Onion Male Sterility: Genetics, Genomics and Breeding

Abstract

Onion, belonging to the Allium genus, is an essential and versatile vegetable crop that plays a pivotal role in culinary traditions worldwide. Renowned for its distinctive flavor and nutritional value, onion is an indispensable ingredient in countless dishes. As the global demand for onion continues to surge, securing a stable supply of high-quality, high-yielding onion varieties becomes ever more pressing. The onion umbel bears numerous tiny flowers that are protandrous in nature. Hybrid breeding is limited in onion due to high inbreeding depression, tedious emasculation and lack of elite inbreds. In this quest for crop improvement, the phenomenon of male sterility stands out as a key tool in modern onion breeding. Male sterility, which is recognized as the incapacity to produce viable pollen grains, inhibition of anther dehiscence and production of non-functional male gametes, has been harnessed as a mechanism to control cross-pollination and escalating hybrid development. The successful utilization of stable male sterile lines in onion holds the promise of producing uniform, high-yielding and disease-resistant hybrids. In recent decades, scientific advances have illuminated the molecular intricacies underlying male sterility systems in onion. Much progress has been made in elucidating the regulation of male sterility systems in the post-genomics era. This review highlights the current status of molecular markers linked with male sterility and provides genetic and molecular insights into its regulation. Additionally, it discusses the role of male sterility as a transformative tool in onion breeding in the genomics era.

1. Introduction

Onion (Allium cepa L.) belongs to the genus Allium, which encompasses over 1100 species with ethno medicinal value including various other well-known members such as garlic (A. sativum), leeks (A. ampeloprasum var. porrum), chives (A. cepa var. aggregatum) and shallots (A. schoenoprasum) [1]. Onion is diploid, with a chromosome number of 2n = 2x = 16 and boasts a notably extensive genome size, approximately 16 Gbp (16,400 Mb/1C) [2]. This genome size ranks it as one of the largest cultivated diploid crops, surpassing the genome sizes of Arabidopsis and tomato by a factor of 100 and 18, respectively, which is also comparable to size of cultivated staple cereal, wheat [3].

Onion is a biennial cross-pollinated crop, but grown as an annual plant for bulb production. It produces a distinctive bulb composed of fleshy leaves designed to store carbohydrates and water for the plant’s sustenance. Onions can be propagated through various means, including seeds, sets (small bulbs) or transplants. They are categorized into long-day or short-day cultivars based on their response to day length. A day length of 14–16 h is required to initiate bulbing in long-day type cultivars, while a shorter day-length of 10–12 h is sufficient in short-day type cultivars for bulb formation [4,5]. On the other hand, the day-neutral types exhibit flexibility in bulb formation across a wide range of day lengths. The onion inflorescence is known as umbel that bears numerous perfect and protandrous flowers [6]. Self-pollination is infrequent, as onions are entomophilous and primarily pollinated by insects. Honeybees are the principal pollinators, and it is a common practice to place beehives in the fields to enhance seed yield.

Beyond its culinary appeal, onion holds a promising position in production and export value globally, ranking second only to tomatoes (http://www.fao.org/faostat/en/, accessed on 1 December 2024). Notably, China and India collectively contribute 47.49% of the world’s onion production. Onions are cherished for their versatility, being consumed in both immature and mature bulb stages, either raw or cooked. In addition to their culinary application, these plants have served as medicinal resources for over 5000 years. Their nutritional and medicinal properties make them a valuable commodity [7]. Onions are rich in bioactive compounds like quercetin and rutin [8]; hence, the regular intake is associated with reduction in different types of cancer, skin and heart ailments and control generation of ROS (reactive oxygen species) [9]. Onions are considered as low-calorie food and are abundant in mineral nutrients, vitamins and flavonoid compounds. Moreover, they contain sulfur compounds known for their anti-inflammatory and anti-cancer properties.

Wide variation is present in onions with respect to bulb shape, bulb color and flavor, bulb size and quality attributes [8,10]. This inherent diversity in onion cultivars led to extensive efforts in onion breeding to enhance desirable traits for both commercial and consumer preferences. Breeders focus on developing cultivars with improved disease resistance, higher yield and adaptability to different growing conditions. One key aspect of onion breeding involves the selection and manipulation of genetic traits to achieve desired outcomes. Traditional breeding methods often rely on controlled pollination to cross plants with specific characteristics, followed by rigorous selection of the resulting progeny. Modern breeding techniques, including MAS (marker-assisted selection), genomics assisted breeding, genome engineering via CRISPR/Cas9 and RNAi tools, have expedited the breeding process by allowing breeders to identify and introduce desired traits more efficiently [11]. Moreover, advancements in molecular biology have facilitated the elucidation of genetics of important commercial traits in onion. The molecular markers linked with desirable traits have been identified which further facilitates for more precise and accelerated breeding programs [12].

In this context, hybrid breeding has become a valuable strategy. Hybrid onions, derived from the cross-breeding of distinct parental lines, often exhibit improved vigor, uniformity and yield compared to their non-hybrid counterparts [13,14]. Male sterility, which refers to a condition wherein plants do not produce viable pollen or are devoid of anthers, have a lack of anther dehiscence, or produce malformed and shriveled anthers, is often exploited in hybrid onion seed production [13]. The advantages of male sterility include preventing self-pollination, assurance of hybrid purity and facilitating the production of F₁ hybrid onions with desirable characteristics [14,15,16]. The cytoplasmic genic male sterility (CGMS) system, which is the result of interaction of sterile cytoplasm and nuclear genes, has been exploited commercially in onion breeding [13]. Since the inception of the CGMS system by Jones and Emsweller [17], a substantial improvement in onion productivity has been recorded [18]. Male sterility has emerged as a pivotal factor in revolutionizing onion crop improvement. Male sterility systems play a crucial role in crop improvement, offering significant advantages in the realm of agricultural productivity and breeding programs to escalate hybrid breeding and overall crop performance [13,19,20].

Male sterility in onions opens a pathway for controlled hybrid seed production, providing breeders with a powerful tool to regulate the pollination process [15]. In onion, CMS-S, CMS-R and CMS-T types of cytoplasm have been reported for the production of elite male sterile parents and enhancing hybrid onion breeding [13,17,21]. This carefully orchestrated breeding approach, involving the strategic crossing of male sterile female parents with fertile lines, has demonstrated remarkable success in boosting onion yields and improving overall crop quality [22].

This review aims at shedding light on the current status of understanding molecular mechanisms of male sterility systems in onion, their utilization in onion breeding and future prospects. Understanding the intricacies of CGMS in onion holds great promise for improving the efficiency and effectiveness of onion breeding programs. This review also highlights the progress of genomic insights and scope of genome editing in onion breeding based on male sterility systems. As we explore the multifaceted role of male sterility in onion breeding programs, we uncover a transformative tool that holds the key to addressing the evolving challenges of modern agriculture.

2. Male Sterility in Onion

2.1. Types of Male Sterility Systems in Onion

Male sterility is widespread in angiosperms and is vital in hybrid breeding of crops like onion, cole vegetables and solanaceae vegetables where flowers are tiny and hand emasculation is cumbersome [13]. Male sterility can be defined as the condition where plants are devoid of male reproductive organs, i.e., stamens, and if anthers are formed, they are deformed or non-dehiscent in nature, the male gametes are non-viable and the female organ is viable [13,19,20,23,24,25]. Hence, male sterility avoids self-pollination and ensures increase in genetic diversity through cross-pollination [26]. Although male sterility rarely occurs in nature, as these plants are eliminated by natural selection forces, it has been maintained during the domestication process, because it could be a valuable tool for onion breeding. In that way, male sterility provides a natural and effective mean for genetic emasculation of plants facilitating hybrid breeding in onion [11]. The maternally inherited sterility controlled by the mitochondrial genome is designated as cytoplasmic male sterility. The sterility encoded by nuclear genes is regarded as nuclear or genic male sterility. Meanwhile, the cytoplasmic-genic male sterility (CGMS) is a type of CMS where sterility is caused by interaction of nuclear genes and sterile cytoplasm [20,27,28,29]. The CGMS system is widespread in onion and has been exploited commercially [13].

The effect of cytoplasmic genes can be masked by dominant fertility restorer genes (Rf genes) [30]. In onion, the CGMS system has been proved instrumental, where male sterility is regulated by the interaction of different sterile cytoplasms (CMS-S, CMS-T, CMS-R) and recessive nuclear genes (

Table 1. Characteristics of sterile cytoplasm types governing CGMS in onion.

Features	CMS-S	CMS-T	CMS-R	CMS-Y
Description	First identified in the material of Italian-Red in 1925	Discovered in the French variety “Jaune Paille Des Vertus” in 1965	Originated from the onion cultivar “Rijnsburge”	Reported in two onion accessions, PI273626 and PI236025
Male sterility mechanism	Increased genomic shift of the mitochondrial gene orf725 along with a reduction in the copy number of coxI gene	Enhanced genomic shift of orf725 gene along with an increase in the copy number of coxI gene	Male sterility is linked to a specific mitochondrial gene, orf725	Male sterility is linked to the mitochondrial gene, orf725 with the cox1 gene
Fertility restoration (Nuclear Rf gene)	Fertility is restored by a single nuclear dominant locus Ms	Three independent nuclear loci are involved in fertility restoration (A, C, D)	Restored by a nuclear dominant Ms locus	Potentially restored by an Rf gene (not fully characterized)
Utility	Stable and widely exploited	Not used commercially	Less common as compared to CMS-S cytoplasm	Unstable and rarerly used

). The first documentation of male sterility in onion was made in the material of Italian Red cultivar by Jones during 1925 [31]. The male sterility in this material was reported to be associated with the interaction of sterile cytoplasm “S” (CMS-S) and recessive nuclear genes, which was recognized as the CGMS system [17,31]. The nuclear gene, when present in dominant form (Ms), leads to the fertile phenotype, while in recessive homozygous state (msms) with sterile cytoplasm it leads to the male sterile phenotype (Figure 1). The male sterile gene can be present in three different genotypes in diploid onion, i.e., dominant homozygous with MsMs genotype, heterozygous as Msms and recessive homozygous state with msms genotype (Figure 1). These genotypes may occur with either sterile “S” or normal “N” cytoplasm, and interaction with nuclear genes and cytoplasm yields respective phenotypes [26]. The male sterile (S msms) lines can be maintained by crossing with maintainer (N msms) [32].

Another cytoplasmic source in the CGMS system of onion, CMS-T, was discovered in France in the material of Jaune-Paille Des-Vertus cultivar by Berninger [33]. This cytoplasm has been used in breeding hybrid cultivars of onion in Europe and Japan [34]. The male sterile phenotype in CMS-T is conditioned by the “T” cytoplasm with three male fertile loci [35]. Plants carrying the “T” cytoplasm along with dominant fertile loci “A” or two complementary dominant fertility restoring loci “C” and “D” are male fertile in nature [35,36]. Among CMS-S and CMS-T sources of sterile cytoplasms in the CGMS system of onion, the CMS-S has been widely exploited widely in onion hybrid breeding due to its greater stability under varying environments and simple inheritance [21,37,38]. Another type of cytoplasm, identified as CMS-R, has been reported in the Rijnsburger cultivar in the Netherlands [39]. The CMS-R can be reverted to male fertility by fertile locus “Ms” [39,40,41,42]. However, recently Kim et al. [43] reported another CMS type in onion, i.e., “CMS-Y”, which was discovered in the “PI273626” accession of onion (Table 1).

2.2. Utility of Male Sterile Lines in Onion Hybrid Breeding

Onion hybrid breeding is limited by lack of stable male sterile lines particularly in the tropical environments. Onion flowers are tiny and protandrous, which favours cross-pollination [6], but this does not completely rule out chances of self-pollination, as a single onion umbel may bear numerous flowers and the anther dehiscence may coincide with stigma receptivity of adjacent flowers of same umbel. Therefore, it is necessary to use methodologies like male sterility systems to ensure hybrid seed production. Heterosis breeding in onion was initiated with the discovery of male sterility in onion [17] that was exploited for developing high yielding hybrids [31]. The CGMS system, governed by interactions between, sterile cytoplasms and nuclear genes, has been instrumental in accelerating hybrid development in onions [44,45,46]. The higher production and productivity of onion in the current scenario is due to the exploitation of heterosis breeding via male sterility. Hence, much progress has been made in onion with respect to generation and utilization of male sterile lines along with identification of suitable maintainers [47,48].

Realizing the value of male sterility, Kazakova and Yakovlev [49] initiated the heterosis breeding in onion by generating twenty male sterile lines. The male sterile lines like Oriental S61, Oriental S 57, Valencia S1, etc., were utilized in the development of 98 hybrid progenies, which depicted heterotic performance for yield and quality traits. Sharma [50] isolated eight different male sterile lines (MS20-MS23, MS34, MS35, MS37 and MS40) and maintainer lines in the material of Hisar-2. Based on combining ability analysis, they identified MS34 and MS40 as best general combiner lines and Pusa Red as best tester for average bulb size and weight. Male sterile plants have been recognized in the material of Nasik White Globe, IIHR-20 and Pusa Red genotypes in India [51]. Their work led to the identification of male sterile S-cytoplasm in Arka Pragti and Red Coral [51]. ICAR-IIHR released two hybrids based on the male sterility system in onion, namely Arka Kirthiman and Arka Lalima [52]; however, these hybrids did not meet much popularity among Indian onion farmers. Recently, in quest of identification of novel male sterile lines in short-day type onion genotypes in India, Manjunathagowda and Anjanappa [47] identified male sterile and maintainer lines using the black card assay (a visual method to assess pollen dispersal) and a pollen viability test employing aceto-carmine dye (used to stain the nuclei of viable pollen). The male sterility was confirmed using the molecular markers which were designed based on the orf725 gene. Development of ideal male sterile lines in onion is of significant value for escalating hybrid breeding in onion.

Onion is highly cross-pollinated crop and suffers from high inbreeding depression in the process of development of inbred lines through continuous inbreeding. Hence, identification and isolation of stable male sterile systems is a boon for onion breeders. Some of the breeding lines of onion carrying different cytoplasms reported from different geographical locations are B1750A (CMS-S), B1750B (CMS-N), RJ70A (CMS-T), RJ70B (CMS-N), OM113, M1111 (Male sterile lines isolated from the material of Nasik White Globe), and CMS-ga614A, CMS-ga8111A and CMS-8152A (ga-cytoplasm from A. galanthum) [31,53,54]. The male sterile lines with the genotype “S msms” can be maintained by repeated backcrossing with maintainer (B line) with genotype, “N msms”, and subsequent cross-pollination of male sterile line with ideal pollen parent [11] (Figure 2). Thus, the CGMS system requires maintenance of three breeding lines in onion: A line (male sterile parent), B line (male fertile maintainer) and R line (fertility restorer) [22].

3. Genetic and Molecular Basis of Male Sterility in Onion

3.1. Genetics of Male Fertility and Sterility in Onion

The critical understanding of genetic and molecular basis of male sterility in variable male sterile lines of onion carrying variable cytoplasms is crucial for enhancing an onion hybrid breeding program. The CMS-S cytoplasm (S msms) has been widely exploited in onion [13] and is investigated extensively using different populations. The inheritance of fertility restoration in onion was investigated using the F₂ mapping population derived from a cross of BYG15–13 (N MsMs) × AC43 (N msms). The results fitted well in the estimated segregation of ratio of 1 (14 N MsMs): 2 (28 N Msms): 1 (13 N msms) and testcross progenies segregated in 1 N MsMs: 1 N msms ratio [55]. Bang et al. [56] developed a F₂ mapping population of 188 plants from a cross of 506L (male sterile parent) × H6 (DH based male fertile parent), and the results fitted well in a segregation ratio of 3 (male fertile): 1 (male sterile), indicating the dominance of fertility locus. Huo et al. [57] derived a backcross population from a three-way cross combination [118 (S msms) × {118 × 12-10 (S MsMs)}] and studied the segregation pattern, which fitted in a ratio of 1:1 with 112 male fertile individuals and 128 male sterile individuals having a differential gene expression for the AcPME gene that was expressed in male fertile lines at the flower bud stage. To verify the inheritance of fertility restoration in CMS-S type male sterility in onion, two backcross populations derived from 118 (S msms) × 12-12 (S MsMs) and 110 (S msms) × 12-12 were investigated [58]. The genotyping of backcross populations with SCAR markers linked with dominant locus “Ms” (DNF-567) and recessive locus “ms” (RNS-375) indicated the segregation ratio of 1:1. These results revealed that fertility restoration in CMS-S types is under the genetic control of the dominant gene [58]. The degree of fertility restoration varies with the source of fertile Ms locus in onion germplasm. The testcross progenies based on three different sources of Ms locus, viz. Sapporo-Ki (Ski), Ailsa Craig (AC) and B2354B, were screened for male fertility through visual phenotyping and acetocarmine staining technique [59]. The results revealed variable degrees of fertility restoration in these three different sources of Ms locus. The segregation ratio fitted in 1:1 proportion for male fertility and male sterility in the testcross progenies derived from AC and Ski sources [59]. The genotyping with molecular marker “AcPms1” confirmed the segregation ratio of 1:1 [59].

The CMS-T cytoplasm exhibits maternal inheritance and fertility restoration in CMS-T cytoplasm depicts a complex inheritance pattern governed by interaction of three Rf genes, while only one Rf gene is required in case of CMS-S cytoplasm [33,35]. The findings revealed by Kim [60], based on the analysis of four F₂ populations developed using CMS-T-like male sterile lines and male fertile lines, suggested perfect co-segregation of the “jnurf13” marker for male fertile phenotypes and genotypes. The segregation ratio based on analysis of these F₂ populations fitted in 3:1 ratio, indicating single dominant inheritance of fertility restoration. These results contradicted earlier findings of inheritance of fertility restoration in the CMS-T cytoplasm by three Rf genes.

CMS-Y, also referred as “cytotype-Y”, is a novel maternally inherited CMS type in onion (PI273626 and PI236025) containing unique combination of mitochondrial genes, “coxI” and “orf725” [43,61]. The inheritance studies based on segregation generation of a single plant from the accession “PI273626” indicated a single dominant gene, “Ms”, for fertility restoration in CMS-Y type male sterility.

3.2. Utility of Molecular Markers in Onion Male Sterility

The cytoplasmic male sterility (CMS) phenomenon is triggered in the crop plants if there is duplication or increase in the copy number of specific regions within the mitochondrial genome containing CMS-linked genes. The higher proportion of copy number of mitochondrial genes is associated with more chances of occurrence of male sterility [62,63,64]. The majority of CMS-linked genes are chimeric in nature [20,28,65,66,67,68] and the ATP synthase encoding genes are mainly responsible for the generation of CMS causing chimeric genes [28]. The frequent recombinations in plant mitochondrial genomes are mediated by short repeat sequences of less than 100 bp, that ultimately leads to the development of chimeric genes inducing CMS [69,70,71,72,73]. However, the CMS-causing genes may be masked by fertility restorer genes (Rf) [27]. These “Rf” genes mainly encode PPR (pentatrico peptide repeat) proteins [20,74], although some of the “Rf” genes encode other proteins like aldehyde dehydrogenase proteins, acyl-carrier proteins, glycine-rich proteins and peptidases [75,76,77,78].

The three main CMS types, CMS-S, CMS-T and CMS-R, are mainly involved in onion hybrid breeding programs. The comparative mitochondrial genome sequence analysis of these CMS types recognized “orf725” as a common CMS-causing gene in onion [61,79,80,81]. The restoration of fertility in CMS-R type is conditioned by Ms and Ms2 loci [41,60,82]. The dominant Ms2 locus with CMS-R confers restoration of male fertility despite Ms locus being in homozygous recessive state [41]. On the other hand, a genomic region located at the 3′ end of the “orf725” gene that exhibits high homology with “orfA501” has been detected in the CMS-T cytoplasm [83]. The genome walking PCR analysis using homologous sequences of “orfA501” followed by sequencing of 5′ PCR products indicated the association of 128-bp of atp1 exon1 sequences with orfA501-homolog sequences. Furthermore, the close linkage of 5′ partial sequences of the nad7 gene with orf725 was revealed via sequencing of 3′ genome walking PCR products. Thus, a novel candidate gene, “orf219”, was suggested to be involved in conferring male sterility by the CMS-T cytoplasm [83].

To facilitate the fast identification of male sterile and fertile lines for enhancing onion hybrid breeding, molecular markers are playing a crucial role. Molecular markers have been designed for discriminating mitotypes in onions [60,79,84]. The development of PCR-based markers such as RFLP, CAPS, SCAR and SNP markers has facilitated the marker-assisted selection of Ms locus in onion [56,85,86]. The other markers reported for identification of Ms locus in onion germplasm of India are cob (PCR marker), MKFR (PCR marker), accD (InDel marker), Jnurf13 (InDel marker) and AcPMS1 (PCR marker) [85,86]. Initially, a molecular marker linked with mitochondrial “cob” gene was developed, which was capable of distinguishing CMS-S and -N cytoplasms in onion [87]. However, this marker was not able to differentiate between CMS-T and -N cytoplasms. A remarkable success was achieved by Engelke et al. [88] who developed a molecular marker, that was a combination of cob and orfA501, to distinguish CMS-S, -T and -N cytoplasms in onion breeding material. Later on, Kim et al. [79] reported another mitochondrial marker “orf725” based on sequences of the “cox1” gene and chimeric “orf725” gene to distinguish CMS-S, -T and -N cytoplasms in onion. However, mitochondrial “orf725” marker was not able to distinguish bona fide T cytoplasm (RJ70A) and N cytoplasm (B1750B), both producing amplicon of 833 bp. The cytoplasm-producing amplicon of both 833 bp and 628 bp was referred to as “R” cytoplasm, tracing its origin from the “Rijnsburger” onion cultivar [79].

Havey [21] stated that the dominant “Ms” locus does not restore male fertility of CMS-T, while Kim [60] documented that fertility in CMS-T is restored by dominant “Ms” locus. This contradiction can be explained by the fact that CMS-R and CMS-S cytoplasms generate 628 bp amplicon of mitochondrial “orf725” marker, and here the dominant “Ms” locus restores the male fertility in both cytoplasms. On the other hand, a 628 bp amplicon of “orf725” is not generated by the CMS-T cytoplasm, and hence the “Ms” locus fails to restore the male fertility [21,39]. A dominant marker “orfA501” was reported to distinguish CMS-T cytoplasm type by Havey and Kim [39]. However, being dominant in nature, “orfA501” is not ideal for screening large numbers of male sterile lines in onion. Hence, a novel molecular marker was developed based on the orf219 candidate gene [83]. In addition, three HRM (high-resolution melting) markers (accD-HRM, orf725-HRM, orf219-HRM) were generated to screen the large breeding material [83,89]. Recently, a two-step technique was devised to distinguish CMS-N, CMS-T, CMS-S and CMS-R cytoplasms based on HRM markers (AcM-HRM-F1, AcM-HRM-R1 and AcM-HRM-R2) [90]. Firstly, a forward primer and two reverse primers based on both cox1 and orf725 gene sequences was designed to differentiate CMS-N, CMS-R and CMS-S cytoplasms. In the second step, to distinguish CMS-N and CMS-T cytoplasms, screening of breeding lines carrying -N cytoplasm was performed using molecular markers developed based on the sequence of the orfA501 gene [90]. The genetic linkage mapping of Ms locus using F₂ mapping population derived from a cross of male sterile and doubled haploid line (506L × H6) located the Ms locus on chromosome 2 linked with CAPS markers (jnurf05 and jnurf17) [86]. Previously, Martin et al. [67] also located the Ms locus on chromosome 2 using genetic linkage mapping of F₂ population (BYG15−23 × AC43) and depicted the role of SNPs, InDel and SSR markers [67].

Onion hybrid breeding using CGMS requires A (male sterile), B (maintainer) and R lines (restorer), where Ms/ms alleles are used in restorer and maintainer lines [15]. Thus, development and identification of R (N MsMs) and B (N msms) lines is crucial in onion hybridization. In line with this, the molecular markers linked with the Ms locus are instrumental in accelerating onion hybrid breeding [15]. Gökçe et al. [55] identified RFLP-based allelic diversity in the Ms locus, which facilitated the determination of maintainer lines in onion germplasm [55]. Furthermore, PCR-based co-dominant oligopeptide-transporter (OPT) markers specific to the Ms locus were reported by Gökçe and Havey [38]. Another PCR-based marker “WHR240” linked with the “AcPME” gene was reported to identify fertility restorer lines [57]. Yang et al. [58] reported SCAR markers (DNF-566 and RNS-357) linked with Ms locus to differentiate between homozygous dominant (MsMs), homozygous recessive (msms) and heterozygous (Msms) phenotypes among varieties, hybrids and OP populations.

4. Phenotypic Features

Male sterility is a crucial trait in onion breeding programs due to its pivotal role in hybrid seed production, ensuring uniformity, vigor and yield stability. Phenotypic observation of stamen morphology and pollen viability remains a traditional yet effective method for identifying male sterility. Indeed, the overall appearance of the inflorescence can be indicative of fertility or sterility (Figure 3).

The phenotype of male sterile plants can be distinguished from their male fertile counterparts based on floral, morphological, reproductive and seed-related traits [24]. The male sterile plants often exhibit deformed flowers, malformed or shriveled anthers (Figure 4), or failure to produce functional pollen grains, anther indehiscence or failure of male reproductive success [13,73,91]. In onion, the plants with the male sterile cytoplasm may exhibits light green, dark green or yellow anthers [92]. In contrast to that, Saini et al. [93] did not record any association between male sterility and color of anthers in onion breeding material; instead, they noticed erratic production of pollen grains in male sterile plants. This could be explained by the influence of environmental conditions, particularly high temperature. Recently, Singh and Khar [94] documented significant phenotypic differences between male sterile and male fertile counterparts in short-day Indian onion lines. The phenotypic observations were made by examining male sterile and fertile flowers, anthers and pollen grains.

However, advancements in molecular techniques have enabled the development of molecular markers linked with cytoplasm types in onion, that revolutionized an onion breeding program [18,21]. As discussed, these markers enable precise identification and selection of sterile lines, facilitating the creation of superior hybrid varieties with desired traits. By integrating both phenotypic and molecular approaches, onion breeders can accelerate the development of high-performing cultivars, ultimately enhancing crop productivity and achieving the goals of food security.

5. Genomic and Transcriptomic Insights into Onion Male Sterility

In the post-genomics era, the increase in availability of genomic resources such as reference genomes, molecular markers, transcriptome data and genomic databases across the crop plants has accelerated plant breeding [95,96]. In the case of onion, the first draft genome assembly of a doubled haploid line “DHCU066619” was of 14.9 Gb genome size [97]. The gene prediction analysis based on ab initio estimated 540,925 genes. The availability of onion genome assembly has significantly facilitated onion breeding. The male sterility in onion is vital for escalating hybrid breeding.

The CMS is governed by mitochondrial genome and, in this context, Kim et al. [80] provided the complete mitochondrial genome sequence of a breeding line carrying S- cytoplasm. Furthermore, the structure of mitochondrial genome and analysis of DNA rearrangements linked with male sterility in onion has been studied extensively [61,81,98]. The next-generation sequencing (NGS) platform was used to sequence the mitochondrial genome of CMS-S type onion cultivar “Momiji-3” [81]. The results indicated that the mitochondrial genome of “Momiji-3” is represented by three circles due to high recombination frequency via repeated sequences. The transcriptome data analysis of mitochondrial genome revealed the presence of 635 RNA-editing positions in the coding regions of the gene. The RNA editing positions indicated the start and stop codons in six genes, viz. nad1, nad4L, atp6, atp9, ccmFC and orf725 [81]. The study did not report any presence of novel orf transcripts and indicated “orf725” as candidate gene for CMS in “Momiji-3” variety [81]. The comparative mitochondrial genome sequence of CMS-T and male fertile counterpart in onion was provided by Kim et al. [61]. They also reported high genomic similarity between CMS-T and normal cytoplasm except for the presence of “orf725” along with “cox1” sequences in CMS-T mitochondrial genome.

Recently, Bishnoi et al. [98] reported mitochondrial genome sequence of a short-day tropical onion CMS line “97A“ and its maintainer line “97B”. The mitochondrial genome of CMS line “97A” was of 3,16,321 bp, while the mitochondrial genome of “97B” was comparatively 15 scaffolds due to repetitive genomic regions. Both male sterile and fertile genomes contained 13 and 20 chloroplast-derived fragments, respectively. Further genome analysis depicted 24 protein coding genes in the mitochondrial genome. The comparative genome analysis revealed high genome similarity between male sterile and fertile mitochondrial genomes, except for the presence of chimeric orf725 gene in CMS 97A line [98].

Various studies employing RNA sequencing and transcriptomic analysis have contributed significantly to the identification of genes exhibiting differential expression between male sterile lines and their male fertile counterparts, thereby shedding light on the intricate physiological and molecular pathways responsible for male sterility. Although limited in number, research specifically conducted on onion has played a crucial role in this regard. In this context, Yuan et al. [99] analyzed the microscopic structure of the anthers in CMS plants (SA2) and their male-fertile maintainers (SB2) in onion. They discovered that, in male sterile plants, pollen production was disrupted at a specific stage called the tetrad stage, which is different from what happens in male-fertile plants. To understand the genetic basis of this differential expression, comparative RNA sequencing of anthers collected from both SA2 and SB2 plants was performed using Illumina HiSeq platform and a large number of 146,413 unique genetic sequences, termed all-unigenes, were identified. Further genomic analysis revealed the role of two cytoplasmic genes, atp9 and cox1, in controlling male sterility. Three nuclear genes, viz. SERK1, AG and AMS, exhibited differential expression between CMS and male-fertile plants. They confirmed these findings using a method called fluorescence quantitative PCR.

In another study, Liu et al. [100] used RNA-seq analysis to determine the differential expression of genes between CMS line “64-2” and its maintainer line “64-1”, in Welsh onion. The study revealed significant differences between two lines demonstrating differential expression of 1504 unigenes in 2013 and 2928 unigenes in 2014. The validation of CMS genes (F-type ATPase, NADH dehydrogenase and cytochrome c oxidase) was performed with qPCR analysis. The study revealed the role of both mitochondrial and nuclear genes in regulating CMS in Welsh onion. Despite differences in species, onion (A. cepa L.) and Welsh onion (A. fistulosum L.) share genetic similarities and physiological traits, suggesting that conclusions drawn from Welsh onion studies could be relevant and informative for understanding aspects including cytoplasmic male sterility.

The transcriptome analysis and the investigation of MADS-box genes in onion male sterility revealed the downregulation of class-B and class-C MADS-box genes, leading to stamen developmental failure and male sterility in onion [101].

Figure 5 integrates genomic and transcriptomic insights to illustrate the complex regulatory network governing male sterility in onion. It provides an overview of the molecular mechanisms underlying this process, highlighting the role of mitochondrial genes (orf725) in mitochondrial dysfunction. Additionally, it emphasizes the differential expression of nuclear genes associated with floral organ identity, particularly the downregulation of MADS-box genes, which leads to stamen developmental failure in male sterile anthers [101]. It also illustrates the interactions between mitochondrial and nuclear genes, detailing functional consequences such as tapetal cell degeneration and pollen abortion.

6. Conclusions

Research on male sterility in onion holds great promise for enhancing crop improvement efforts in the future. Male sterility systems serve as a powerful tool for hybrid seed production, enabling the development of high-performing onion hybrids. Male sterility, primarily governed by mitochondrial genes, plays a crucial role in hybrid breeding. With the advances in molecular tools and genomics, the complete mitochondrial genome has been sequenced, shedding light on the genetic basis of male sterility in onion. This knowledge has opened new opportunities for harnessing male sterility in onion breeding programs, allowing breeders to exploit male sterility mechanisms to develop hybrid onion varieties with enhanced yield potential and improved agronomic traits. Genomic studies have facilitated the development and identification of molecular markers linked to male sterility, particularly the mitochondrial genes orf725, orf219 and cox1, enabling marker-assisted selection of male-sterile lines in onion. Furthermore, transcriptomic studies have revealed the differential expression of nuclear genes, which plays a role in the regulation of male sterility in onion. These findings highlight the complex mitochondrial–nuclear interactions underlying this trait.

By exploiting male sterility mechanisms, breeders can overcome the limitations of intensive manual emasculation, facilitating efficient hybrid seed production. This approach also helps mitigate inbreeding depression and achieve hybrid vigor, resulting in increased yield potential and improved uniformity in onion crops. Continued research on male sterility will be essential for advancing onion hybrid breeding strategies and addressing challenges in sustainable agriculture.