Wax Genes in Broccoli Curd: Primary Localization and Identification Based on BSA-seq Technology

doi:10.11924/j.issn.1000-6850.casb2023-0901

Abstract

Abstract:

In order to explore the related genes regulating the synthesis of wax in broccoli flower bulbs, the F₂ population was constructed using the broccoli waxy wild type (WT20) as the male parent and the waxless mutant (MT20) as the female parent. Based on F₂, extreme wax-pool and no-wax-pool were constructed. The parent pools and F₂ extreme pools were sequenced by BSA-seq technology. The intervals associated with wax synthesis were initially located by theΔSNP-index algorithm. Candidate genes were predicted based on genes annotation information, and the expression of the candidate genes in the parents was analyzed by qRT-PCR. The results showed that the segregation ratio of wax-powdered plants to no-wax-powdered plants in the F₂ segregation population was 2.87:1, which conformed to the genetic law of single-gene recessive nuclear genes. A total of 2,045 high-quality SNP loci and 687 InDel loci were obtained between the two pools. Based on SNP-index association analysis, the candidate genes were located in three intervals on chromosomes 4, 7 and 8, including 93 genes. Further analysis of genes in the candidate regions in KEGG, GO, and GOC databases revealed that 92 genes in the 0.12 Mb and 1.07 Mb segments were annotated. Quantitative analysis was performed on 11 genes that might be related to wax synthesis in the intervals, and 4 differentially significantly expressed genes, BoAPC3, BoCDH1, BoEEF1A, and BoAHP4, were screened out. The results of this study can provide new ideas for further research on the wax synthesis pathway of broccoli, and lay a foundation for the breeding of high-quality broccoli.

Key words: broccoli, wax, BSA-seq, initial positioning, identification, candidate gene

TAO Meiqi, ZHANG Zhenchao, PAN Yongfei, SUN Guosheng, XU Dingfan, SHAN Xi, YAO Yuemei. Wax Genes in Broccoli Curd: Primary Localization and Identification Based on BSA-seq Technology[J]. Chinese Agricultural Science Bulletin, 2024, 40(33): 131-140.

Figures/Tables 13

References 23

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引物名称	引物序列(5’-3’)	引物名称	引物序列(5’-3’)
Actin-F	AGGCTACACGTTCGGACAAG	qRAD51L3-F	GAAGGAGGGTATCACTGTGA
Actin-R	TGGGGCACTAAACACAGTCA	qRAD51L3-R	TGTCAGCTGACCCTCACGGT
qAHP4-F	GTTGAAGAAGTTGCCACATT	qECA1-F	TCTCACCTACGGATATCGAC
qAHP4-R	TACTCGCACCAATGCTGGTG	qECA1-R	CTACGGAGATAACACTAGCG
qCDH1-F	GTAACTGAGTTCAGTTCAGT	qCNOT2-F	TGAGCAGGGCTTTGATCTCT
qCDH1-R	CGCAAGGAACATAAATCGGT	qCNOT2-R	CCTGCTCATTAAGCCTCCAC
qAPC3-F	CTCGCTCATCAAAAGTCTCC	qAPX1-F	CGCTGAGAAGCACTGCACAC
qAPC3-R	CTAGAGATCCTCCTCGGTCT	qAPX1-R	ACACCAGCAAGCTGATGGAA
qNUP35-F	GGTTCAGAGGCAGGTTATAC	qEEF1A-F	ATGGGTAAAGAGAAGTTTCA
qNUP35-R	TCCTTGGACCAGGAACATGT	qEEF1A-R	GACCTCTTGTTCATCTCAGC
qRABG3E-F	GATATGGGACACGGCTGGTC	qNQR-F	GCGGTTACATGCCCATGGAC
qRABG3E-R	CTGGATCAGAAACTCTTCCC	qNQR-R	GTTCTTTTGCGGGAACCTGA

引物名称	引物序列(5’-3’)	引物名称	引物序列(5’-3’)
Actin-F	AGGCTACACGTTCGGACAAG	qRAD51L3-F	GAAGGAGGGTATCACTGTGA
Actin-R	TGGGGCACTAAACACAGTCA	qRAD51L3-R	TGTCAGCTGACCCTCACGGT
qAHP4-F	GTTGAAGAAGTTGCCACATT	qECA1-F	TCTCACCTACGGATATCGAC
qAHP4-R	TACTCGCACCAATGCTGGTG	qECA1-R	CTACGGAGATAACACTAGCG
qCDH1-F	GTAACTGAGTTCAGTTCAGT	qCNOT2-F	TGAGCAGGGCTTTGATCTCT
qCDH1-R	CGCAAGGAACATAAATCGGT	qCNOT2-R	CCTGCTCATTAAGCCTCCAC
qAPC3-F	CTCGCTCATCAAAAGTCTCC	qAPX1-F	CGCTGAGAAGCACTGCACAC
qAPC3-R	CTAGAGATCCTCCTCGGTCT	qAPX1-R	ACACCAGCAAGCTGATGGAA
qNUP35-F	GGTTCAGAGGCAGGTTATAC	qEEF1A-F	ATGGGTAAAGAGAAGTTTCA
qNUP35-R	TCCTTGGACCAGGAACATGT	qEEF1A-R	GACCTCTTGTTCATCTCAGC
qRABG3E-F	GATATGGGACACGGCTGGTC	qNQR-F	GCGGTTACATGCCCATGGAC
qRABG3E-R	CTGGATCAGAAACTCTTCCC	qNQR-R	GTTCTTTTGCGGGAACCTGA

材料	株高/cm	开展度/cm	外叶数	最大叶宽×长(cm×cm)	花球直径/cm	单球重/g
WT20	48.3±2.6	63.4±2.4	16.5±1.5	25×48	14.6±1.4	502.6±2.6
MT20	34.6±1.7	41.8±2.1	10.7±1.3	15×32	4.8±0.6	96.5±3.1

材料	株高/cm	开展度/cm	外叶数	最大叶宽×长(cm×cm)	花球直径/cm	单球重/g
WT20	48.3±2.6	63.4±2.4	16.5±1.5	25×48	14.6±1.4	502.6±2.6
MT20	34.6±1.7	41.8±2.1	10.7±1.3	15×32	4.8±0.6	96.5±3.1

世代	总数	花球有蜡粉	花球无蜡粉	分离比率	卡平方测验	卡平方测验
P₁(MT20)	20		20
P₂(WT20)	20	20
F₁	30	30
F₂	267	198	69	2.87:1	0.101	3.84