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.

Assessment of drought resistance in sorghum CMS lines based on various sterility sources

Background. Global climate changes have recently led to a more frequent occurrence of adverse factors and a decrease in the productivity of major crops. Sorghum is a highly drought-resistant crop that can tolerate long-term soil and air droughts with much lower harvest losses than wheat or barley. It is important to understand physiological mechanisms affecting drought tolerance when breeding efforts are aimed at improving the adaptability to abiotic conditions and productivity of sorghum hybrids.Materials and methods. Twenty sterile lines of grain sorghum with 8 types of CMS were studied in 2019 and 2020 in the arid conditions of Saratov Province. Indicators of the leaf water regime were assessed according to VIR’s guidelines. Statistical processing of the research results was performed using the AGROS 2.09 software.Results. The indicators of the leaf water regime that reflected differentiated responses of the CMS-line plants to the prevailing water and temperature stressors during the critical flowering period for sorghum were analyzed. Four CMS lines were identified according to the chosen set of indicators: they manifested 71.13–72.02% of total water content, 5.26–9.08% of water deficit, and 57.40–83.17% of water retention capacity on average for the two years of research. For the first time, the effect of CMS in sorghum on the manifestation of water regime indicators was registered. In isonuclear CMS lines, the greatest effect on drought resistance was shown by cytoplasm A3 versus A4 (with the Zheltozernoe 10 genome), cytoplasm A5 versus A1 (with the Karlik 4v genome), and M35-1A versus the analog on cytoplasm 9E (with the Pischevoe 614 genome).Conclusion. It is shown that genetically different types of sterility can be used in breeding practice to increase the resistance to abiotic stressors in components of F1 crosses and hybrids.

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.

Improvement ability of male parent by gibberellic acid application to enhancing the outcrossing of cytoplasmic male sterility rice lines

<p class="042abstractstekst">The study quantified the effect of gibberellic acid (GA₃) as a pre-flowering treatment for male parent Giza 178 R and the influence of male to female ratio (2R:10A, 2R:12A, 2R:14A and 2R:16A) between male (R) to female (A) for two Cytoplasmic Male Sterility (CMS) lines (‘IR69625’ and ‘G46’) on hybrid rice seed production. The main plots were occupied by CMS lines while; GA3 application for male parent Giza 178R were arranged in the sub plots and male to female ratio was arranged in the sub-sub plots. The results indicated that, the, duration of floret opening, angle of floret opening, filaments exsertion, filaments length<strong>, </strong>anther length, plant height and number of tiller hill^-1of male parent Giza 178R were significantly at 300 g GA₃ ha^-1 concentration. Plant height, panicle exsertion, panicle length, flag leaf angle and 1000-grain mass of CMS were not significantly affected by the GA₃ application for male parent and male to female ratio, while, number of fertile panicles hill^-1, panicle mass, seed set, seed yield and harvest index of CMS lines were highly significantly affected. The highest seed yield (2.880 and 2.950 t ha^-1) was obtained by CMS line IR69625A using 300 g GA₃ ha^-1 of male parent Giza 178R with male to female ratio of 2R:14A during both seasons.</p>

Improvement ability of male parent by gibberellic acid application to enhancing the outcrossing of cytoplasmic male sterility rice lines

<p class="042abstractstekst">The study quantified the effect of gibberellic acid (GA₃) as a pre-flowering treatment for male parent Giza 178 R and the influence of male to female ratio (2R:10A, 2R:12A, 2R:14A and 2R:16A) between male (R) to female (A) for two Cytoplasmic Male Sterility (CMS) lines (‘IR69625’ and ‘G46’) on hybrid rice seed production. The main plots were occupied by CMS lines while; GA3 application for male parent Giza 178R were arranged in the sub plots and male to female ratio was arranged in the sub-sub plots. The results indicated that, the, duration of floret opening, angle of floret opening, filaments exsertion, filaments length<strong>, </strong>anther length, plant height and number of tiller hill^-1of male parent Giza 178R were significantly at 300 g GA₃ ha^-1 concentration. Plant height, panicle exsertion, panicle length, flag leaf angle and 1000-grain mass of CMS were not significantly affected by the GA₃ application for male parent and male to female ratio, while, number of fertile panicles hill^-1, panicle mass, seed set, seed yield and harvest index of CMS lines were highly significantly affected. The highest seed yield (2.880 and 2.950 t ha^-1) was obtained by CMS line IR69625A using 300 g GA₃ ha^-1 of male parent Giza 178R with male to female ratio of 2R:14A during both seasons.</p>

Heterosis and Combining Ability For Floral and Yield Characters in Rice Using Cytoplasmic Male Sterility System

Developing high-yielding rice genotypes has become more urgent to ensure global food security with continuing population growth and the threat of environmental pressures. Cytoplasmic male sterility (CMS) system provides a valuable approach for commercial exploitation of heterosis and producing high-yielding and quality hybrid rice. Three CMS lines and ten diverse restorers were crossed using line × tester mating design. The obtained thirty F1 hybrids and their thirteen parents were evaluated. Yield traits as well as certain floral traits characters that influence the efficiency of crossing and hybrid seed production as duration of floret opening (min), stigma exsertion (mm), stigma length (mm), opening floret angle, and anther length (mm) were assessed. Highly significant variations were detected among parents, crosses, and parents vs. crosses for all the studied traits. The CMS line L2 and the restorer T5 were identified as good combiners for stigma exsertion, stigma length, opining floret angle, and duration of floret opening. Moreover, the hybrids L1×T1, L1×T3, L2×T2, L2×T5, L3×T4, L3×T5, and L3×T9 had positive SCA effects for most floral traits. Besides, the CMS lines L1 and L3 as well as the restorers T1, T2, T3, T6, and T9 proved to be the best general combiners for grain yield and certain contributing traits. The hybrids L1×T1, L1×T5, L1×T7, L2×T3, L2×T4, L2×T5, L2×T10, L3×T1, L3×T2, and L3×T6 displayed positive SCA effects for grain yield and one or more of its attributes. Both additive and non-additive gene effects are involved in the governing inheritance of all evaluated traits. The biochemical variations among the certain evaluated genotypes were further studied. The esterase and peroxidase isozymes were applied for verifying the genetic diversity at the protein level among the used CMS lines, restorers, and their crosses. All the applied isozymes displayed polymorphism for the parents and their crosses. The banding pattern and intensity differences provided accurate results on the reliable variability among the tested genotypes.

Unraveling the Genetic Basis of Fertility Restoration for Cytoplasmic Male Sterile Line WNJ01A Originated From Brassica juncea in Brassica napus

Crosses that lead to heterosis have been widely used in the rapeseed (Brassica napus L.) industry. Cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) systems represent one of the most useful tools for rapeseed production. Several CMS types and their restorer lines have been identified in rapeseed, but there are few studies on the mechanisms underlying fertility restoration. Here, we performed morphological observation, map-based cloning, and transcriptomic analysis of the F2 population developed by crossing the CMS line WNJ01A with its restorer line Hui01. Paraffin-embedded sections showed that the sporogenous cell stage was the critical pollen degeneration period, with major sporogenous cells displaying loose and irregular arrangement in sterile anthers. Most mitochondrial electron transport chain (mtETC) complex genes were upregulated in fertile compared to sterile buds. Using bulked segregant analysis (BSA)-seq to analyze mixed DNA pools from sterile and fertile F2 buds, respectively, we identified a 6.25 Mb candidate interval where Rfw is located. Using map-based cloning experiments combined with bacterial artificial chromosome (BAC) clone sequencing, the candidate interval was reduced to 99.75 kb and two pentatricopeptide repeat (PPR) genes were found among 28 predicted genes in this interval. Transcriptome sequencing showed that there were 1679 DEGs (1023 upregulated and 656 downregulated) in fertile compared to sterile F2 buds. The upregulated differentially expressed genes (DEGs) were enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) lysine degradation pathway and phenylalanine metabolism, and the downregulated DEGs were enriched in cutin, suberine, and wax biosynthesis. Furthermore, 44 DEGs were involved in pollen and anther development, such as tapetum, microspores, and pollen wall development. All of them were upregulated except a few such as POE1 genes (which encode Pollen Ole e I allergen and extensin family proteins). There were 261 specifically expressed DEGs (9 and 252 in sterile and fertile buds, respectively). Regarding the fertile bud-specific upregulated DEGs, the ubiquitin–proteasome pathway was enriched. The top four hub genes in the protein–protein interaction network (BnaA09g56400D, BnaA10g18210D, BnaA10g18220D, and BnaC09g41740D) encode RAD23d proteins, which deliver ubiquitinated substrates to the 26S proteasome. These findings provide evidence on the pathways regulated by Rfw and improve our understanding of fertility restoration.

Genetic editing of GS3 via CRISPR/Cas9 Accelerates the Breeding of Three-line Hybrid Rice With Superior Yield and Grain Quality

Grain size is one of the major traits that control rice grain yield and quality. The GS3 gene, a major QTL regulating grain length and weight, is the first one been identified. A mutation occurred in its N-terminal organ size regulating domain (OSR)results in a loss of function of the mutant allele gs3 and rice varieties carry this gs3 allele often produce longer grains. In this study, we exploited the CRISPR/Cas9 gene editing technology to introduce an edited gs3 allele, two guide RNAs(gRNA) targeted at the OSR of the GS3 gene were transformed into one of our indica maintainer lines, GM1B, for its grain yield and quality improvement. Through molecular analysis and sequencing, in T1 generation, a homologous edited-gs3 mutant line without tansgene was obtained and name as GM2B, then converted to CMS line GM1A by backcrossing to obtain another superior male sterile line GM2A for further tests. GM2B showed improved grain quality and yield compared to the WT GM1B, with grain length increased by 7.9%, length/width ratio increased from 3.89 to 4.19, TGW increased by 6.7%, and grain yield per plant increased by 14.9%. Meanwhile, genetic improvement of other quality traits including rice length (6.83mm), rice grain length/width ratio (3.61), matched the appearance standards set for traditional Simiao (silk seedling) type cultivars. Two restorer lines were outcrossed to both GM1A and GM2A to produce hybrid rice. Compared to GM1A’s two hybrids, the hybrids of GM2A had longer grains, higher length/width ratio, higher TGW and yield per plant. In addition, hybrids of GM2A showed better performance on grain appearance including better translucency, lower chalky rice rate and chalkiness degree than hybrids of GM1A.These data strongly demonstrate that the introduction of an elite gs3 allele into GM1A via CRISPR/Cas9 gene editing technology leads to significant genetic improvement. The resultant CMS line GM2A(gs3) performs much better than the original GM1A on grain quality and yield. Thus, our study proves that targeted genetic improvement using gene editing technology can expedite rice breeding, especially the breeding of three-line hybrid rice.

Complete Mitochondrial Genome Sequence and Identification of a Candidate Gene Responsible for Cytoplasmic Male Sterility in Celery (Apium graveolens L.)

Celery (Apium graveolens L.) is an important leafy vegetable worldwide. The development of F1 hybrids in celery is highly dependent on cytoplasmic male sterility (CMS) because emasculation is difficult. In this study, we first report a celery CMS, which was found in a high-generation inbred line population of the Chinese celery “tanzhixiangqin”. Comparative analysis, following sequencing and assembly of the complete mitochondrial genome sequences for this celery CMS line and its maintainer line, revealed that there are 21 unique regions in the celery CMS line and these unique regions contain 15 ORFs. Among these ORFs, only orf768a is a chimeric gene, consisting of 1497 bp sequences of the cox1 gene and 810 bp unidentified sequences located in the unique region, and the predicted protein product of orf768a possesses 11 transmembrane domains. In summary, the results of this study indicate that orf768a is likely to be a strong candidate gene for CMS induction in celery. In addition, orf768a can be a co-segregate marker, which can be used to screen CMS in celery.