Genic Male Sterile Line Research Articles

Transcriptomics Analysis on Fertility Conversion in Thermosensitive Genetic Male Sterility Line Zhu1S under High Temperature

Zhu1S is a thermosensitive genic male-sterile (TGMS) line of rice possessing outstanding combining ability and low critical temperature, which has been extensively utilized as a female parent in two-line hybrid ricebreeding. However, the fertility of Zhu 1S during hybrid seed production is frequently affected by high temperature, thus leading to its fertility alteration and aborted hybrid seed production. To understand its fertility conversion mechanism under high temperature, we employed transcriptomics analyses on the anthers of young panicles of Zhu 1S during the fertility alternation sensitivity stage under high (Zhu 1S-H) and low (Zhu 1S-L) temperatures. The results showed that a total of 1119 differentially expressed genes (DEGs) were identified between Zhu 1S-H and Zhu1S-L anthers, including 680 up-regulated and 439 down-regulated genes. Bioinformatics analysis of these DEGs revealed that the high temperature induction caused fertility-sterility conversion in Zhu1S, mainly by decreasing the mRNA abundances of important genes closely related to plant hormone and MAPK signal pathway and transcriptional regulation factors, thereby impeding the growth and development of the anther of Zhu 1S, which ultimately affected the fertility transition of Zhu 1S under high temperature. The protein–protein interaction network analysis indicates that transcription factor OsTIFY11C possibility plays a central role in the fertility transition of Zhu 1S under high temperature.The present studies offer a theoretical foundation for further research into the molecular mechanism underlying fertility conversion in TGMS line Zhu 1S.

Analysis of blast resistance genes in Longliangyou and Jingliangyou hybrid rice varieties

<p id="C3">Longke 638S and Jing 4155S, developed by Yuan Longping High-Tech Agriculture Co., Ltd. in 2014, were two thermo-sensitive genic male sterile (TGMS) lines with disease resistance, high grain quality and combining ability for producing mid-season<italic> indica</italic> hybrid rice. In this study, we analyzed the blast resistance evaluation data from the regional trials of the Longliangyou and Jingliangyou hybrid rice varieties approved by the state from 2015 to 2019. Meanwhile, to provide theoretical basis for distribution and further improvement of these hybrid rice varieties, a genotyping panel containing 16 rice blast resistance (<italic>R</italic>) genes based on KASP (Kompetitive Allele-Specific PCR) technology was developed and used for molecular detection of these hybrid rice varieties. The results showed that 43.92% of Longliangyou and Jingliangyou hybrid rice varieties conferred moderate resistance to high resistance to blast disease. The mean value of integrated disease index (IDI) and highest scale of panicle blast severity (HSPBS) was 3.3 and 4.7, respectively. These hybrid rice varieties carried different number of <italic>R</italic> genes, ranging from 3 to 7. The average number of <italic>R</italic> genes in each variety was 5.1. The distribution frequency of the five genes including <italic>Pia</italic>, <italic>Pita</italic>, <italic>Pi2</italic>, <italic>Pi5</italic>, and <italic>Piz </italic>were higher by more 50%, among which, it was 100% for <italic>Pia</italic> gene. In contrast,<italic> Pi9</italic>, <italic>Pi35</italic>,<italic> Pi36</italic>, and <italic>Pb1</italic> genes were not detected in Longliangyou and Jingliangyou hybrid rice varieties. With the increase of the number of <italic>R</italic> genes in the varieties, the mean values of IDI and HSPBS were generally decreased. In conclusion, we suggested that introduction of <italic>Pi9 </italic>into Longke 638S and Jing 4155s might lead to the further improvement of blast resistance of Longliangyou and Jingliangyou hybrid rice varieties.

A natural allele of OsMS1 responds to temperature changes and confers thermosensitive genic male sterility

Changes in ambient temperature influence crop fertility and production. Understanding of how crops sense and respond to temperature is thus crucial for sustainable agriculture. The thermosensitive genic male-sterile (TGMS) lines are widely used for hybrid rice breeding and also provide a good system to investigate the mechanisms underlying temperature sensing and responses in crops. Here, we show that OsMS1 is a histone binding protein, and its natural allele OsMS1wenmin1 confers thermosensitive male sterility in rice. OsMS1 is primarily localized in nuclei, while OsMS1wenmin1 is localized in nuclei and cytoplasm. Temperature regulates the abundances of OsMS1 and OsMS1wenmin1 proteins. The high temperature causes more reduction of OsMS1wenmin1 than OsMS1 in nuclei. OsMS1 associates with the transcription factor TDR to regulate expression of downstream genes in a temperature-dependent manner. Thus, our findings uncover a thermosensitive mechanism that could be useful for hybrid crop breeding.

MALE STERILITY 3 encodes a plant homeodomain-finger protein for male fertility in soybean.

Male‐sterile plants are used in hybrid breeding to improve yield in soybean (Glycine max (L.) Merr.). Developing the capability to alter fertility under different environmental conditions could broaden germplasm resources and simplify hybrid production. However, molecular mechanisms potentially underlying such a system in soybean were unclear. Here, using positional cloning, we identified a gene, MALE STERILITY 3 (MS3), which encodes a nuclear‐localized protein containing a plant homeodomain (PHD)‐finger domain. A spontaneous mutation in ms3 causing premature termination of MS3 translation and partial loss of the PHD‐finger. Transgenetic analysis indicated that MS3 knockout resulted in nonfunctional pollen and no self‐pollinated pods, and RNA‐seq analysis revealed that MS3 affects the expression of genes associated with carbohydrate metabolism. Strikingly, the fertility of mutant ms3 can restore under long‐d conditions. The mutant could thus be used to create a new, more stable photoperiod‐sensitive genic male sterility line for two‐line hybrid seed production, with significant impact on hybrid breeding and production.

The regulatory role of CARBON STARVED ANTHER-mediated photoperiod-dependent male fertility in rice.

Environmental signals, especially daylength, play important roles in determining fertility in photoperiod-sensitive genic male sterile (PGMS) lines that are critical to sustain production of high-yielding hybrid rice (Oryza sativa) varieties. However, the mechanisms by which PGMS lines perceive changes in photoperiod and transmit those signals to elicit downstream effects are not well understood. In this study, we compared the transcriptomes from the leaves and anthers of carbon starved anther (csa), a PGMS line, to wild-type (WT) tissues under different photoperiods. Components of circadian clock in the leaves, including Circadian Clock-Associated 1 and Pseudo-Response Regulator (PRR95), played vital roles in sensing the photoperiod signals. Photoperiod signals were weakly transduced to anthers, where gene expression was mainly controlled by the CSA allele. CSA played a critical role in regulating sugar metabolism and cell wall synthesis in anthers under short-day conditions, and transcription of key genes inducing csa-directed sterility was upregulated under long-day (LD) conditions though not to WT levels, revealing a mechanism to explain the partial restoration of fertility in rice under LD conditions. Eight direct targets of CSA regulation were identified, all of which were genes involved in sugar metabolism and transport (cell wall invertases, SWEETs, and monosaccharide transporters) expressed only in reproductive tissues. Several hub genes coordinating the effects of CSA regulation were identified as critical elements determining WT male fertility and further analysis of these and related genes will reveal insights into how CSA coordinates sugar metabolism, cell wall biosynthesis, and photoperiod sensing in rice anther development.

Transcript Profiling Analysis and ncRNAs' Identification of Male-Sterile Systems of Brassica campestris Reveal New Insights Into the Mechanism Underlying Anther and Pollen Development.

Male-sterile mutants are useful materials to study the anther and pollen development. Here, whole transcriptome sequencing was performed for inflorescences in three sterile lines of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis), the genic male-sterile line (A line), the Polima cytoplasmic male-sterile (CMS) line (P line), and the Ogura CMS line (O line) along with their maintainer line (B line). In total, 7,136 differentially expressed genes (DEGs), 361 differentially expressed long non-coding RNAs (lncRNAs) (DELs), 56 differentially expressed microRNAs (miRNAs) (DEMs) were selected out. Specific regulatory networks related to anther cell differentiation, meiosis cytokinesis, pollen wall formation, and tapetum development were constructed based on the abortion characteristics of male-sterile lines. Candidate genes and lncRNAs related to cell differentiation were identified in sporocyteless P line, sixteen of which were common to the DEGs in Arabidopsis spl/nzz mutant. Genes and lncRNAs concerning cell plate formation were selected in A line that is defected in meiosis cytokinesis. Also, the orthologs of pollen wall formation and tapetum development genes in Arabidopsis showed distinct expression patterns in the three different sterile lines. Among 361 DELs, 35 were predicted to interact with miRNAs, including 28 targets, 47 endogenous target mimics, and five precursors for miRNAs. Two lncRNAs were further proved to be functional precursors for bra-miR156 and bra-miR5718, respectively. Overexpression of bra-miR5718HG in B. campestris slowed down the growth of pollen tubes, caused shorter pollen tubes, and ultimately affected the seed set. Our study provides new insights into molecular regulation especially the ncRNA interaction during pollen development in Brassica crops.

Development of the PARMS Marker of the Dominant Genic Male Sterility (DGMS) Line and Its Utilization in Rapeseed (Brassica napus L.) Breeding.

The 8029AB line is a dominant genic male sterility (DGMS) two-type line in Brassica napus L., which can be used in a three-line approach for the seed production of rapeseed hybrids. Genetic analyses have demonstrated that the sterility of 8029A is controlled by a single dominant nuclear gene (BnMS5e) interacting with one recessive gene (BnMS5c). Six pairs of penta-primer amplification refractory mutation system (PARMS) markers were designed according to the sequence of BnMS5a, BnMS5c and BnMS5e. Two pairs of these PARMS markers were successfully identified and validated. The PARMS markers MS5-1Fc/MS5-1Ft/MS5-1R12 could distinguish BnMS5c from BnMS5a/BnMS5e, and the PARMS markers MS5-2Ft/MS5-2Fa/MS5-1R12 could genotype BnMS5a and BnMS5c/BnMS5e. The combination of these two pairs of PARMS markers could be used to identify the presence or absence of BnMS5a/BnMS5c/BnMS5e effectively. Consequently, marker-assisted selection can be carried out in the early generation to shorten the breeding period and improve the breeding efficiency.

Identification of Heterotic Groups and Patterns Based on Genotypic and Phenotypic Characteristics Among Rice Accessions of Diverse Origins.

Identification of the right parental combinations to maximize heterosis is the major goal of hybrid breeding, which could be achieved through identification of heterotic groups. The main objective of this study was to identify promising heterotic groups for future rice breeding programs. A collection of 359 rice genotypes of diverse origins of China and abroad, composed of inbreds, maintainers, restorers, and temperature-sensitive genic male sterile (TGMS) lines were genotyped using 10K SNP chips. The SNP data set was subjected to genomic analyses for estimation of genetic divergence and diversity. Significant variations were observed in the germplasm with the identification of six different genetic groups. These lines were assigned to the genetic groups independent of their origin. Taking an account of commercially used heterotic groups present in each cluster, three cytoplasmic male sterile (CMS) lines and 14 inbred and restorer lines with moderate to high genetic distances selected from five heterotic patterns were crossed and obtained 42 F1 hybrids. A total of 14 hybrids were found with significant maximum mid- and better-parent heterosis, namely, TaifengA × Guang122, TaifengA × Wushansimiao, and TaifengA × Minghui63 for earliness; Guang8A × Huazhan for dwarf stature; and Guang8A × Huanghuzhan-1, TaifengA × Yuexiangzhan, Guang8A × Minhui3301, TianfengA × Guang122, Guang8A × Yahui2115, TianfengA × Huanghuazhan, TianfengA × Minghui63, TianfengA × Minhui3301, TaifengA × Gui99, and Guang8A × Yuenongsimiao for yield and yield-related traits. Mid-parent and better-parent heterotic F1 hybrids were in positive correlation with the genetic distances as that manifested by commercially used heterotic groups, encouraging the use of genotypic data for identification of heterotic groups. Our study provides an informative strategy for the development of early maturing, lodging resistant and high-yielding commercial hybrids and cultivars in future heterosis breeding programs.

Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize

Male sterility represents an important trait for hybrid breeding and seed production in crops. Although the genes required for male fertility have been widely studied and characterized in many plant species, most of them are single genic male-sterility (GMS) genes. To investigate the role of multiple homologous genes in anther and pollen developments of maize, we established the CRISPR/Cas9-based gene editing method to simultaneously mutate the homologs in several putative GMS gene families. By using the integrated strategies of multi-gene editing vectors, maize genetic transformation, mutation-site analysis of T0 and F1 plants, and genotyping and phenotyping of F2 progenies, we further confirmed gene functions of every member in ZmTGA9-1/-2/-3 family, and identified the functions of ZmDFR1, ZmDFR2, ZmACOS5-1, and ZmACOS5-2 in controlling maize male fertility. Single and double homozygous gene mutants of ZmTGA9-1/-2/-3 did not affect anther and pollen development, while triple homozygous gene mutant resulted in complete male sterility. Two single-gene mutants of ZmDFR1/2 displayed partial male sterility, but the double-gene mutant showed complete male sterility. Additionally, only the ZmACOS5-2 single gene was required for anther and pollen development, while ZmACOS5-1 had no effect on male fertility. Our results show that the CRISPR/Cas9 gene editing system is a highly efficient and convenient tool for identifying multiple homologous GMS genes. These findings enrich GMS genes and mutant resources for breeding of maize GMS lines and promote deep understanding of the gene family underlying pollen development and male fertility in maize.

Transcriptional and physiological data revealed cold tolerance in a photo-thermo sensitive genic male sterile line Yu17S

BackgroundRice is highly sensitive to chilling stress during the seedling stage. However, the adaptable photo-thermo sensitive genic male sterile (PTGMS) rice line, Yu17S, exhibits tolerance to low temperatures. Currently, the molecular characteristics of Yu17S are unclear.ResultsTo evaluate the molecular mechanisms behind cold responses in rice seedlings, a comparative transcriptome analysis was performed in Yu17S during seedling development under normal temperature and low temperature conditions. In total, 9317 differentially expressed genes were detected. Gene ontology and pathway analyses revealed that these genes were involved mostly in photosynthesis, carotenoid biosynthesis, carbohydrate metabolism and plant hormone signal transduction. An integrated analysis of specific pathways combined with physiological data indicated that rice seedlings improved the performance of photosystem II when exposed to cold conditions. Genes involved in starch degradation and sucrose metabolism were activated in rice plants exposed to cold stress treatments, which was accompanied by the accumulation of soluble sugar, trehalose, raffinose and galactinol. Furthermore, chilling stress induced the expression of phytoene desaturase, 15-cis-ζ-carotene isomerase, ζ-carotene desaturase, carotenoid isomerase and β-carotene hydroxylase; this was coupled with the activation of carotenoid synthase activity and increases in abscisic acid (ABA) levels in rice seedlings.ConclusionsOur results suggest that Yu17S exhibited better tolerance to cold stress with the activation of carotenoid synthase activity and increasing of ABA levels, and as well as the expression of photosynthesis-related genes under cold condition in rice seedlings.

Heterosis and Combining Ability for Fruit Yield, Sweetness, β-Carotene, Ascorbic Acid, Firmness and Fusarium Wilt Resistance in Muskmelon (Cucumis melo L.) Involving Genetic Male Sterile Lines

Ten genetically diverse inbred lines, including two genic male sterile lines, of muskmelon (Cucumis melo L.) were crossed in a half-diallel to generate 45 F1 hybrids. These hybrids, along with the parental lines and commercial check, were evaluated for their fruit yield, level of phytochemicals and Fusarium wilt resistance. Both additive and non-additive genetic variances were important in governing the expression of all of the traits; however, the additive gene action for the fruit weight (g), flesh thickness (cm), rind thickness (mm), firmness (lb inch−2), β-carotene content (mg/100 g), non-additive variance for fruit yield (t ha−1), fruit number, total soluble solids (TSS, °Brix), ascorbic acid (mg/100 g) and reaction to Fusarium wilt were comparatively more important. The parental line MM-625 was the best general combiner for fruit yield, rind thickness and β-carotene content (mg/100 g). The exotic line Riogold was the best combiner for flesh thickness and firmness. The netted inbred line MM-610 was the best general combiner for fruit weight, ascorbic acid and reaction to Fusarium wilt. The inbred lines KP4HM-15 and MM-916 were the best general combiners for the number of fruits per vine and TSS. The best cross-combinations for fruit yield ha−1 and TSS were MS-1×M-610 and Kajri×MM-904, respectively. The hybrids KP4HM-15×MM Sel-103 and KP4HM-15×MM-1831 recorded the highest standard heterosis for fruit yield and TSS. The landrace-derived inbred lines Kajri, MM Sel-103 and KP4HM-15 produced moderate-to-highly FW-resistant hybrids. Out of the 121 SSR markers applied, 70 exhibited parental polymorphism. The markers DM0561, CMAAAGN14, TJ147, CMMS35_3, CMAGN45 and DE1337 identified specific/unique alleles in certain parental genotypes. Thus, the findings of this study revealed that the novel inbred lines can effectively be combined to generate heterotic F1 hybrids for yield and other traits, such as rind and flesh thickness, TSS, β-carotene content and firmness. Furthermore, SSR markers can potentially be utilized to confirm the genetic diversity among the parental lines, and for the DNA fingerprinting of F1 hybrids.

Reproductive tissue-specific translatome of a rice thermo-sensitive genic male sterile line

Translational regulation, especially tissue- or cell type-specific gene regulation, plays essential roles in plant growth and development. Thermo-sensitive genic male sterile (TGMS) lines have been widely used for hybrid breeding in rice (Oryza sativa). However, little is known about translational regulation during reproductive stage in TGMS rice. Here, we use translating ribosome affinity purification (TRAP) combined with RNA sequencing to investigate the reproductive tissue-specific translatome of TGMS rice expressing FLAG-tagged ribosomal protein L18 (RPL18) from the germline-specific promoter MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1). Differentially expressed genes at the transcriptional and translational levels are enriched in pollen and anther-related formation and development processes. These contain a number of genes reported to be involved in tapetum programmed cell death (PCD) and lipid metabolism during pollen development and anther dehiscence in rice, including several encoding transcription factors and key enzymes, as well as several long non-coding RNAs (lncRNAs) that potentially affect tapetum and pollen-related genes in male sterility. This study represents the comprehensive reproductive tissue-specific characterization of the translatome in TGMS rice. These results contribute to our understanding of the molecular basis of sterility in TGMS rice and will facilitate further genetic manipulation of TGMS rice in two-line breeding systems.

Engineering of rice varieties with enhanced resistances to both blast and bacterial blight diseases via CRISPR/Cas9.

SummaryRice blast and bacterial blight represent two of major diseases having devastating impact on the yield of rice in most rice‐growing countries. Developments of resistant cultivars are the most economic and effective strategy to control these diseases. Here, we used CRISPR/Cas9‐mediated gene editing to rapidly install mutations in three known broad‐spectrum blast‐resistant genes, Bsr‐d1, Pi21 and ERF922, in an indica thermosensitive genic male sterile (TGMS) rice line Longke638S (LK638S). We obtained transgene‐free homozygous single or triple mutants in T1 generations. While all single and triple mutants showed increased resistance to rice blast compared with wild type, the erf922 mutants displayed the strongest blast resistance similar with triple mutants. Surprisingly, we found that Pi21 or ERF922 single mutants conferred enhanced resistance to most of tested bacterial blight. Both resistances in mutants were attribute to the up‐regulation of SA‐ and JA‐pathway associated genes. Moreover, phenotypic analysis of these single mutants in paddy fields revealed that there were no trade‐offs between resistances and main agricultural traits. Together, our study provides a rapid and effective way to generate rice varieties with resistance to both rice blast and bacterial blight.

Comparative study on the fertility and outcrossing characteristics of new dual-purpose genic male-sterile rice in China

The widespread application of ‘two-line’ hybrid rice, based on temperature- and photoperiod-sensitive genic male-sterile lines, plays an important role in ensuring global food security. Such lines are fertile in short-day/low-temperature conditions when used for self-breeding, and sterile in long-day/high-temperature conditions when used for hybrid seed production. However, fertility instability and low hybrid seed production of sterile lines are urgent problems. The aim of this study was to increase the stability of pollen sterility by studying the critical sterile-inducing temperature and stigma vitality of 97 newly selected male-sterile lines in China from 2017 to 2020. Sterility gene source analysis showed that 89.7% of the materials contained the tms5 sterile gene. After 6 days of treatment at 23.5°C during the reproductive period, 42 male-sterile lines were sterile. After 10 days of treatment at 23.5°C, the sterility of 12 sterile lines remained stable, indicating a high level of safety for hybrid seed production. After 10 days of low-temperature treatment at 19°C, the fertile pollen rate of 47 of the 97 sterile lines was &gt;70%, indicating a high level of seed reproduction safety. Only six of these 47 male-sterile lines could maintain low fertility after 10 days of treatment at 23.5°C, indicating that they could be used safely for both hybrid seed production and sterile line reproduction. The results of delayed pollination showed that among the six lines, only Zhong 7S had high stigma vitality, and BYXA1S and H10S-2 had medium stigma vitality. Therefore, those three sterile lines have the potential for safe hybrid seed production and high-yield seed reproduction. Based on our research, we propose a new breeding standard of dual-purpose male-sterile lines to promote the development of two-line hybrid rice.

Slow development allows redundant genes to restore the fertility of rpg1, a TGMS line in Arabidopsis.

Slow development has been shown to be a general mechanism to restore the fertility of thermo-sensitive and photoperiod-sensitive genic male sterile (TGMS and PGMS) lines in Arabidopsis. rpg1 is a TGMS line defective in primexine, which is essential for pollen wall pattern formation. Here, we showed that RPG1-GFP was highly expressed in microsporocytes, microspores, and pollen grains but not in the tapetum in the complemented transgenic line, suggesting that microsporocytes are the main sporophytic cells for primexine formation. Further cytological observations showed that primexine formation in rpg1 was partially restored under slow growth conditions, leading to its fertility restoration. RPG2 is the homolog of RPG1 in Arabidopsis. We revealed that the fertility recovery of rpg1 rpg2 was significantly reduced compared with that of rpg1 under low temperature. The RPG2-GFP protein was also expressed in microsporocytes in the RPG2-GFP (WT) transgenic line. These results suggest that RPG2 plays a redundant role in rpg1 fertility restoration. rpg1 plants were male sterile at the early growth stage, while their fertility was partially restored at the late developmental stage. The fertility of the rpg1 lateral branches was also partially restored. Further growth analysis showed that slow growth at the late reproductive stage or on the lateral branches led to fertility restoration. This work reveals the importance of gene redundancy in fertility restoration for TGMS lines and provides further insight into pollen wall pattern formation.

Improving the resistance of the rice PTGMS line Feng39S by pyramiding blast, bacterial blight, and brown planthopper resistance genes

Knowledge of rice (Oryza sativa L.) genes and various DNA markers can be used in genomic breeding programs aimed at developing improved elite rice cultivars. We used an efficient genomic breeding approach to pyramid four resistance genes (Pi2, Xa23, Bph14, and Bph15) in the popular photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice line Feng39S. We performed foreground selection for the target genes, followed by recombinant selection and background selection. This process reduced the sizes of the genomic segments harboring the target genes (566.8 kb for Pi2, 1143.9 kb for Xa23, 774.7 kb for Bph14, and 1574.9 kb for Bph15) and accelerated the recovery of the recurrent parent genome to proportions ranging from 98.77% to 99.16%, thus resulting in four near-isogenic lines. To assemble the four resistance genes in Feng39S, we performed a double-way cross combined with foreground and background selection to generate two improved lines of Feng39S (Pi2 + Xa23 + Bph14 + Bph15) with a recurrent parent genome recovery of 98.98%. The two lines showed agronomic performance, grain quality, and fertility–sterility transition characteristics similar to those of the original Feng39S line. The newly developed PTGMS lines and corresponding hybrid combinations were resistant to various field blast isolates and seven representative isolates of bacterial blight. At the seedling stage, the lines also showed resistance against brown planthopper. This study provides an efficient and accurate genomic breeding approach for introducing desirable traits into PTGMS lines.

Phytochrome interacting factor 3 regulates pollen mitotic division through auxin signalling and sugar metabolism pathways in tomato.

Summary The development of viable pollen determines male fertility, and is crucial for reproduction in flowering plants. Phytochrome interacting factor 3 (PIF3) acts as a central regulator of plant growth and development, but its relationship with pollen development has not been determined.Through genetic, histological and transcriptomic analyses, we identified an essential role for SlPIF3 in regulating tomato (Solanum lycopersicum) pollen development. Knocking out SlPIF3 using clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 resulted in pollen mitosis I arrest, and a failure to form viable pollen. We further demonstrated that both glutamate synthase 1 (SlGLT1) and cell wall invertase 9 (SlCWIN9), involved in auxin and sugar homeostasis, respectively, colocalised with SlPIF3 in the anthers and were directly regulated by SlPIF3. Knockout of either SlGLT1 or SlCWIN9 phenocopied the pollen phenotype of SlPIF3 knockout (Slpif3) lines. Slpif3 fertility was partially restored by exogenous auxin indole‐3‐acetic acid in a dose‐dependent manner.This study reveals a mechanism by which SlPIF3 regulates pollen development and highlights a new strategy for creating hormone‐regulated genic male sterile lines for tomato hybrid seed production.

Comparative transcriptome and DNA methylation analysis in temperature-sensitive genic male sterile wheat BS366

BackgroundKnown as the prerequisite component for the heterosis breeding system, the male sterile line determines the hybrid yield and seed purity. Therefore, a deep understanding of the mechanism and gene network that leads to male sterility is crucial. BS366, a temperature-sensitive genic male sterile (TGMS) line, is male sterile under cold conditions (12 °C with 12 h of daylight) but fertile under normal temperature (20 °C with 12 h of daylight).ResultsDuring meiosis, BS366 was defective in forming tetrads and dyads due to the abnormal cell plate. During pollen development, unusual vacuolated pollen that could not accumulate starch grains at the binucleate stage was also observed. Transcriptome analysis revealed that genes involved in the meiotic process, such as sister chromatid segregation and microtubule-based movement, were repressed, while genes involved in DNA and histone methylation were induced in BS366 under cold conditions. MethylRAD was used for reduced DNA methylation sequencing of BS366 spikes under both cold and control conditions. The differentially methylated sites (DMSs) located in the gene region were mainly involved in carbohydrate and fatty acid metabolism, lipid metabolism, and transport. Differentially expressed and methylated genes were mainly involved in cell division.ConclusionsThese results indicated that the methylation of genes involved in carbon metabolism or fatty acid metabolism might contribute to male sterility in BS366 spikes, providing novel insight into the molecular mechanism of wheat male sterility.

Global survey of alternative splicing and gene modules associated with fertility regulation in a thermosensitive genic male sterile wheat

Thermosensitive genic male sterile (TGMS) wheat lines are the core of two-line hybrid systems. Understanding the mechanism that regulates male sterility in TGMS wheat lines is helpful for promoting wheat breeding. Several studies have obtained information regarding the mechanisms associated with male sterility at the transcriptional level, but it is not clear how the post-transcriptional process of alternative splicing might contribute to controlling male sterility. In this study, we performed genome-wide analyses of alternative splicing during the meiosis stage in TGMS line BS366 using PacBio and RNA-Seq hybrid sequencing. Cytological observations indicated that cytoskeleton assembly in pollen cells, calcium deposition in pollen and tapetal cells, and vesicle transport in tapetal cells were deficient in BS366. According to our cytological findings, 49 differentially spliced genes were isolated. Moreover, 25 long non-coding RNA targets and three bHLH transcription factors were identified. Weighted gene co-expression network analysis detected four candidate differentially spliced genes that had strong co-relation with the seed setting percentage, which is the direct representation of male sterility in BS366. In this study, we obtained comprehensive data regarding the alternative splicing-mediated regulation of male sterility in TGMS wheat. The candidates identified may provide the molecular basis for an improved understanding of male sterility.

Molecular Breeding of a Novel PTGMS Line of WDR for Broad-Spectrum Resistance to Blast Using Pi9, Pi5, and Pi54 Genes

BackgroundThe two-line method based on the photoperiod and thermo-sensitive genic male sterile (PTGMS) lines is more cost-effective, simple, and efficient than the three-line system based on cytoplasmic male-sterility. Blast and drought are the most prevalent biotic and abiotic stress factors hampering rice production. Molecular techniques demonstrate higher efficacy in the pyramiding of disease resistance genes, providing green performance under the background of water-saving and drought-resistance rice.ResultsThis study employed molecular marker-assisted selection, conventional hybridization, and high-intensity stress screening to integrate three broad-spectrum blast resistance genes Pi9, Pi5, and Pi54 into Huhan 1S. Subsequently, a novel water-saving and drought-resistance rice (WDR) PTGMS line Huhan 74S was developed. The drought resistance of the new PTGMS line Huhan 74S was comparable to that of Huhan 1S. Pathogenicity assays involving the inoculation of 14 blast prevalent isolates in the glasshouse showed that the blast resistance frequency of Huhan 74S was 85.7%. Further evaluation under natural blast epidemic field conditions showed that Huhan 74S and its hybrids were resistant to leaf and neck blast. The critical temperature point of fertility-sterility alteration of Huhan 74S was 23 °C daily mean temperature. The complete male sterility under natural growth conditions in 2017 at Shanghai lasted for 67 days. Also, both the agronomic and grain quality traits met the requirement for two-line hybrid rice production.ConclusionThese results indicate that the newly bred PTGMS line Huhan 74S can be used to breed high-yielding, good-quality, disease-resistant two-line hybrid water-saving and drought-resistance rice (WDR), hence promoting sustainable rice production in China.