
有Yes 是No
BnaC02g01030.1D2 TPR1 AT1G80490 0.465 TPL相关的基因1 TOPLESS-related 1 无No 否No
BnaC02g01120.1D2 AGAL2 AT5G08370 0.493 α-半乳糖苷酶2 Alpha-galactosidase 2 无No 否No
BnaC02g00270.1D2 AIL6 AT5G10510 0.996 ANT基因6
AINTEGUMENTA-LIKE 6
有Yes 否No
BnaC02g02670.1D2 TFL1 AT5G03840 1.10 磷脂酰乙醇胺结合蛋白
Phosphatidylethanolamine-binding-protein
无No 是Yes
BnaC02g08680.1D2 COL1 AT5G15850 4.83 CO基因1 CONSTANS-like 1 有Yes 否No
BnaC02g08890.1D2 ELF9 AT5G16260 4.96 RNA结合(RRM/RBD/RNP基序)家族蛋白RNA binding (RRM/RBD/RNP motifs) family protein 有Yes 否No
BnaC02g09480.1D2 AIM1 AT4G29010 5.23 烯酰辅酶A水合酶/异构酶家族
Enoyl-CoA hydratase/isomerase family
有Yes 否No
BnaC02g10490.1D2 CHR17 AT5G18620 5.97 染色质重塑因子17
Chromatin remodeling factor 17
无No 是Yes
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