Integrated cyto-physiological and proteomic analyses reveal new insight into CMS mechanism in a novel upland cotton CMS line LD6A

Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we elucidated molecular pathways regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) line were analyzed for physiological and proteomic changes during the development process. Significant differential expression of proteins, such as Abrin, malate dehydrogenase, malic enzyme, isocitrate dehydrogenase, histone acetyltransferase was observed in CMS and its maintainer line. Transmission electron micrographs of anther tapetum showed that inner ridge of CMS mitochondria was relatively indistinct than that of LD6B with narrower membranous space at tetrad stage. Further, relatively higher reactive oxygen species were accumulated in the anther of CMS than its maintainer line at pollen mother cell and tetrad stage. We suggest that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein gene Abrin in CMS line damaged mitochondrial membrane and consequently induced pollen sterility. These data provide new insight into CMS mechanism in cotton crops and a tool to develop new CMS germplasm resources.

Combined Transcriptome and Proteome Analysis of Anthers of AL-type Cytoplasmic Male Sterile Line and Its Maintainer Line Reveals New Insights into Mechanism of Male Sterility in Common Wheat

Cytoplasmic male sterility (CMS) plays an essential role in hybrid seeds production. In wheat, orf279 was reported as a CMS gene of AL-type male sterile line (AL18A), but its sterility mechanism is still unclear. Therefore, transcriptomic and proteomic analyses of the anthers of AL18A and its maintainer line (AL18B) were performed to interpret the sterility mechanism. Results showed that the electron transport chain and ROS scavenging enzyme expression levels changed in the early stages of the anther development. Biological processes, i.e., fatty acid synthesis, lipid transport, and polysaccharide metabolism, were abnormal, resulting in pollen abortion in AL18A. In addition, we identified several critical regulatory genes related to anther development through combined analysis of transcriptome and proteome. Most of the genes were enzymes or transcription factors, and 63 were partially homologous to the reported genic male sterile (GMS) genes. This study provides a new perspective of the sterility mechanism of AL18A and lays a foundation to study the functional genes of anther development.

Comparative Transcriptome Analysis of the Anthers from the Cytoplasmic Male-Sterile Pepper Line HZ1A and Its Maintainer Line HZ1B

Cytoplasmic male-sterility (CMS) is important for the utilization of crop heterosis and study of the molecular mechanisms involved in CMS could improve breeding programs. In the present study, anthers of the pepper CMS line HZ1A and its maintainer line HZ1B were collected from stages S1, S2, and S3 for transcriptome sequencing. A total of 47.95 million clean reads were obtained, and the reads were assembled into 31,603 unigenes. We obtained 42 (27 up-regulated and 15 down-regulated), 691 (346 up-regulated and 345 down-regulated), and 709 (281 up-regulated and 428 down-regulated) differentially expressed genes (DEGs) in stages S1, S2, and S3, respectively. Through Gene Ontology (GO) analysis, the DEGs were found to be composed of 46 functional groups. Two GO terms involved in photosynthesis, photosynthesis (GO:0015986) and photosystem I (GO:0009522), may be related to CMS. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, oxidative phosphorylation (ko00190) and phenylpropanoid biosynthesis (ko00940) were significantly enriched in the S1 and S2 stages, respectively. Through the analysis of 104 lipid metabolism-related DEGs, four significantly enriched KEGG pathways may help to regulate male sterility during anther development. The mitochondrial genes orf470 and atp6 were identified as candidate genes of male sterility for the CMS line HZ1A. Overall, the results will provide insights into the molecular mechanisms of pepper CMS.

Organelle Comparative Genome Analysis Reveals Novel Alloplasmic Male Sterility with orf112 in Brassica oleracea L.

B. oleracea Ogura CMS is an alloplasmic male-sterile line introduced from radish by interspecific hybridization and protoplast fusion. The introduction of alien cytoplasm resulted in many undesirable traits, which affected the yield of hybrids. Therefore, it is necessary to identify the composition and reduce the content of alien cytoplasm in B. oleracea Ogura CMS. In the present study, we sequenced, assembled, and compared the organelle genomes of Ogura CMS cabbage and its maintainer line. The chloroplast genome of Ogura-type cabbage was completely derived from normal-type cabbage, whereas the mitochondrial genome was recombined from normal-type cabbage and Ogura-type radish. Nine unique regions derived from radish were identified in the mitochondrial genome of Ogura-type cabbage, and the total length of these nine regions was 35,618 bp, accounting for 13.84% of the mitochondrial genome. Using 32 alloplasmic markers designed according to the sequences of these nine regions, one novel sterile source with less alien cytoplasm was discovered among 305 materials and named Bel CMS. The size of the alien cytoplasm in Bel CMS was 21,587 bp, accounting for 8.93% of its mtDNA, which was much less than that in Ogura CMS. Most importantly, the sterility gene orf138 was replaced by orf112, which had a 78-bp deletion, in Bel CMS. Interestingly, Bel CMS cabbage also maintained 100% sterility, although orf112 had 26 fewer amino acids than orf138. Field phenotypic observation showed that Bel CMS was an excellent sterile source with stable 100% sterility and no withered buds at the early flowering stage, which could replace Ogura CMS in cabbage heterosis utilization.

Marker-assisted breeding of onion (A. cepa L.) maintainer line resistant to Downy mildew

Relevance. F1 hybrids breeding programs of onion are based on the use of nuclear cytoplasmic male sterility (NCMS). Breeding and seed production based on NCMS suggest a development of a female sterile maternal line, a maintainer line and a paternal component. The development of an isogenic pair of sterile line – maintainer line is one of the most labor-, time-consuming and intellectually-intensive stages of a breeding program, the implementation of which becomes more complicated with an increase in the number of traits / genes for which selection is carried out.Methods and results. This paper presents a genetic scheme for the development of a Downy mildewresistant (pathogen P. destructor) maintainer line of onion using marker-assisted selection (MAS). F2 populations were obtained from the cross of the female maintainer inbred line, Bn1- (13) (genotype – cytN msms pdpd) and male inbred line resistant to Downy mildew, No. 136 (genotype – cytT MsMs PdPd). DNA marker DMR1 were used for Downy mildew resistance gene Pd, jnurf13 – for maintainer gene ms and marker system 5`cob: orfA501 – for N, S and T cytoplasms. It was shown that the MAS application allow to develop a stable Downy mildew resistant maintainer line cytN msms PdPd in two generations.

MicroRNAs Involved in Regulatory Cytoplasmic Male Sterility by Analysis RNA-seq and Small RNA-seq in Soybean

Cytoplasmic male sterility (CMS) is an important plant characteristic for exploiting heterosis to enhance crop traits during breeding. However, the CMS regulatory network remains unclear in plants, even though researchers have attempted to isolate genes associated with CMS. In this study, we performed high-throughput sequencing and degradome analyses to identify microRNAs (miRNAs) and their targets in a soybean CMS line (JLCMS9A) and its maintainer line (JLCMS9B). Additionally, the differentially expressed genes during reproductive development were identified using RNA-seq data. A total of 280 miRNAs matched soybean miRNA sequences in miRBase, including mature miRNAs and pre-miRNAs. Of the 280 miRNAs, 30, 23, and 21 belonged to the miR166, miR156, and miR171 families, respectively. Moreover, 410 novel low-abundant miRNAs were identified in the JLCMS9A and JLCMS9B flower buds. Furthermore, 303 and 462 target genes unique to JLCMS9A and JLCMS9B, respectively, as well as 782 common targets were predicted based on the degradome analysis. Target genes differentially expressed between the CMS line and the maintainer line were revealed by an RNA-seq analysis. Moreover, all target genes were annotated with diverse functions related to biological processes, cellular components, and molecular functions, including transcriptional regulation, the nucleus, meristem maintenance, meristem initiation, cell differentiation, auxin-activated signaling, plant ovule development, and anther development. Finally, a network was built based on the interactions. Analyses of the miRNA, degradome, and transcriptome datasets generated in this study provided a comprehensive overview of the reproductive development of a CMS soybean line. The data presented herein represent useful information for soybean hybrid breeding. Furthermore, the study results indicate that miRNAs might contribute to the soybean CMS regulatory network by modulating the expression of CMS-related genes. These findings lay the foundation for future studies on the molecular mechanisms underlying soybean CMS.

Comparative analysis of chloroplast genomes of kenaf cytoplasmic male sterile line and its maintainer line

Kenaf is a great source of bast fiber and possesses significantly industrial interests. Cytoplasmic male sterility (CMS) is the basis of heterosis utilization in kenaf. Chloroplast, an important organelle for photosynthesis, could be associated with CMS. To understand the phylogenetic position and molecular basis of kenaf CMS from the perspective of chloroplast, the chloroplast (cp) genomes of the CMS line P3A and its maintainer line P3B were characterized and their comparative analysis was also performed. In this study, the chloroplast genomes of P3B and P3A were sequenced with 163,597 bp and 163,360 bp in length, respectively. A total of 131 genes including 85 protein coding genes (PCGs), 38 transfer RNA (tRNA) genes, and 8 ribosome RNA (rRNA) genes were annotated in P3B, while 132 genes containing 83 PCGs, 41 tRNA genes, and 8 rRNA genes were found in P3A. The phylogenetic tree revealed that kenaf was closely related to Hibiscus syriacus and Abelmoschus esculentus. Further analysis of single nucleotide polymorphism (SNP) and insertion and deletion (InDel) showed that compared with P3B, a total of 22 SNPs and 53 InDels were detected in gene coding region, gene intron, and intergenic regions of P3A. Remarkably, a total of 9 SNPs including 6 synonymous SNPs and 3 nonsynonymous SNPs were found in psbK, atpA, rpoC2, atpB, rpl20, clpP, rpoA, and ycf1. The present study provided basic information for further study of kenaf CMS mechsnism.

Search for cytoplasmic male sterility plants and its maintainer plants of carrots in Rogneda variety

Relevance. Hybrid seed production of carrots is carried out on a three-line basis, which suggests the presence of a male sterile maternal line and a fertile paternal line. For the reproduction of a male sterile line, a maintainer line is needed. The process of creating an isogenic pair of a manly sterile line – a maintainer line is of great complexity. As a maintainer line, it is necessary to use only a plant – a homozygote Cyt N msms for the core genes providing sterility, but with a normal cytoplasm. The purpose of the work is the creation of maintainer line for the sterility of carrots from the population of the Rogneda variety.Methods. Research were carried out: in 2016, at the experimental base of the Voronezh Vegetable Experimental Station (Voronezh Region); in 2017 - 2019, at the breeding center of All-Russian Research Institute of Vegetable Growing – branch of Federal State Budgetary Scientific Institution Federal Scientific Vegetable Center (Moscow Region). The material for the work was seed plants and roots of the Rogneda variety and hybrid, inbred, backcross progenies obtained from this population. Breeding methods: hybridization, inbreeding. Crosses used: simple direct and complex return (backcrosses), inbreeding.Results.Research revealed the possibility of searching for a maintainer line in the Rogneda variety. The P43f inbred progeny is potential line B, which maintains the CMS trait of the petaloid type at 100%. Of the 67 hybrid progenies, only P43st x P43f was left, in which all plants with male sterility. 65 hybrid progenies contained both sterile and fertile plants. Associated 65 inbred progenies had sterile and fertile plants, i.e. it makes no sense to try to obtain a maintainer line is needed. The process of creating an isogenic pair of a manly sterile line – a maintainer line from such fertile plants. Hybrid progeny of P4st x P4f had 100% fertile plants. The inbred progeny of P4f was represented only by fertile plants.

Discovery of Four Novel ORFs Responsible for Cytoplasmic Male Sterility (CMS) in Cotton (Gossypium hirsutum L.) through Comparative Analysis of the Mitochondrial Genomes of Four Isoplasmic Lines

Cytoplasmic male sterility (CMS) is an important feature for achieving heterosis in the development of hybrid crops. Mitochondria contribute to CMS, especially via mitochondrial DNA (mtDNA) rearrangements and chimeric genes. However, the mechanisms of CMS have not been fully elucidated, and the isonuclear alloplasmic lines used in previous studies have limited utility in cotton CMS research. In this study, three CMS lines (J4A-1, J4A-2 and J4A-3) and their isoplasmic maintainer line (J4B) were analyzed for mtDNA structural differences via high-throughput sequencing. The results showed that mtDNA was conserved (with similarities higher than 99%) among the three CMS lines and their isoplasmic maintainer line. All lines harbored 36 known protein-coding genes, 3 rRNAs, and 15 tRNAs. The protein-coding genes with non-synonymous mutations mainly encoded two types of proteins: ATPase and ribosomal proteins. Four new open reading frames (ORFs) (orf116b, orf186a-1, orf186a-2 and orf305a) were identified as candidate ORFs responsible for CMS. Two of the ORFs (orf186a-1 and orf186a-2) were identified as orf4 and orf4-2 of the upland cotton CMS line 2074A (a line with Gossypium harknessii Brandegee CMS-D2-2 cytoplasm), respectively. These findings provide a reference for CMS research in cotton or other crops.