Cytoplasmic Male Sterile Lines Research Articles

Genetic Analysis of Combining Ability and Gene Action in Sunflower (Helianthus annuus L.) Hybrids through Line × Tester Design

This study was conducted to evaluate the combining ability of six cytoplasmic male sterile (CMS) lines and nine testers using the line × tester method. Biometrical data were recorded for ten agronomic traits: days to 50% flowering, days to maturity, plant height, head diameter, seed filling percentage, 100-seed weight, volume weight, hull content, oil content, and seed yield per plant. The results indicated that specific combining ability (SCA) variance was higher than general combining ability (GCA) variance for most traits, except for days to 50 percent flowering and seed filling percentage, suggesting that non-additive gene action predominantly governed the inheritance of these traits. The GCA of parents did not always align with their per se performance, indicating that GCA should be the primary criterion for selecting parents in breeding programs. Three CMS lines, CMS-103A, CMS-10A and CMS-112A, along with testers EC-75717, EC-502036, EC-279309-1 and R-271-1, were identified as good general combiners for seed yield and yield-contributing traits. Among the hybrids, CMS-243A × EC-279309-1, CMS-243A × EC-75717 and CMS-103A × EC-75717 exhibited the highest SCA effects for seed yield per plant. These findings underscore the importance of selecting superior general combiners for traits like oil content and seed yield to develop high-yielding sunflower hybrids.

Genetic Diversity Analysis of Monogerm Cytoplasmic Male Sterile and Maintainer Lines of Sugar Beet

Genetic Diversity Analysis of Monogerm Cytoplasmic Male Sterile and Maintainer Lines of Sugar Beet

Morphological and cytological assessments reveal pollen degradation causes pollen abortion in cotton cytoplasmic male sterility lines

BackgroundUnderstanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources. However, only a few cytoplasmic male sterility (CMS) lines of cotton have been produced due to several challenges, like inadequate variation of agronomic traits, incomplete sterility, weak resilience of restorer lines, and difficulty in combining strong dominance. Therefore, the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding.ResultsTwo F2 segregating populations of cotton were constructed from cytoplasmic male sterile lines (HaA and 01A, maternal) and restorer lines (HaR and 26R, paternal). Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants. Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants. However, sterile plants exhibited smaller floral organs, shortened filament lengths, and anther atrophy on the flowering day in comparison with the fertile plants. When performed scanning electron microscopy (SEM), the two F2 populations revealed morphological variations in the anther epidermis. Cellular analysis showed no significant differences in pollen development before pollen maturation. Interestingly, between the pollen maturation and flowering stages, the tapetum layer of sterile plants degenerated prematurely, resulting in abnormal pollen grains and gradual pollen degradation.ConclusionThe results of this study suggest that fertility-restoring genes are controlled by a single dominant gene. Sterile plants exhibit distinctive floral morphology, which is characterized by stamen atrophy and abnormal anthers. Pollen abortion occurs between pollen maturity and flowering, indicating that premature tapetum degradation may be the primary cause of pollen abortion. Overall, our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations, mechanisms of sterility, and the role of sterile and restorer genes.

Estimation of heterosis on CGMS based pigeonpea

Thirty-five hybrids generated from seven cytoplasmic male sterile lines and five fertility restorer lines were tested for determining heterosis. All studied traits except pod length, number of seeds per pod, and leaf area showed significant variations between the parents indicating a sufficient variability amongthe parents (lines and testers). The mean sum of squares of the hybrids suggested high heterosis for studied traits except for test weight and number of branches per plant. The findings showed noteworthy and affirmative relative heterosis for eaj hybrid. Top three hybrids viz. CMS GT 1615 A × GTR 105 (28.34% and 38.97%), CMS GT 1615 A × GTR 18 (24.36% and 34.66%) and CMS GT 1615 × GTR 23 (22.23% and 32.35%) identified for seed yield and its contributing traits on the basis of standard heterosis against standard checks GTH 1 and GT 101, respectively. These can be exploited commercially for heterosis breeding in pigeonpea.

Estimation of Heterosis and Inbreeding Depression for Yield Related and Quality Traits in Forage Sorghum [Sorghum bicolor L. (Moench)

The present study was conducted at GBPUAT Pantnagar to study the heterosis and inbreeding depression for nine yield related and three quality traits which consisted of seventy F1 crosses developed through line x tester mating design involving ten cytoplasmic male sterile (female) lines and seven pollinator (male) lines during Kharif 2018 and 70 F2s developed by selfing of F1 crosses during Kharif 2019 in random block design (R.B.D.) with three replications. Observations were recorded on plant height, stem girth, number of leaves per plant, leaf length, leaf width, leaf area, leaf: stem ratio, total soluble solids, HCN content, green fodder yield per plot, dry fodder yield per plot, and protein percent. The observed F1data was analyzed for mid parent heterosis, heterobeltiosis and standard heterosis whereas F2data was analyzed for inbreeding depression. The large number of crosses exhibited heterosis and inbreeding depression in desired direction for all yield related and quality traits under study which indicated the presence of additive as well as non-additive gene actions. The crosses ICSA 271 x PC 5, ICSA 276 x CSV-15, 993100 A x 04K 668, 11 A2 x 04 K 693, ICSA 469 x 04 K 668, ICSA 293 x 01 K 733, SPA2 94012 x 04 K 700, ICSA 293 x CSV-15, SP 55609 A x UPMC-8, and 993100 A x 04 K 700 exhibited high mean performance for all the traits under study coupled with high heterosis in desired direction.

Functional analysis of the CaPIPLC5 gene in the regulation of the fertility restoration in pepper.

Cytoplasmic male sterility (CMS) is a very important factor to produce hybrid seeds, and the restoration of fertility involves the expression of many fertility-related genes. Our previous study showed that the expression of CaPIPLC5 was significantly up-regulated in pepper restorer accessions and minimally expressed in sterile accessions, speculating that CaPIPLC5 is related to the restoration of fertility. In this study, we further validated the function of CaPIPLC5 in the restoration of fertility. The results showed that CaPIPLC5 was specifically expressed in the anthers of the restorer accessions with the subcellular localization in the cytoplasm. Furthermore, the expression of CaPIPLC5 was significantly higher in restorer lines and restorer combinations than that in CMS lines and their maintainer lines. Silencing CaPIPLC5 led to the number of pollen decreased, pollen grains wrinkled, and the ratio of pollen germination reduced. In addition, the joint analysis of Yeast One-Hybrid (Y1H) and Dual-Luciferase (dual-LUC) assays suggested that transcription factors such as CaARF5, CabZIP24 and CaMYB-like1, interacted with the promoter regions of CaPIPLC5, which regulated the expression of CaPIPLC5. The present results provide new insights into the study of CaPIPLC5 involved in the restoration of fertility in pepper.

Pyramiding of bacterial blight resistance genes into promising restorer BRRI31R line through marker-assisted backcross breeding and evaluation of agro-morphological and physiochemical characteristics of developed resistant restorer lines.

BRRI31R is one of the Bangladesh's most promising restorer lines due to its abundant pollen producing capacity, strong restoring ability, good combining ability, high outcrossing rate and genetically diverse from cytoplasmic male sterile (CMS) line. But the drawback of this line is that it is highly susceptible to bacterial blight (BB) disease of rice caused by Xanthomonas oryzae pv. oryzae. The present study highlighted the pyramiding of effective BB resistance genes (xa5, xa13 and Xa21) into the background of BRRI31R, through marker-assisted backcrossing (MABC). Backcross progenies were confirmed and advanced based on the foreground selection of target genes. Pyramided lines were used for pathogenicity test against five Bangladeshi Xanthomonas oryzae (BXo) races (BXo93, BXo220, BXo822, BXo826, BXo887) and confirmed the dominant fertility restore genes, Rf3 and Rf4 and further validated against SNP markers for more confirmation of target resistance genes. All pyramided restorer lines consisted of Xa4 (in built), xa5, xa13, Xa21, and Chalk5 with two fertility restorer genes, Rf3, Rf4. and these restorer lines showed intermediate amylose content (<25%). Restorer lines BRRI31R-MASP3 and BRRI31R-MASP4 showed high levels of resistance against five virulent BXo races and SNP genotyping revealed that these lines also contained a blast resistance gene Pita races. Gene pyramided restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 can directly be used as a male parent for the development of new BB resistant hybrid rice variety or could be used as a replacement of restorer line of BRRI hybrid dhan5 and 7 to enhance the quality of hybrid seeds as well as rice production in Bangladesh.

Re-Sequencing the Mitochondrial Genome Unveils a Novel Isomeric Form of NWB CMS Line in Radish and Functional Verification of Its Candidate Sterile Gene

Radish (Raphanus sativus L.) is a globally significant vegetable and relies on cytoplasmic male sterile (CMS) lines for hybrid seed production. The NWB CMS type is favored over Ogura CMS for its ease in maintainer screening. Despite its varied mitochondrial configurations and unvalidated sterile gene, we re-sequenced the mitochondrial genome of NWB CMS Tibet A and verified the function of the sterility gene via genetic transformation of Arabidopsis thaliana. The mitochondrial genomes of Tibet A could be assembled into circular DNA molecules, with a mitochondrial genome size of 239,184 bp. Our analysis indicated that the specific orf463a was the CMS-associated gene in Tibet A, sharing sequence consistency with the CMS gene in DCGMS and NWB CMS YB-A. Collinearity analysis showed that the mitochondrial genomes of NWB CMS Tibet A, DCGMS, and NWB CMS YB-A share the same mitotype, with structural variations due to recombination via 9731 bp long repeat sequences and 508 bp short repeat sequences. Driven by the Ap3 promoter, transgenic Arabidopsis with orf463a exhibited male sterility, confirming the gene’s potential role in CMS. In this study, we assembled a new isomeric form of NWB CMS mitochondrial genome and proved the function of the candidate sterile gene.

The R2R3 MYB gene TaMYB305 positively regulates anther and pollen development in thermo-sensitive male-sterility wheat with Aegilops kotschyi cytoplasm.

The loss of TaMYB305 function down-regulated the expression of jasmonic acid synthesis pathway genes, which may disturb the jasmonic acid synthesis, resulting in abnormal pollen development and reduced fertility. The MYB family, as one of the largest transcription factor families found in plants, regulates plant development, especially the development of anthers. Therefore, it is important to identify potential MYB transcription factors associated with pollen development and to study its role in pollen development. Here, the transcripts of an R2R3 MYB gene TaMYB305 from KTM3315A, a thermo-sensitive cytoplasmic male-sterility line with Aegilops kotschyi cytoplasm (K-TCMS) wheat, was isolated. Quantitative real-time PCR (qRT-PCR) and promoter activity analysis revealed that TaMYB305 was primarily expressed in anthers. The TaMYB305 protein was localized in the nucleus, as determined by subcellular localization analysis. Our data demonstrated that silencing of TaMYB305 was related to abnormal development of stamen, including anther indehiscence and pollen abortion in KAM3315A plants. In addition, TaMYB305-silenced plants exhibited alterations in the transcriptional levels of genes involved in the synthesis of jasmonic acid (JA), indicating that TaMYB305 may regulate the expression of genes related to JA synthesis and play an important role during anther and pollen development of KTM3315A. These results provide novel insight into the function and molecular mechanism of R2R3-MYB genes in pollen development.

Commercial suitability and characterization of newly developed Erucastrum canariense (Can) sterile cytoplasm based cytoplasmic male sterile (CMS) lines in Indian cauliflower

The study presents the first to characterize novel Erucastrum canarianse Webb and Berthel (or Can) sterile cytoplasm-based CMS lines in Indian cauliflower (Brassica oleracea var. botrytis L.) and investigating their commercial suitability. Eleven Can-based CMS lines were examined for 12 agro-morphological and yield traits,18 floral traits, four seed yield traits together with three each of the Ogura (source: wild Japanese Radish) and Tour (Source: Brassica tournefortii) cytoplasms. All of the recorded floral and seed traits showed significant (P > 0.05) differences between the CMS lines of each group. Agro-morphological and yield traits in CMS lines and their maintainers, however, were non-significantly different. All the Can- and Ogura-based CMS lines showed flowering and appropriate seed formation by natural cross-pollination. Only two Tour cytoplasm-based CMS lines, Tour (DC-41-5) and Tour (DC-67), produced the smallest malformed flowers and stigma. The highest seed yield per plant in CMS lines was in Ogu (DC-98-4) and the lowest in Tour (DC-67). P14 and P15, two polymorphic mtDNA markers, were discovered for the Can CMS system for early detection. Five primers (ITS5a-ITS4, atpF-atpH, P16, rbeL and trnL), along with their maintainers, were sequenced and aligned to detect nucleotide changes including as additions and or deletions at different positions. The newly introduced E. canariense sterile cytoplasm-based CMS system in cauliflower is the subject of the first comprehensive report, which emphasises their potential as a further stable and reliable genetic mechanism for hybrid breeding.

PMAT: an efficient plant mitogenome assembly toolkit using low-coverage HiFi sequencing data.

Complete mitochondrial genomes (mitogenomes) of plants are valuable resources for nucleocytoplasmic interactions, plant evolution, and plant cytoplasmic male sterile line breeding. However, the complete assembly of plant mitogenomes is challenging due to frequent recombination events and horizontal gene transfers. Previous studies have adopted Illumina, PacBio, and Nanopore sequencing data to assemble plant mitogenomes, but the poor assembly completeness, low sequencing accuracy, and high cost limit the sampling capacity. Here, we present an efficient assembly toolkit (PMAT) for de novo assembly of plant mitogenomes using low-coverage HiFi sequencing data. PMAT has been applied to the de novo assembly of 13 broadly representative plant mitogenomes, outperforming existing organelle genome assemblers in terms of assembly accuracy and completeness. By evaluating the assembly of plant mitogenomes from different sequencing data, it was confirmed that PMAT only requires 1× HiFi sequencing data to obtain a complete plant mitogenome. The source code for PMAT is available at https://github.com/bichangwei/PMAT. The developed PMAT toolkit will indeed accelerate the understanding of evolutionary variation and breeding application of plant mitogenomes.

Identification of heterosis and combining ability in the hybrids of male sterile and restorer sorghum [Sorghum bicolor (L.) Moench] lines.

In sorghum [Sorghum bicolor (L.) Moench], combining ability and heterosis analysis are commonly used to evaluate superior parental lines and to screen for strongly heterotic hybrids, which helps in sorghum variety selection and breeding. In this context, combining ability and heterosis analysis were assessed using 14 restorer lines and seven cytoplasmic male sterile (CMS) lines in 2019 and 2020. The analysis of variance of all cross combinations had highly significant differences for all characters studied, which indicated a wide variation across the parents, lines, testers, and crosses. Combining ability analysis showed that the general combining ability (GCA) and specific combining ability (SCA) of the different parents were differed significantly among different traits. Most combinations with high SCA also showed high GCA in their parent lines. The heritability in the narrow sense of grain weight per panicle and grain yield was relatively low, indicating that the ability of these traits to be directly inherited by offspring was weak, that they were greatly affected by the environment. The better-parent heterosis for plant height, grain weight per panicle, panicle length, and 1000-grain weight was consistent with the order of mid-parent heterosis from strong to weak. The GCA effects of two lines 10480A, 3765A and three testers 0-30R, R111, and JY15R were significant for the majority of the agronomic traits including grain yield and might be used for improving the yield of grains in sorghum as parents of excellent specific combining ability. Seven strongly heterotic F1 hybrids were screened; of these, hybrids 3765A × R111, 1102A × L2R, and 3765A × JY15R showed significant increases in seed iristectorigenin A content and will feature into the creation of new sorghum varieties rich in iristectorigenin A.

Scanning Electron Microscopy Investigation of Cytoplasmic Male Sterile Line Rice : Exploring Treatment Effects on Plant Components and their Impact on Hybrid Rice Seed Production

In addressing the challenge of inflorescence emergence in cytoplasmic male sterile line (CMS) rice, specifically the CMS A-line, GA3 has conventionally been utilized. However, the high cost of GA3 poses financial constraints for middle-class and marginal farmers. This study aims to identify cost-effective alternatives to GA3 for CMS breeding. Utilizing IR58025A as the A-line and KMR3 as the R-line, obtained from Rice Research Station, Chinsurah, Hooghly, the experiment was conducted following CMS breeding layout design at the Crop Research Farm under the Department of Botany, the University of Burdwan. Various chemicals, including GA3 , penicillin, sulfonamide, and gentamicin, were applied as foliar spray treatments, alongside a control group, during flowering to produce F1 seeds from the A-line in the boro season. The primary objectives include exploring microfeatures of different plant organs such as leaves, stems, androecium, and gynoecium through SEM analysis to evaluate the effectiveness of the diverse treatments. This research contributes valuable insights for rice breeders and the scientific community, offering potential alternatives to GA3 in CMS breeding technology.

Safflower heterosis for yield and its contributed characters

Twenty two hybrids were tested using line x tester design involving two cytoplasmic male sterile lines during rabi 2018, using Randomized block design with three replications. Twenty two along with three checks 'A-1,PBNS-12, PKV Pink' were evaluated at the field of Oilseeds Research Unit, Dr. PDKV, Akola during rabi 2019- 2020 to estimate extent of heterosis in safflower genotypes.Among all the crosses, AKS CMS 2A x GMU 1183 (46.15 % and 44.30%) exhibited highly significant and positive average heterosis for number of seeds/plant over mid and better parent. crosses, AKS CMS 2A x GMU 184 (49.26% and 34.44%) exhibited highly significant and positive average heterosis for 100 seed weight, whereas, cross AKS CMS 2A x GMU 5149 (15.28% and 9.32%) exhibited highly significant and positive average heterosis for volume weight. The highest heterosis and heterobeltiosis in desirable direction were recorded for seed yield/plant in AKS CMS 2A x GMU 1654 (246.96%) and the cross AKS CMS 3A x GMU 1183 (206.75%) showed highest and significantly positive standard heterosis over the checks i.e. PKV PINK, AKS 207 and PBNS-12.

Safflower heterosis for yield and its contributed characters

Safflower (Carthamus tinctorius L.) is one of the major edible oilseed crops grown in winter season in India. The first safflower hybrid released in India for commercial cultivation in all safflower growing regions was based on genetic male sterility system. Twenty two hybrids were tested using line x tester design involving two cytoplasmic male sterile lines during rabi 2018, using Randomized block design with three replications. Twenty two along with three checks 'A-1, PBNS-12, PKV Pink' were evaluated at the field of Oilseeds Research Unit, Dr. PDKV, Akola during rabi 2019-2020 to estimate extent of heterosis in safflower genotypes. Among all the crosses, AKS CMS 2A x GMU 1183 (46.15 % and 44.30%) exhibited highly significant and positive average heterosis for number of seeds per plant over mid and better parent. crosses, AKS CMS 2A x GMU 184 (49.26% and 34.44%) exhibited highly significant and positive average heterosis for 100 seed weight, whereas, cross AKS CMS 2A x GMU 5149 (15.28% and 9.32%) exhibited highly significant and positive average heterosis for volume weight. The highest heterosis and heterobeltiosis in desirable direction were recorded for seed yield per plant in AKS CMS 2A x GMU 1654 (246.96. %) and the cross AKS CMS 3A x GMU 1183 (206.75%) showed highest and significantly positive standard heterosis over the checks i.e. PKV PINK, AKS 207 and PBNS-12.Biotechnological tools, such as genetic engineering need to be perfected for incorporation of mitochondrial male sterility causing factor of sunflower into safflower to develop safflower hybrids which can be more easily produced.

Transcriptomics reveals a core transcriptional network of K-type cytoplasmic male sterility microspore abortion in wheat (Triticum aestivum L.)

BackgroundCytoplasmic male sterility (CMS) plays a crucial role in hybrid production. K-type CMS, a cytoplasmic male sterile line of wheat with the cytoplasms of Aegilops kotschyi, is widely used due to its excellent characteristics of agronomic performance, easy maintenance and easy restoration. However, the mechanism of its pollen abortion is not yet clear.ResultsIn this study, wheat K-type CMS MS(KOTS)-90-110 (MS line) and it’s fertile near-isogenic line MR (KOTS)-90-110 (MR line) were investigated. Cytological analysis indicated that the anthers of MS line microspore nucleus failed to divide normally into two sperm nucleus and lacked starch in mature pollen grains, and the key abortive period was the uninucleate stage to dinuclear stage. Then, we compared the transcriptome of MS line and MR line anthers at these two stages. 11,360 and 5182 differentially expressed genes (DEGs) were identified between the MS and MR lines in the early uninucleate and binucleate stages, respectively. Based on GO enrichment and KEGG pathways analysis, it was evident that significant transcriptomic differences were “plant hormone signal transduction”, “MAPK signaling pathway” and “spliceosome”. We identified 17 and 10 DEGs associated with the IAA and ABA signal transduction pathways, respectively. DEGs related to IAA signal transduction pathway were downregulated in the early uninucleate stage of MS line. The expression level of DEGs related to ABA pathway was significantly upregulated in MS line at the binucleate stage compared to MR line. The determination of plant hormone content and qRT-PCR further confirmed that hormone imbalance in MS lines. Meanwhile, 1 and 2 DEGs involved in ABA and Ethylene metabolism were also identified in the MAPK cascade pathway, respectively; the significant up regulation of spliceosome related genes in MS line may be another important factor leading to pollen abortion.ConclusionsWe proposed a transcriptome-mediated pollen abortion network for K-type CMS in wheat. The main idea is hormone imbalance may be the primary factor, MAPK cascade pathway and alternative splicing (AS) may also play important regulatory roles in this process. These findings provided intriguing insights for the molecular mechanism of microspore abortion in K-type CMS, and also give useful clues to identify the crucial genes of CMS in wheat.

Copy number variation of the restorer Rf4 underlies human selection of three-line hybrid rice breeding

Cytoplasmic male sterility (CMS) lines are important for breeding hybrid crops, and utilization of CMS lines requires strong fertility restorer (Rf) genes. Rf4, a major Rf for Wild-Abortive CMS (CMS-WA), has been cloned in rice. However, the Rf4 evolution and formation of CMS-WA/Rf system remain elusive. Here, we show that the Rf4 locus emerges earlier than the CMS-WA gene WA352 in wild rice, and 69 haplotypes of the Rf4 locus are generated in the Oryza genus through the copy number and sequence variations. Eight of these haplotypes of the Rf4 locus are enriched in modern rice cultivars during natural and human selections, whereas non-functional rf4i is preferentially selected for breeding current CMS-WA lines. We further verify that varieties carrying two-copy Rf4 haplotype have stronger fertility restoration ability and are widely used in three-line hybrid rice breeding. Our findings increase our understanding of CMS/Rf systems and will likely benefit crop breeding.

Maintainer/restorer identification for different CMS lines in sunflower (Helianthus annuus L.)

Thirty one uniform and stable germplasm accessions of economic importance of sunflower were crossed with five cytoplasmic male sterile lines viz., CMS-234A, CMS-17A, CMS-338A, CMS-851A and ARM-243A in a Line x Tester fashion to study their maintainer or restorer reaction during kharif-2012 in a Randomized Block Design in two replications. The accessions TSG-22, TSG-24, ID-7, SCG-37, RHA-464 and ID-25 acted asrestorersfor allfive CMS lines. It was found that ID-33 of the restorer for ARM-243A was maintainer for other CMS lines. The use of stable germplasm with restorer behaviour in heterosis breeding programmes and those with maintainer behaviour in the development of new CMS lines through conversion has been suggested. The identification of new restorers to the good combiner CMS sources assembled should receive priority for hybrid synthesis.

Flavonoid Biosynthesis Pathway May Indirectly Affect Outcrossing Rate of Cytoplasmic Male-Sterile Lines of Soybean.

(1) Background: Cytoplasmic male sterility (CMS) is important for exploiting heterosis. Soybean (Glycine max L.) has a low outcrossing rate that is detrimental for breeding sterile lines and producing hybrid seeds. Therefore, the molecular mechanism controlling the outcrossing rate should be elucidated to increase the outcrossing rate of soybean CMS lines; (2) Methods: The male-sterile soybean lines JLCMS313A (with a high outcrossing rate; HL) and JLCMS226A (with a low outcrossing rate; LL) were used for a combined analysis of the transcriptome (RNA-seq) and the targeted phenol metabolome; (3) Results: The comparison between HL and LL detected 5946 differentially expressed genes (DEGs) and 81 phenolic metabolites. The analysis of the DEGs and differentially abundant phenolic metabolites identified only one common KEGG pathway related to flavonoid biosynthesis. The qRT-PCR expression for eight DEGs was almost consistent with the transcriptome data. The comparison of the cloned coding sequence (CDS) regions of the SUS, FLS, UGT, and F3H genes between HL and LL revealed seven single nucleotide polymorphisms (SNPs) only in the F3H CDS. Moreover, five significant differentially abundant phenolic metabolites between HL and LL were associated with flavonoid metabolic pathways. Finally, on the basis of the SNPs in the F3H CDS, one derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to distinguish between HL and LL soybean lines; (4) Conclusions: The flavonoid biosynthesis pathway may indirectly affect the outcrossing rate of CMS sterile lines in soybean.

A small heat shock protein GmHSP18.5a improves the male fertility restorability of cytoplasmic male sterility-based restorer line under high temperature stress in soybean

Small heat shock protein (sHSP) is involved in high temperature (HT) stress response. However, the function of sHSPs in regulating male fertility of soybean under HT stress remains largely unknown. Here, we identified a sHSP gene, GmHSP18.5a, which was responded to HT stress during flowering in cytoplasmic male sterility (CMS)-based restorer line of soybean. Moreover, GmHSFA6b turned out to directly activated the expression of GmHSP18.5a by binding to the heat shock cis-element in its promoter. Overexpression of GmHSP18.5a increased male fertility in transgenic Arabidopsis, soybean CMS-based restorer line and its hybrid F1 with CMS line under HT stress. Reactive oxygen species (ROS) content detection revealed that GmHSP18.5a promoted the ROS scavenging ability of Arabidopsis inflorescence and soybean flower bud under HT stress. Enzyme activity assay and gene expression analysis indicated that GmHS18.5a mainly increased the activity of antioxidant enzymes and the expression level of ROS metabolism-related genes under HT stress. Our results indicated that GmHSP18.5a improved the male fertility restorability of CMS-based restorer line in soybean by regulating ROS metabolic pathway and reducing ROS accumulation. Our findings not only revealed the molecular mechanism of sHSP regulating the male fertility of soybean under HT stress, but also provided a theoretical basis for creating strong restorer line with thermotolerance.