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Genome-wide analysis of mRNA and lncRNA expression and mitochondrial genome sequencing provide insights into the mechanisms underlying a novel cytoplasmic male sterility system, BVRC-CMS96, in Brassicarapa

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Abstract

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Characterization of a novel and valuable CMS system in Brassicarapa.

Abstract

Cytoplasmic male sterility (CMS) is extensively used to produce F1 hybrid seeds in a variety of crops. However, it has not been successfully used in Chinese cabbage (Brassicarapa L. ssp. pekinensis) because of degeneration or temperature sensitivity. Here, we characterize a novel CMS system, BVRC-CMS96, which originated in B.napus cybrid obtained from INRAE, France and transferred by us to B.rapa. Floral morphology and agronomic characteristics indicate that BVRC-CMS96 plants are 100% male sterile and show no degeneration in the BC7 generation, confirming its suitability for commercial use. We also sequenced the BVRC-CMS96 and maintainer line 18BCM mitochondrial genomes. Genomic analyses showed the presence of syntenic blocks and distinct structures between BVRC-CMS96 and 18BCM and the other known CMS systems. We found that BVRC-CMS96 has one orf222 from ‘Nap’-type CMS and two copies of orf138 from ‘Ogu’-type CMS. We analyzed expression of orf222, orf138, orf261b, and the mitochondrial energy genes (atp6, atp9, and cox1) in flower bud developmental stages S1-S5 and in four floral organs. orf138 and orf222 were both highly expressed in S4, S5-stage buds, calyx, and the stamen. RNA-seq identified differentially expressed mRNAs and lncRNAs (long non-coding RNAs) that were significantly enriched in pollen wall assembly, pollen development, and pollen coat. Our findings suggest that an energy supply disorder caused by orf222/orf138/orf261b may inhibit a series of nuclear pollen development-related genes. Our study shows that BVRC-CMS96 is a valuable CMS system, and our detailed molecular analysis will facilitate its application in Chinese cabbage breeding.

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Data availability

Sequencing data has been deposited in the NCBI Sequence Read Archive under the Accession Number PRJNA595648.

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Acknowledgements

This research was supported by the National Key Research and Development Program of China (2016YFD0101701), the scientist training program of BAAFS (JKZX201906), the Science and Technology Innovation Capacity Project (KJCX20170710), the collaborative innovation center of BAAFS (KJCX201907-2), the National Natural Science Foundation of China (No. 31801852, No. 3180110043), the Key Program of Beijing Municipal Science and Technology Committee (Z191100004019010), and the earmarked fund for China Agriculture Research System (CARS-23-A-05).

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  1. Peirong Li and Deshuang Zhang have contributed equally to this work.

Authors and Affiliations

  1. Beijing Vegetable Research Center (BVRC), Beijing Academy of Agriculture and Forestry Sciences (BAAFS), Beijing, 100097, China

    Peirong Li, Deshuang Zhang, Tongbing Su, Weihong Wang, Yangjun Yu, Xiuyun Zhao, Zhenxing Li, Shuancang Yu & Fenglan Zhang

  2. Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture, Beijing, 100097, China

    Peirong Li, Deshuang Zhang, Tongbing Su, Weihong Wang, Yangjun Yu, Xiuyun Zhao, Zhenxing Li, Shuancang Yu & Fenglan Zhang

  3. Beijing Key Laboratory of Vegetable Germplasm Improvement, Beijing, 100097, China

    Peirong Li, Deshuang Zhang, Tongbing Su, Weihong Wang, Yangjun Yu, Xiuyun Zhao, Zhenxing Li, Shuancang Yu & Fenglan Zhang

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  1. Peirong Li
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Contributions

PRL led the sequencing and data analysis, and wrote the manuscript. DSZ provided the CMS materials and did the backcross work. TBS, WHW, XYZ, and YJY provided comments relating to the manuscript. ZXL performed the qRT-PCR analysis. PRL, SCY, and FLZ conceived and designed the study. All authors read and approved the final manuscript.

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Correspondence to Shuancang Yu or Fenglan Zhang.

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Figure S1

The five floral bud developmental stages and four floral organs used for RT-PCR. (a) Flower buds of BVRC-CMS96 (left) and the maintainer line 18BCM (right) were divided into five stages: S1 (1.5-2.5 mm), S2 (2.5-3.5 mm), S3 (3.5-4.5 mm), S4 (4.5-5.5 mm) and S5 (>6 mm). (b) The four floral organs are calyx, petal, stamen, and stigma. The white scale bar = 0.5 centimeter.

Figure S2

Synteny comparison between the mitochondrial genomes of BVRC-CMS96 and 18BCM.

Figure S3

Predicted protein sequence alignments of orf224a, orf261a, and orf261b from several CMS systems in B. rapa. (a) Predicted protein sequence alignments between BVRC-CMS96 orf224 and Pol orf224. (b) Predicted protein sequence alignments between BVRC-CMS96 orf261a and 18BCM orf261a. (c) Predicted protein sequence alignments between BVRC-CMS96 orf261b and Nap orf261.

Table S1

Oligonucleotide primers used for orf138 validation and qRT-PCR assays. (DOCX 16 kb)

Table S2

Floral characters of Chinese cabbage backcross 7- (BC7)-generation plants of BVRC-CMS96, Ogu CMS, Pol CMS, and the maintainer lines. (DOCX 15 kb)

Table S3

Agronomic traits of BC7 Chinese cabbage lines with BVRC-CMS96, Pol CMS, Ogu CMS, and the maintainer lines. (DOCX 16 kb)

Table S4

Gene function analysis in the BVRC-CMS96 mitochondrial genome. (XLSX 14 kb)

Table S5

Gene function analysis in the 18BCM mitochondrial genome. (XLSX 13 kb)

Table S6

Summary of structural variations in the five mitochondrial genomes. (DOCX 14 kb)

Table S7

Unique ORFs in the mitochondrial genomes of BVRC-CMS96 and 18BCM in Chinese cabbage. (DOCX 16 kb)

Table S8

Repeat sequences in the mitochondrial genomes of Chinese cabbage BVRC-CMS96 and 18BCM. (XLSX 10 kb)

Table S9

The differentially expressed genes (DEGs) between BVRC-CMS96 and 18BCM quantified using the FPKM method. (XLSX 84 kb)

Table S10

The differentially expressed lncRNAs (DELs) between BVRC-CMS96 and 18BCM. (XLSX 38 kb)

Table S11

The bracketing genes between BVRC-CMS96 and 18BCM. (XLSX 15 kb)

Table S12

The predicted antisense lncRNA-mRNA interactions between BVRC-CMS96 and 18BCM. (XLSX 20 kb)

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Li, P., Zhang, D., Su, T. et al. Genome-wide analysis of mRNA and lncRNA expression and mitochondrial genome sequencing provide insights into the mechanisms underlying a novel cytoplasmic male sterility system, BVRC-CMS96, in Brassicarapa. Theor Appl Genet 133, 2157–2170 (2020). https://doi.org/10.1007/s00122-020-03587-z

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