Cytoplasmic Male Sterile Lines Research Articles

Characterization and utilization of a cytoplasmic male sterility line of Wucai (Brassica campestris L.)

The novel male sterility line 12-14A of Wucai (Brassica campestris) was generated by backcrossing. In this study, morphological, molecular, and cytological analyses were performed to characterize this line. The phenotypes of flower organs of 12-14A were different from those of its maintainer line 12-14B, as the flowers of 12-14A were completely infertile. Molecular analysis confirmed that the male sterility line 12-14A was classified as the ogu CMS type. An analysis of anther development in the CMS line demonstrated that two types of abortion occur simultaneously: pollen mother cell abortion and tetrad-to-mononuclear microspore abortion. In addition, the mature anthers of 12-14A showed typical abortive features, such as appearing dry, shrunken, and scaly. A heterosis test of hybrids from CMS 12-14A and four inbred lines was carried out, and the F1 hybrids exhibited clear heterosis in plant weight, as well as in the protein, soluble sugar, and vitamin C content, suggesting high yields and high quality potential. These results indicate that this novel CMS line may have powerful potential in Wucai hybrid breeding.

Identification of Differentially Expressed miRNAs between a Wheat K-type Cytoplasmic Male Sterility Line and Its Near-Isogenic Restorer Line.

K-type cytoplasmic male sterility (KCMS) lines were ideal material for three-line hybrid wheat system due to the major role in hybrid wheat production. In this study, the morphology of developing microspore and mature pollen was compared between a KCMS line and its near-isogenic restorer line (KCMS-NIL). The most striking difference is that the microspore was unable to develop into tricellular pollen in the KCMS line. MicroRNA plays vital roles in flowering and gametophyte development. Small RNA sequencing identified a total of 274 known and 401 novel miRNAs differentially expressed between two lines or two developmental stages. Most of miRNAs with high abundance were differentially expressed at the uninucleate stage, and their expression level recovered or remained at the binucleate stage. Further degradome sequencing identified target genes which were mainly enriched in transcription regulation, phytohormone signaling and RNA degradation pathways. Combining with the transcriptome data, a correlation was found between the abnormal anther development, such as postmeiotic mitosis cessation, deformative pollen wall and the chromosome condensation of the vegetative cell, and the alterations in the related miRNA and their targets expression profiles. According to the correlation and pathway analysis, we propose a hypothetic miRNA-mediated network for the control of KCMS restoration.

Effect of male sterile and fertile cytoplasm on nuclear DNA methylation in hybrid rice

Nucleus-controlled fertility restoration and cytoplasmic male sterility are important mechanisms to exploit heterosis. However, the effect of DNA methylation on cytoplasmic-nuclear interaction is not well understood yet. The current study used a methylation-sensitive amplified polymorphism to characterize polymorphism in nuclear DNA methylation among cytoplasmic male sterile line (D62A), corresponding maintainer line (D62B), and two F1 hybrids (D62A × R527 and D62B × R527). In results, 495 fragments were amplified between the parental D62A and D62B lines. The total methylation (double + single-stranded) and full methylation (double-stranded) rates of D62A (33.13%, 24.24%) both were found to be lower than that of corresponding maintainer D62B (33.94%, 24.85%). Analysis of methylation revealed that male sterile line D62A was less methylated than that of corresponding maintainer line D62B in all methylation types I, II and III. A total of 516 fragments were amplified between two F1 hybrids (D62A × R527 and D62B × R527). The total methylation in both hybrids (D62A × R527 and D62B × R527) was identical (34.69%). While full methylation rates for D62A × R527 and D62B × R527 were 25.78% and 25.58%, respectively, that is non-significant. Moreover, polymorphism in DNA methylation was found higher in F1 hybrids (5.43%) than parents (4.24%). These results implied that different cytoplasm leads to changes in nuclear DNA methylation and sterile cytoplasm has reduced the effect on nuclear methylation than non-sterile cytoplasm. Current study explains the interaction between cytoplasmic male sterility and DNA methylation which may contribute to further research.

Estimation of seed yield in oilseed rape to identify the potential of semi-resynthesized parents for the development of new hybrid cultivars.

Resynthesized (RS) Brassica napus can be used to increase the genetic diversity of this important crop plant and to develop the heterotic gene pool required for successful hybrid breeding programmes. The level of heterosis in F1 hybrids depends on the individual performance of the parents and on the degree of genetic difference between them. However, RS forms obtained from crosses of B. rapa ssp. with B. oleracea ssp. possess many undesirable agronomic traits, such as low quality of seeds, low yield and seed oil content, high erucic acid level in the oil and high glucosinolate content in seed meal. Therefore, RS oilseed rape needs to be improved by crossing with natural double-low oilseed rape, leading to selected double-low quality semi-RS lines that can be used for breeding. In this study, we evaluated the seed yield potential of F1 hybrids derived from crosses between Ogura cytoplasmic male-sterility (CMS) lines and doubled haploid (DH) semi-RS lines with restorer gene in three locations in Poland. The genotype by environment interaction (GE interaction) and general combining ability (GCA) of the restorer and CMS line effects, as well as the effects of heterosis, were also assessed. The results of the study provide the first insights into the use of semi-RS lines as components for the development of new hybrid cultivars. Even the introduction of 50% of the RS oilseed rape genotype to natural restorer lines resulted in a marked heterosis effect, with seed yield ranging from 4.56% to 90.17% more than that of the better parent. The yield of the best hybrid amounted to 108.6% of the seed yield of the open-pollinated cultivar Monolit and 94.4% of that of the hybrid cultivar Arsenal. The best DH semi-RS line S1, which had a significantly positive GCA for seed yield, can be recommended as a possible parent for inclusion in breeding programmes aimed at developing new hybrid cultivars.

Registration of DB 199313, Cytoplasmic Male Sterile Grain Amaranth Genetic Stock

Grain amaranth (Amaranthus spp.) seed stocks are customarily from self‐pollinating plants that open pollinate and are not F1 hybrids. However, cytoplasmic male sterility (CMS) traits are available for hybrid seed production. DB 199313 (Reg. No. GS‐10, PI 686465) grain amaranth (A. hypochondriacus L.) is the first publicly available CMS line in Amaranthus. It is an F1 hybrid from a cross between male sterile plants of DB 921 (PI 568125) and male fertile PI 568179. Fertility can be restored by crossing DB 199313 with K432 (PI 538323). DB 199313 is adapted for seed production in Ames, IA, from direct seeding in the field, but it has not been selected for competitive agronomic performance. It provides a new publicly available genetic resource for improved grain amaranth crossing.

Comparative acetylomic analysis reveals differentially acetylated proteins regulating anther and pollen development in kenaf cytoplasmic male sterility line.

Cytoplasmic male sterility (CMS) is widely used in plant breeding and represents a perfect model to understand cyto-nuclear interactions and pollen development research. Lysine acetylation in proteins is a dynamic and reversible posttranslational modification (PTM) that plays an important roles in diverse cell processes and signaling. However, studies addressing acetylation PTM regarding to anther and pollen development in CMS background are largely lacking. To reveal the possible mechanism of kenaf (Hibiscus cannabinus L.) CMS and pollen development, we performed a label-free-based comparative acetylome analysis in kenaf anther of a CMS line and wild-type (Wt). Using whole transcriptome unigenes of kenaf as the reference genome, we identified a total of 1204 Kac (lysin acetylation) sites on 1110 peptides corresponding to 672 unique proteins. Futher analysis showed 56 out of 672 proteins were differentially acetylated between CMS and Wt line, with 13 and 43 of those characterized up- and downregulated, respectively. Thirty-eight and 82 proteins were detected distinctively acetylated in CMS and Wt lines, respectively. And evaluation of the acetylomic and proteomic results indicated that the most significantly acetylated proteins were not associated with abundant changes at the protein level. Bioinformatics analysis demonstrated that many of these proteins were involved in various biological processes which may play key roles in pollen development, inculding tricarboxylic acid (TCA) cycle and energy metabolism, protein folding, protein metabolism, cell signaling, gene expression regulation. Taken together, our results provide insight into the CMS molecular mechanism and pollen development in kenaf from a protein acetylation perspective.

Transcriptomic and Proteomic Analysis of Shaan2A Cytoplasmic Male Sterility and Its Maintainer Line in Brassica napus.

Cytoplasmic male sterility (CMS) lines are widely used for hybrid production in Brassica napus. The Shaan2A CMS system is one of the most important in China and has been used for decades; however, the male sterility mechanism underlying Shaan2A CMS remains unknown. Here, we performed transcriptomic and proteomic analysis, combined with additional morphological observation, in the Shaan2A CMS. Sporogenous cells, endothecium, middle layer, and tapetum could not be clearly distinguished in Shaan2A anthers. Furthermore, Shaan2A anther chloroplasts contained fewer starch grains than those in Shaan2B (a near-isogenic line of Shaan2A), and the lamella structure of chloroplasts in Shaan2A anther wall cells was obviously aberrant. Transcriptomic analysis revealed differentially expressed genes (DEGs) mainly related to carbon metabolism, lipid and flavonoid metabolism, and the mitochondrial electron transport/ATP synthesis pathway. Proteomic results showed that differentially expressed proteins were mainly associated with carbohydrate metabolism, energy metabolism, and genetic information processing pathways. Importantly, nine gene ontology categories associated with anther and pollen development were enriched among down-regulated DEGs at the young bud (YB) stage, including microsporogenesis, sporopollenin biosynthetic process, and tapetal layer development. Additionally, 464 down-regulated transcription factor (TF) genes were identified at the YB stage, including some related to early anther differentiation such as SPOROCYTELESS (SPL, also named NOZZLE, NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), MYB80 (formerly named MYB103), and ABORTED MICROSPORES (AMS). These results suggested that the sterility gene in the Shaan2A mitochondrion might suppress expression of these TF genes in the nucleus, affecting early anther development. Finally, we constructed an interaction network of candidate proteins based on integrative analysis. The present study provides new insights into the molecular mechanism of Shaan2A CMS in B. napus.

Identifying the genes around Rf5 and Rf6 loci for the fertility restoration of WA-type cytoplasmic male sterile japonica rice (Oryza sativa) lines

Wild abortive (WA)-type, Honglian (HL)-type, and Chinsurah Boro II-type cytoplasm are three typical sterile cytoplasms used to generate three-line hybrid rice, and the fertility restorer (Rf) genes are considered to have specificity for fertility restoration of cytoplasmic male sterility (CMS) lines. ‘93-11’, an HL-type indica restorer line used widely in China, shows a weak ability to restore the fertility of WA-type CMS lines. Rf5 and Rf6, the fertility restorer genes for HL-type CMS, are members of a multigene cluster that encodes pentatricopeptide repeat proteins in ‘93-11’. In the present study, we studied the function of Rf genes around Rf5 and Rf6 loci on fertility restoration to WA-type CMS lines. We generated plants carrying WA-type cytoplasm and different genotypes at the Rf5 and Rf6 loci. All plants exhibited no seed setting on bagged panicles but had different anther and pollen grain morphologies. Plants with the genotypes of Rf5rf5rf6rf6, Rf5Rf5rf6rf6, rf5rf5Rf6rf6, rf5rf5Rf6Rf6, and Rf5rf5Rf6rf6 exhibited degraded anthers and typical abortive pollen grains, which were same as those of WA-NipA plants (rf5rf5rf6rf6); however, plants with the genotypes Rf5rf5Rf6Rf6, Rf5Rf5Rf6rf6, and Rf5Rf5Rf6Rf6 displayed restored anthers and pollen grains. These results indicated that Rf genes around the Rf5 and Rf6 loci had minor effects on the fertility restoration of WA-type CMS lines, which were mediated by dosage effects. Furthermore, these Rf genes functioned to decrease the WA352 (the mitochondrial gene conferring CMS-WA) transcript levels. Our findings will promote the development of three-line hybrids.

RNA editing analysis of ATP synthase genes in the cotton cytoplasmic male sterile line H276A

BackgroundPollen development is an energy-consuming process that particularly occurs during meiosis. Low levels of adenosine triphosphate (ATP) may cause cell death, resulting in CMS (cytoplasmic male sterility). DNA sequence differences in ATP synthase genes have been revealed between the N- and S-cytoplasms in the cotton CMS system. However, very few data are available at the RNA level. In this study, we compared five ATP synthase genes in the H276A, H276B and fertile F1 (H276A/H268) lines using RNA editing, RNA blotting and quantitative real time-PCR (qRT-PCR) to explore their contribution to CMS. A molecular marker for identifying male sterile cytoplasm (MSC) was also developed.ResultsRNA blotting revealed the absence of any novel orf for the ATP synthase gene sequence in the three lines. Forty-one RNA editing sites were identified in the coding sequences. RNA editing showed that proteins had 32.43% higher hydrophobicity and that 39.02% of RNA editing sites had proline converted to leucine. Two new stop codons were detected in atp6 and atp9 by RNA editing. Real-time qRT-PCR data showed that the atp1, atp6, atp8, and atp9 genes had substantially lower expression levels in H276A compared with those in H276B. By contrast, the expression levels of all five genes were increased in F1 (H276A/H268). Moreover, a molecular marker based on a 6-bp deletion upstream of atp8 in H276A was developed to identify male sterile cytoplasm (MSC) in cotton.ConclusionsOur data substantially contributes to the understanding of the function of ATP synthase genes in cotton CMS. Therefore, we suggest that ATP synthase genes might be an indirect cause of cotton CMS. Further research is needed to investigate the relationship among ATP synthase genes in cotton CMS.

Abnormal tapetum development and energy metabolism associated with sterility in SaNa-1A CMS of Brassica napus L.

Abnormal tapetum degradation and anther development in cytoplasmic male sterility SaNa-1A are the main reasons for the anther abortion. SaNa-1A is a novel cytoplasmic male sterility (CMS) line of Brassica napus derived from somatic hybrids of B. napus-Sinapis alba, and SaNa-1B is the corresponding maintainer line. Ultrastructural comparison between developing anthers of sterile and maintainer lines revealed abnormal subcellular structure of pollen mother cells (PMCs) in the CMS line. The PMC volume and size of nucleus and nucleolus in the CMS line were smaller than those in the maintainer line. The abnormal tapetum cell development and delayed tapetum degradation inhibited microspore development. Finally, anther abortion in the CMS line occurred. Physiological and biochemical analyses of developing anthers and mitochondria revealed that over-accumulation of reactive oxygen species (ROS) in the SaNa-1A and deficiency in antioxidant enzyme system aggravated the oxidization of membrane lipids, resulting in malondialdehyde (MDA) accumulation in anthers. High MDA content in the CMS line was toxic to the cells. ROS accumulation in SaNa-1A also affected anther development. Abnormal structure and function of terminal oxidase, which participates in the electron transport chain of mitochondrial membrane, were observed and affected the activity of cytochrome c oxidase and F1F0-ATPase, which inhibited ATP biosynthesis. Proline deficiency in SaNa-1A also affected anther development. Few hybridization signals of programmed cell death (PCD) in tetrads of SaNa-1A were identified using TdT-mediated dUTP Nick-End Labeling assay. PCD was not obvious in tapetum cells of SaNa-1A until the unicellular stage. These results validated the cytological differences mentioned above, and proved that abnormal tapetum degradation and anther development in SaNa-1A were the main reasons for the anther abortion.

Undesired fertility restoration in msm1 barley associates with two mTERF genes

The novel Rfm3 locus causing undesired fertility restoration in the msm1 cytoplasm of winter barley is located on the short arm of chromosome 6H. Undesired fertility restoration of cytoplasmic male sterile (CMS) mother lines in absence of the functional Rfm1 restorer gene is a significant problem for hybrid breeding in winter barley. Here, we describe that a novel restorer locus on the short arm of chromosome 6H, designated Rfm3, is closely linked to two mitochondrial transcription termination factor family (mTERF) protein coding genes. Genome-wide association studies in a multiparental mapping population revealed that two of the most significantly associated markers are located very close to these genes, with one marker lying directly within one mTERF gene sequence. Sequences of the candidate genes in the parental lines, segregating individuals and an independent set of breeding lines clearly revealed haplotypes discriminating completely sterile, partially fertile and Rfm1-restorer lines. The haplotypes segregate for several single nucleotide polymorphisms, a 6bp insertion-deletion (InDel) polymorphism and another 2bp InDel. CMS-unstable genotypes carrying haplotypes associated with undesired fertility restoration showed significantly higher grain setting on bagged spikes when plants were subjected to elevated temperatures during anthesis, indicating a temperature influence on pollen fertility. SNPs associated with desirable Rfm3 haplotypes can be implemented in marker-assisted selection of stable CMS mother lines.

Identification of genes related to the regulation of anther and pollen development in Mu-type cytoplasmic male sterile wheat (Triticum aestivum) by transcriptome analysis

Cytoplasmic male sterile (CMS) lines are important tools for hybrid production but they cannot produce viable pollen. Breeding new CMS lines and studying their sterility mechanism in wheat (Triticum aestivum L.) greatly facilitates the process of hybrid wheat breeding. We conducted transcriptome sequencing for a recently identified Mu-CMS line with Aegilops uniaristata Vis. cytoplasm, named U706A, and its isonuclear maintainer line (706B) at the binucleate stage, which was a critical period when abortion occurred. We found that most of the genes involved in phosphatidylinositol metabolism and pectin degradation were downregulated, as well as genes encoding the MYB21 and MYC2 transcription factors, in U706A compared with 706B. In addition, pectin contents indicated that the production of pectin has been enhanced from the binucleate stage to the trinucleate stage, owing to the downregulation of pectin-degradation-related genes in U706A at the binucleate stage, which confirmed the reliability of the sequencing results. We also discovered that the accumulation period of pectin content in U706A is abnormal compared with 706B, which may be an important reason for abortion. Some differentially expressed genes that might be related to the sterile phenotype were verified by quantitative RT-PCR. Therefore, we suggest that the downregulation of these genes possibly leads to the anther not to crack; the tapetum and microspore membrane system is less metabolised, and the abnormal pectin accumulation results in microspore nutrient deficiencies and abnormal development. These findings provide novel insights into the mechanism responsible for pollen abortion in CMS, which may facilitate hybrid wheat breeding.

Study on the combining ability and gene action in sunflower through line × tester mating design

This study was conducted to evaluate gene action and combining ability for yield, agronomic traits through line X tester analysis with 36 hybrids, developed from nine testers and four lines. The experiment was conducted in randomized complete block design with three replicates for two years 2015-16 and 2016-17 at AICRP-Sunflower, Nimpith Centre. Gene action and combining ability for seed yield and other yield attributing agronomic traits were studied in the sunflower hybrids. The cytoplasmic male sterile lines were used as female lines while some new male inbreds were introduced as testers in the form of fertility restorers. The inbred lines and their F1hybrids differed significantly in their mean values of the traits under the present study. For each of the traits, highly significant positive and negative GCA values were recorded which was reflected from the hybrid combinations. Highly significant positive effects of the GCA for seed oil content were observed in the Rf-lines R-138-2, R-104 and EC-601978 and in Pet-89-1A. On the basis of analysis, SCA: GCA ratio variances indicated that the most of the total genetic variation for head diameter, oil content (%), seed yield per plant or seed yield in kg per hectare, volume weight (g/100 cc), 100 seed weight(g), 100 seed kernel weight, hull content and oil yield in kg per hectare were governed by the non-additive gene action or non-additive component of genetic variance had a more significant role in the inheritance of the above mentioned traits in the F1 hybrid combinations in sunflower. The study’s results indicate that the additive as well as non-additive components had a more or less equal role for the inheritance of plant height trait in sunflower. Highly significant positive effects of the GCA for seed yield (kg/ha) were observed in the Rf-lines R-12-96, R-104, EC-601978 and R-630 and in Pet-2-7-1A and CMS-107A. For early maturity and semi-dwarfness, the best general combiners were CMS P-2-7-1A and CMS-10A and EC-601958 (restorer line). Five parents, R-104, R-12-96, EC-601978, EC-601958 and P-2-7-1A exhibited positive high gca effect for seed yield and some other yield components like head diameter, 100 seed weight, volume weight (g/100cc) and oil %, therefore, these five parents could be adjudged good general combiners for high seed oil content as well as the high oil yielder sunflower hybrids in near future. It also depicted that the above genotypes appeared to possess high concentration of non-additive genes for seed yield and component traits. The results of this study may be used for the identification of new high yielding sunflower hybrids based on the hybridization between the best combiners.

General and Specific Combining Ability Estimates for Morphological, Yield and its Attributes and Seed Traits in Sunflower (Helianthus annuus L.)

The improvement in sunflower breeding requires exploitation of combining ability of divergent male and female inbreds. Six cytoplasmic male sterile (CMS) lines and three testers were crossed in line ´ tester design, thus 18 F1 hybrids were developed for evaluation of general combining ability (GCA) and specific combining ability (SCA) of inbred parents for days to 90% maturity, stem girth, head size, achenes/plant, 1000-seed weight, achene yield kg/ha, oil and protein%. The significant variances due to lines and testers both determined GCA variances revealed the predominance of additive genes whilst significance of lines ´ tester interactions indicated the importance of SCA variances and the involvement of non-additive genes in the expression of traits studied. The foremost role of non-additive genes was apparent when ratio s2 SCA/s2GCA was above 1.0. These results suggested the prevalence of dominant genes and possibility of hybrid crop development. The GCA effects indicated that CMS parents SF-187, 64-A-93 and ARG-0405 and tester RHP-46 were high general combiners, thus may be chosen for crossing and selection programmes, whereas F1 hybrids SF-187 ´ RHP-46, 64-A-93 ´ RHP-46, PAC-ARG-0405 PAC-ARG-0106, 64-A-93 ´ RHP-46 and PSF-025 ´ RHP-64 which used parents with good ´ good and good ´ poor GCA estimates revealed higher positive SCA estimates for achene yield, oil and protein traits yet manifested desirable negative effects for 90% maturity. Such results suggested that these hybrids are desirable for the exploitation of hybrid crop development or selection of desirable plants from earlier filial generations.

Development of High Yielding Glutinous Cytoplasmic Male Sterile Rice (Oryza sativa L.) Lines through CRISPR/Cas9 Based Mutagenesis of Wx and TGW6 and Proteomic Analysis of Anther

Development of high yielding and more palatable glutinous rice is an important goal in breeding and long-standing cultural interaction in Asia. In this study, the TGW6 and Wx, major genes conferring 1000 grain weight (GW) and amylose content (AC), were edited in a maintainer line by CRISPR/Cas9 technology. Four targets were assembled in pYLCRISPR/Cas9Pubi-H vector and T0 mutant plants were obtained through Agrobacterium mediated transformation with 90% mutation frequency having 28% homozygous mutations without off-target effects in three most likely sites of each target and expression level of target genes in mutant lines was significantly decreased (P < 0.01), the GW and gel consistency (GC) were increased, and the AC and gelatinization temperature (GT) were decreased significantly and grain appearance was opaque, while there was no change in starch content (SC) and other agronomic traits. Mutations were inheritable and some T1 plants were re-edited but T2 generation was completely stable. The pollen fertility status was randomly distributed, and the mutant maintainer lines were hybridized with Cytoplasmic Male Sterile (CMS) line 209A and after subsequent backcrossing the two glutinous CMS lines were obtained in BC2F1. The identified proteins from anthers of CMS and maintainer line were closely associated with transcription, metabolism, signal transduction, and protein biosynthesis. Putative mitochondrial NAD+-dependent malic enzyme was absent in CMS line which caused the pollen sterility because of insufficient energy, while upregulation of putative acetyl-CoA synthetase and Isoamylase in both lines might have strong relationship with CMS and amylose content. High yielding glutinous CMS lines will facilitate hybrid rice breeding and investigations of proteins linked to male sterility will provide the insights to complicated metabolic network in anther development.

Comparative Transcriptome Analysis between Fertile and CMS Flower Buds in Wucai (Brassica campestris L.)

BackgroundWucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variant of nonheading Chinese cabbage (Brassica campestris L.), which is one of the major vegetables in China. Cytoplasmic male sterility (CMS) has been used for Wucai breeding in recent years. However, the underlying molecular mechanism of Wucai CMS remains unclear. In this study, the phenotypic and cytological features of Wucai CMS were observed by anatomical analysis, and a comparative transcriptome analysis was carried out to identify genes related to male sterility using Illumina RNA sequencing technology (RNA-Seq).ResultsMicroscopic observation demonstrated that tapetum development was abnormal in the CMS line, which failed to produce fertile pollen. Bioinformatics analysis detected 4430 differentially expressed genes (DEGs) between the fertile and sterile flower buds. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these DEGs. Among the DEGs, 35 genes (53 DEGS) were implicated in anther and pollen development, and 11 genes were involved in pollen cell wall formation and modification; most of these showed downregulated expression in sterile buds. In addition, several genes related to tapetum development (A6, AMS, MS1, MYB39, and TSM1) and a few genes annotated to flowering (CO, AP3, VIN3, FLC, FT, and AGL) were detected and confirmed by qRT-PCR as being expressed at the meiosis, tetrad, and uninucleate microspore stages, thus implying possible roles in specifying or determining the fate and development of the tapetum, male gametophyte and stamen. Moreover, the top four largest transcription factor families (MYB, bHLH, NAC and WRKY) were analyzed, and most showed reduced expression in sterile buds. These differentially expressed transcription factors might result in abortion of pollen development in Wucai.ConclusionThe present comparative transcriptome analysis suggested that many key genes and transcription factors involved in anther development show reduced gene expression patterns in the CMS line, which might contribute to male sterility in Wucai. This study provides valuable information for a better understanding of CMS molecular mechanisms and functional genome studies in Wucai.

Cytoplasmic male sterile and doubled haploid lines with desirable combining ability enhances the concentration of important antioxidant attributes in Brassica oleracea

The Brassica vegetable crops are rich source of important antioxidant compounds having anticancer and health promoting properties. Development of F1 hybrids with better nutritional traits is one of the main breeding objectives in different vegetable crops. Our study is the first report of determining heterotic combinations utilizing cytoplasmic male sterile (CMS) and doubled haploid (DH) inbred lines for antioxidant compounds in snowball cauliflower. Twenty genetically diverse Ogura CMS lines of cauliflower and six DH male fertile inbred lines were crossed to develop 120 F1 hybrids in line × tester mating design. The resulting 120 test cross progenies along with 26 parents and 4 standard checks were evaluated in 10 × 15 alpha lattice design with three replications during next cropping season. The CMS lines Ogu33-1A, Ogu122-5A and Ogu119-1A were good general combiner and CMS line Ogu118-6A was poor general combiner for majority of traits. Most of the heterotic hybrids were associated with high positive SCA effects. The proportions of σ2A/D and $$\upsigma^{2}_{\text{gca}} /\upsigma^{2}_{\text{sca}}$$ ratios were less than unity in all the cases indicating preponderance of non-additive gene action in the genetic control of all the traits. Highest number of heterotic hybrids with SCA effects in desired positive direction was recorded for ascorbic acid content and phenolic content followed by total carotenoid content. The F1 hybrids with better combining ability and better per se performance could be useful in accumulation of favourable allele for higher concentration of important anti-oxidant compounds.

Characterization for Drought Tolerance and Physiological Efficiency in Novel Cytoplasmic Male Sterile Sources of Sunflower (Helianthus annuus L.)

Sunflower is sensitive to drought, and furthermore, sunflower hybrids display limited cytoplasmic diversity. In addition, the wild cytoplasmic sources of sunflower are not well explored for their potential to introduce drought tolerance into newly developed hybrids. Therefore here, we carried out a Line × Tester-based genetic study using 19 sunflower genotypes representing, 13 cytoplasmic male sterile (CMS) lines from wild and conventional sources, 2 maintainer lines, and 4 restorer lines. The CMS and maintainer lines were crossed with restorer lines to develop sixty F1 hybrids. The parents and their hybrids were evaluated under two water regimes, normal irrigation and drought stress (i.e., withholding water). A total of twelve important plant descriptors were studied over a period of two years and the significant differences between parents and hybrids are reported here. More specifically, hybrid lines were higher in average values for all the descriptors. The contribution of female parent was more prominent in the expression of traits in hybrids as compared to male parents. The CMS sources varied significantly regarding seed yield per plant and other physiological traits. Proline content in the leaves of all the genotypes was three times higher in the water stress regime. Accession CMS-PKU-2A was identified as the best general combiner for leaf area and specific leaf weight., whereas CMS-234A was the best general combiner for biological yield and photosynthetic efficiency under both conditions. The cross combinations CMS-ARG-2A × RCR-8297, CMS-234A × P124R, and CMS-38A × P124R were found significant for biological yield, seed yield and oil content under both environments. Overall, this study provides useful information about the cytoplasmic effects on important sunflower traits and drought stress tolerance.

ITRAQ-Based Proteomic Analysis of Ogura-CMS Cabbage and Its Maintainer Line.

Ogura cytoplasmic male sterility (CMS) contributes considerably to hybrid seed production in Brassica crops. To detect the key protein species and pathways involved in Ogura-CMS, we analysed the proteome of the cabbage Ogura-CMS line CMS01-20 and its corresponding maintainer line F01-20 using the isobaric tags for the relative and absolute quantitation (iTRAQ) approach. In total, 162 differential abundance protein species (DAPs) were identified between the two lines, of which 92 were down-accumulated and 70 were up-accumulated in CMS01-20. For energy metabolism in the mitochondrion, eight DAPs involved in oxidative phosphorylation were down-accumulated in CMS01-20, whereas in the tricarboxylic acid (TCA) cycle, five DAPs were up-accumulated, which may compensate for the decreased respiration capacity and may be associated with the elevated O2 consumption rate in Ogura-CMS plants. Other key protein species and pathways involved in pollen wall assembly and programmed cell death (PCD) were also identified as being male-sterility related. Transcriptome profiling revealed 3247 differentially expressed genes between the CMS line and the fertile line. In a conjoint analysis of the proteome and transcriptome data, 30 and 9 protein species/genes showed the same and opposite accumulation patterns, respectively. Nine noteworthy genes involved in sporopollenin synthesis, callose wall degeneration, and oxidative phosphorylation were presumably associated with the processes leading to male sterility, and their expression levels were validated by qRT-PCR analysis. This study will improve our understanding of the protein species involved in pollen development and the molecular mechanisms underlying Ogura-CMS.

Genome-wide analysis of DNA methylation to identify genes and pathways associated with male sterility in soybean

DNA methylation is an epigenetic modification, which is important for gene expression regulation. Although genome-wide DNA methylation studies have been reported in several plant species, the difference in the methylation pattern between the cytoplasmic male sterile (CMS) line and its maintainer in soybean remains unclear. We compared genome-wide DNA methylation between the soybean CMS line NJCMS1A and its maintainer NJCMS1B using methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) technology. The results showed that the methylation level was higher in transposable elements (TEs) than promoter and intron; however, the methylation levels varied among different types of TEs with the highest level for long terminal repeats (LTRs) and the lowest for transcription start sites (TSS) and transcription termination sites (TTS). We observed 178 differentially methylated genes (DMGs) between NJCMS1A and NJCMS1B, including 156 hypo-methylated and 22 hyper-methylated genes in NJCMS1A compared to NJCMS1B. Gene Ontology (GO) analysis showed that 114 DMGs were annotated to one or more GO categories, among which four GO terms were significantly enriched. KEGG pathway analysis showed that 18 DMGs were significantly enriched in 10 metabolism pathways, including homologous recombination, purine metabolism, proteasome, non-homologous end-joining, and pyrimidine metabolism. Further analysis indicated that the male sterility of NJCMS1A might be caused by the altered DNA methylation and disturbed metabolism pathways of some key DMGs, involved in carbohydrate and energy metabolism, transcriptional regulation, male gametophyte development, encoding mitochondrion protein, etc. However, the relationship between the DMGs and the CMS of NJCMS1A still need further research.