Comparative Transcriptome Analysis of Cytoplasmic Male Sterile Line and Its Restorer Line in Soybean

doi:10.13560/j.cnki.biotech.bull.1985.2024-0219

Abstract

Abstract:

【Objective】 The “three line” method is the main way to breed hybrid soybeans. At present, only a few soybean restoration genes have been cloned. Thus it will be helpful in the breeding strong restorer lines with multi genes aggregation, thereby improving the fertility stability of hybrid F_1,if more new Rf genes can be discovered. 【Method】 Using soybean cytoplasmic male sterile line JLCMS5A and its restorer line JLR2 as materials, transcriptome sequencing technology was used to analyze the transcriptional level changes of JLCMS5A and JLR2 flower buds at different stages, and to explore the relevant genes and pathways regulating restorer of fertility and flower bud development processes. Further gene annotation, sequence difference analysis, RT-qPCR validation, phylogenetic analysis, and protein structure prediction were performed on differentially expressed genes（DEGs） with Rf gene features encoding PPR（pentatrioptide repeat）proteins to explore Rf related genes. 【Result】 Transcriptome sequencing identified 17 181 DEGs, of which 3 856 were related to developmental stage and 2 808 were related to fertility. GO（gene ontology）functional annotation indicated that fertility-related DEGs were mainly enriched in functional categories such as ADP binding, nucleic acid binding transcription factor activity, and protein kinase activity, while developmental stage-related DEGs were mainly enriched in functional categories such as protein heterodimerization activity, DNA replication, and DNA binding. KEGG（kyoto encyclopedia of genes and genes）enrichment analysis showed that fertility-related DEGs mainly participate in metabolic pathways closely related to protein, carbohydrates, and signal transduction, such as endoplasmic reticulum protein processing, glucoside biosynthesis, and plant hormone signal transduction. Development-related DEGs mainly participated in metabolic pathways closely related to DNA replication, mismatch repair, starch and sucrose metabolism, and cell division and energy degradation. Analysis of candidate Rf genes revealed that the Glyma.09G176400 in JLR2 may play a certain role in regulating the restorer-of-fertility process of soybean cytoplasmic male sterility. 【Conclusion】 3 856 DEGs and 2 808 DEGs which respectively related to developmental stage and fertility were identified by transcriptomic techniques and molecular biological methods. Whereafter, a total of 15 DEGs encoding PPR protein were identified, which had the characteristics of restorer-of-fertility gene. Finally, a restorer-of-fertility related gene Glyma.09G176400 was excavated.

Key words: soybean, cytoplasmic male sterility, restorer-of-fertility gene, transcriptome sequencing, differentially expressed gene

GAO Meng-meng, ZHAO Tian-yu, JIAO Xin-yue, LIN Chun-jing, GUAN Zhe-yun, DING Xiao-yang, SUN Yan-yan, ZHANG Chun-bao. Comparative Transcriptome Analysis of Cytoplasmic Male Sterile Line and Its Restorer Line in Soybean[J]. Biotechnology Bulletin, 2024, 40(7): 137-149.

Figures/Tables 11

Fig. 1 Flower bud phenotypes at two developmental stages

Table 1 Information of RT-qPCR primer sequences

基因名称Gene name	上游引物Forward primer（5'-3'）	下游引物Reverse primer（5'-3'）
Cons4	GATCAGCAATTATGCACAACG	CCGCCACCATTCAGATTATGT
Glyma.01G235800	CGTGAGAGATGTGGTGACATAC	ACGACCCATCTTCCATTTCC
Glyma.08G233900	TGAGAGATGTGGTGCTTTGG	:GTTTCCCTCCATCCTCCTAAAC
Glyma.09G160300	GATTGGTAGAAGAGCGACTGAA	CGACCGAGAAGATCGATCAAA
Glyma.09G176400	GGTCAACCCGCAGATGTAAT	CGGTCGAATTCCCTGATCTTT
Glyma.09G176600	GTCGCTGCAAACAAGGTTAAG	GGCTGAGAGTGAAAGTGAGAAT
Glyma.09G209700	CAGGACAAGTCTCAACCACAA	CTGTGACATGTCTGGCGTATAA
Glyma.10G047150	CTTCGTGAAGGAGAGTGGAATC	CAAACAGCTCCCGCGTATAA
Glyma.13G135000	CGCGGGTTGTACTCTGATATT	GTTCCACGTTACGATATCTCTCTC
Glyma.15G019900	TGAGCCGGATATTGTGGTTTAC	ACCTCTTCCTCCTCATCTCTTT
Glyma.15G091700	GTTGTATGGGTGTGAGCCTAAT	CCTTCCCTCTCATCTCCCTATAA
Glyma.16G034600	CCGAGGAGTTCAAGTCTTCTTC	CCTGGTTCTTTCGCTGGTAATA
Glyma.16G195700	CTAGGAGATGCCTGTGATTTGT	AAAGCCATGGATCAGAGTAGTG
Glyma.16G195900	CCGGATGCAATTACCCTCAA	ACCCTTGAGCTACGACCTTA
Glyma.17G220100	GTGCCCATAGGAGTCAGAAATAC	CGCGGAGATTCTTCGTTACTT
Glyma.18G108202	GCGGCTTGAGGAAGGTAATAA	TCATGCATCCAGCCTCTTAATC

Fig. 2 Result of total RNA in flower bud by agarose gel electrophoresis M: Maker Ⅲ. Lane 1-3: FLB_1 - FLB_3; respectively. Lane 4-6: FSB_1 - FSB_3; respectively. Lane 7-9: SLB_1 -SLB_3; respectively. Lane 10-12: SSB_1 - SSB_3; respectively

Fig. 3 Correlation analysis among samples A: Pearson correlation analysis of fertile and sterile samples at different developmental stages. B: PCA analysis of gene expression levels in fertile and sterile samples at different developmental stages. C: Heat map of gene expressions among samples

Fig. 4 Statistics and analysis of differentially expressed genes A: Statistics of differentially expressed genes. B: Venn diagram of differentially expressed genes

Fig. 5 Rich distribution point diagram for GO A：F_LB vs F_SB；B：S_LB vs S_SB；C：F_LB vs S_LB；D：F_SB vs S_SB；E：F_LB vs S_LB和F_SB vs S_SB；F：F_LB vs F_SB和S_LB vs S_SB, the same below

Fig. 6 Rich distribution point diagram for KEGG

Fig. 7 Statistical analysis of differentially expressed genes encoding PPR proteins A: Statistics of differentially expressed genes encoding PPR protein. B: Venn diagram of differentially expressed genes encoding PPR protein. C: Map of GO enrichment of up-regulated DEGs in F_LB vs S_LB. D: Map of GO enrichment of down-regulated DEGs in F_LB vs S_LB. E: Map of GO enrichment of up-regulated DEGs in F_SB vs S_SB. F: Map of GO enrichment of down-regulated DEGs in F_SB vs S_SB

Fig. 8 RNA-Seq and RT-qPCR analysis of candidate genes

Fig. 9 Phylogenetic analysis of candidate genes encoding PPR proteins The red branches indicate PPR proteins in leguminous plants. Phylogenetic analysis was constructed using Maximum-Likelihood, and Bootstrap values of 500 were used

Fig. 10 Structure prediction for Glyma.09G176400 encoded protein Green graphic indicates signal peptides. The orange graphics indicate pentatricopeptide repeat. The yellow graphic indicate amino acid mutation site

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