Introduction

The bulb onion (Allium cepa L., 2n = 2x = 16), which belongs to the Amaryllidaceae family, is one of the most important crops and has been domesticated and cultivated in the Old World for more than 5000 years. Due to their peculiar properties as food and their therapeutic and ethnopharmacological value, onion plants are preferred for consumption across the globe and are cultivated under all climates (Brewster 2008). Although it is an important vegetable crop, breeders have not focused much on its genetics, breeding and genomic research. The main reason for this limited research is due to its biennial nature, highly cross-pollinating nature, high degree of inbreeding depression and giant genome size (Khar 2022).

In India, the phenomenal increase in onion production from 3.23 to 26.74 million tonnes reflects the enhanced awareness of onion consumption over the last three decades i.e., from 1990 to 2020. As the leading country in terms of harvested acreage and total production, India harvested 26.7 MMTs from an area of 1.62 million hectares. However, Indian farmers are getting experiencing comparatively lower productivity (16.4 t/ha) than farmers in other regions such as Korea (79.62 t/ha), the USA (71.10 t/ha), Japan (49.32 t/ha) and China (21.86 t/ha) (FAOSTAT 2023). The foremost reasons for the lower productivity are the cultivation of open-pollinated varieties (OPVs) or locally produced seeds by farmers who have no or less quality control or regulation over seed production. Several scientific reports have demonstrated that compared with open-pollinated varieties, hybrid cultivars exhibit great heterosis and display greater uniformity in terms of their horticultural and morphological characteristics (Nunes et al. 2014; Netrapal and Singh 1999). In India, Shashikanth et al. (2007) observed higher heterosis of 45.31% over better parent and 27.40% over the standard check in one of its hybrid whereas Singh and Bhonde (2011) observed higher gross yield in hybrids ‘Mercedes’ (72.5 t/ha), Linda Vista (71.6 t/ha), Cougar (67.5 t/ha) compared to the open pollinated variety ‘ALR’ (42.7 t/ha). Henceforth, the bulb productivity of Indian onions might also be improved by selecting and developing well-adopted high-yielding F1 hybrids.

Onion hybrid breeding started after the reports of cytoplasmic male sterility induced by S-cytoplasm (Jones and Emsweller 1936) and complete restoration of fertility through the single nuclear dominant (Ms) restorer gene (Jones and Clarke 1943). The development of onion hybrids using this system is the only feasible and economical method for increasing onion productivity and bulb uniformity while maintaining desirable quality and disease resistance. The S-cytotype is popular for hybrid development worldwide due to its complete stability under diverse and varying environmental conditions and further this is inherited simply by a single dominant nuclear gene (Havey 2000). Nevertheless, the hindrances in developing onion hybrids remain because of their biennial habit and male sterility mechanism, which requires 4 to 8 years for identification of the cytoplasm and genotype of nuclear Ms alleles through progeny testing (Havey 2000). Henceforth, specific molecular markers were supposed to be the ideal tool to speed up this time-consuming and laborious procedure of identifying maintainers for hybrid development. Specific PCR-based molecular markers used to distinguish N and S types of cytoplasm (Havey 1995; Sato 1998; Von Kohn et al. 2013) and all three N, S and T cytotypes (Kim et al. 2009) have been tested. Identification of male sterile onion lines based on visual and/or microscopic examination is easy. However, the isolation of maintainer lines requires progeny testing for genotyping of the nuclear Ms locus. Molecular markers closely linked with the Ms locus were initially reported by Gokce and Havey (2002) followed by other marker types (Bang et al. 2011; Kim 2014; Kim et al. 2015; Huo et al. 2015) and single nucleotide polymorphisms (SNPs) (Havey 2013) tightly associated with the Ms locus. To date, no commercial Indian onion hybrids from the public sector are available at the national scale. Despite the fact that Indian onion researchers have been working for the last 60 years, the development of onion hybrid cultivars has not gained momentum. A positive step in this direction are some reports of deployment of PCR based markers for cytoplasm and Ms locus identification in Indian onions (Chaurasia et al. 2010; Saini et al. 2015; Khar and Saini 2016; Khar et al. 2022). However, these reports are not comprehensive enough to provide an overall picture of the cytoplasmic and Ms locus distributions in Indian onion populations. To accelerate hybrid breeding programme, the first step is to identify male sterile and maintainer lines in different genetic backgrounds to serve as base materials. Hence, this work aimed to identify cytoplasm types and nuclear fertility restorer (Ms) locus in populations of commercially grown short-day Indian onion open-pollinated varieties.

Materials and methods

Plant materials

The plant material comprised 35 open-pollinated commercially cultivated short-day Indian varieties (OPVs) representing more than 10 states. The varieties represent diversity present in Indian onions and bulb colour varied from white to dark red (Table 1). These varieties are being maintained at the Division of Vegetable Science, ICAR-Indian Agricultural Research Institute, New Delhi.

Table 1 List of the open pollinated varieties of Indian onion used in the present study
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Morphological assessment of anthers

A visual assessment of flowers on the basis of anther colour could lead to the use of morphological markers for the identification of male sterile and fertile plants. Phenotypically, male sterile flowers and fertile plants were distinguished based on anther colour. The initial hypothesis was that light green anthers would be considered as male sterile and that dark green anthers would be considered as male fertile plants (Pathak 1997).

Microscopic assessment of pollens

For pollen viability, flowers were collected in the morning from umbels which had 60% dehisced flowers. Anthers were collected and pollens were dispersed on a microscope slide and stained with 0.5% acetocarmine solution (Khar and Saini 2016). Pollens were observed under a microscope and classified as viable if the pollens were elliptical in shape with two stained nuclei and if the cytoplasm was pink. Pollen that was clumped, and transparent with an unusual shape was identified as non-viable.

Collection of samples for DNA extraction

Twenty-four plants from each variety were tagged and assigned serial numbers ranging from 1 to 24. After three to four weeks of bulb planting, tender leaf tips of the 1–2 cm portion were cut with a sterilized pair of scissors, immediately wrapped in aluminum foil and put in liquid nitrogen for extraction of DNA. A total of 840 samples were taken from 35 OPVs.

Genomic DNA extraction and PCR amplification

Genomic DNA was isolated, from 840 individual plants of 35 OPVs, using the CTAB method with minor modifications (Murray and Thompson 1980). For the determination of cytoplasm, two specific PCR based molecular markers were explored. The mitochondrial orf725 gene mentioned as a MKFR marker (Kim et al. 2009) and an indel in the accD gene in chloroplast genome denoted as the accD marker (Von Kohn et al. 2013) were used (Supplementary Table 1). To identify the male fertility (Ms) locus, four identified PCR markers, namely, OPT (Bang et al. 2011), AcSKP1 (Huo et al. 2015), jnurf13 (Kim 2014) and AcPMS1 (Kim et al. 2015), were used (Supplementary Table 2).

Results and discussion

The CMS mechanism is an excellent model for examining the nuclear and cytoplasmic interactions since fertility restoration depends upon nuclear genes that govern the cytoplasmic dysfunction in terms of the viability of pollen. This genetic system is the most preferred approach for the commercial and wide exploitation of heterosis in various field and horticultural crops and is characterized by the genetic expression of developmental differences among hybrids and their respective parents. Onion hybrids exhibit higher heterosis over open pollinated varieties in terms of agronomical characteristics (Netrapal and Singh 1999; Nunes et al. 2014). Hybrids are quite popular among onion growers worldwide due to their ability to yield higher quantities of onions with uniform bulb, morphological and maturity traits. This crop is strictly cross-pollinated and is classified as a crop showing steep inbreeding depression.

Morphological assessment and pollen sterility status

The fertility and sterility status of the flower buds were assessed by examining anther colour visually and examining pollen dust by touching the flowers. It was observed that visual examination led to the identification of 789 (93.93%) plants as fertile whereas 51 (6.07%) were observed to be sterile. A wide range of anther and pollen colour was also observed and it varied from light creamish yellow to dark green colour (Fig. 1).

Fig. 1
figure 1

Variation in anther colour of different onion varieties from dark green to yellow

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It was observed that the colour of anther or pollen could not decide the sterility status of the pollen. In Pusa Sona, anthers were light green and visually sterile but upon microscopic examination, they were not sterile. Similar discrepancies were observed in PWF, L-819, Bhima Safed, Bhima Shweta, RO252, Punjab Naroya and KRR (Supplementary Table 3). Earlier, it has been documented that light green coloured anthers possess sterile pollen in onion flowers (Pathak 1997; Santos et al. 2010; Saini et al. 2015). Further, Santos et al. (2010) elicited that light green anthers in onion flowers were male-sterile. The present investigation revealed that anther and pollen colour alone cannot decide the sterility level of pollen, since dark green and other coloured anthers contain sterile pollen. Khar and Saini (2016) also hypothesized that male sterility has no correlation with the anther colour, and our results confirm their findings.

Microscopic assessment of pollen

In plant species, the use of staining chemicals to separate viable and nonviable pollen grains has been exploited for decades (Peterson et al. 2010). Acetocarmine staining for onion ideally works to assess aborted or non-aborted pollens (Khar and Saini 2016; Lee and Havey 2020). In the present study, considerable variation in anther and pollen colour was observed (Supplementary Table 4) and it was cumbersome to determine the fertility versus sterility level of plants on the basis of colour visually. Hence, acetocarmine staining solution (0.5%) was used to distinguish the sterility status of the pollen grains. The scoring of viable pollen grains was based on the strongly stained nucleus and cytoplasm, whereas, no or lightly stained pollen grains were scored as non-viable (Fig. 2). Apart from stain, abnormal or misshapen pollens were also recorded in the aborted category. A total of 24 plants per variety were tagged for assessing viability via visual and microscopic studies. There was discrepancy between the visual and microscopic studies for most of the varieties. e.g., In Pusa Shobha, 21 fertile and 3 sterile plants were observed visually but microscopic observation led to the detection of 23 fertile and 1 sterile plant. This was the case for most of the varieties. This implies that the selection of plants based on visual examination for sterility is not advisable. Microscopic studies are important for a valid assessment of sterility. Male sterility was also detected in at least eleven varieties that can be used for the development of male sterile and maintainer lines in different genetic backgrounds. Based on the visual examination, 93.9% (789) were fertile and 6.1% (51) were sterile whereas microscopic studies revealed that out of 840 plants, 804 (95.71%) were fertile, and 36 (4.29%) were sterile plants. The highest percentage (62.5%) of sterile plants was observed in Arka Bheem followed by KRR (25.0%) and Pusa Red (20.8%). The colour of anthers and pollen might be influenced by the prevailing environmental conditions of the specific region. With increasing temperature and plant age, continuous variation in colour was observed. Another observation was that the fertile pollen possessed some adhesive traits whereas, the sterile pollen grains showed dryness and dullness. It might be due to the manipulation in the synthesis in the glucose, fatty acids, pectin and cellulose as described in the cotton by Wu et al. (2015). Further in potato, mature sterile pollens were recorded with significant decline in the carbohydrate pool and augmentation in the amino acid concentration. Various alterations in lipophilic compounds and fatty acid pool led to defective cell wall morphology of pollen grain depending upon the deposition of callose and sporopollenin quality (Shishova et al. 2019).

Fig. 2
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Microscopic visualization of a male fertile and b male sterile pollens

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Cytoplasm identification

In onion, the sterile (S) type of cytoplasm is extensively exploited and preferred globally for the development of F1 hybrids in the onion crop owing to its greater stability under diverse climatic and environmental conditions. The polymorphism in mtDNA for differentiation of N and S type of cytoplasm are significantly faster than test crosses and saves abundant time to accelerate hybrid development. Identification of the plants that possess male sterile cytoplasm and the apt fertility restorer nuclear gene(s) is the critical step for the initiation of a heterosis breeding program.

In the present investigations, all 840 individual plants of thirty-five genotypes were analyzed using two cytoplasmic specific markers viz., accD and MKFR. Based on accD marker, out of 840 plants, 803 (95.6%) were having normal (N) and 37 (4.4%) had sterile (S) cytoplasm. None of the plants was recorded with T-cytoplasm. The same results were observed by using MKFR markers for identification of the N and S cytoplasm. The chloroplast based InDel primer accD determined 37 (4.4%) individuals possessing S-cytoplasm in the eight commercial cultivars that included Pusa Shobha (1), Pusa Red (10), GJWO-11 (1), Bhima Shweta (1), Arka Bheem (15), Kalyanpur Red Round, PRO-6 (1) and Punjab Naroya (1). Similarly, the mitochondrial marker MKFR followed the same trend and validated the results obtained from accD. The MKFR too couldn’t predict any plant possessing T-cytoplasm. T cytoplasm was reported by Khar et al. (2022) in the hybrid ‘T821’ being grown under Indian conditions. Recently, the ‘T’ like cytoplasm has been designated as ‘R’ since the sterility is restored by single dominant Ms allele (Havey and Kim 2021). The OPVs popularly grown in Northern Indian conditions for the last 30 years namely Pusa Red and Kalyanpur Red Round exhibited 17 (35.41%) plants having S-cytoplasm. Pusa Red has earlier been reported to have S cytoplasm (Khar and Saini 2016; Khar et al. 2022). On the other hand, Arka Bheem, a tri-parental synthetic variety having pinkish red and elongated globe bulbs possessed a maximum number (15/24) of plants containing S-cytoplasm. This can be expected since the initial work on onion male sterility started at IIHR, Bengaluru (Pathak et al. 1980) from where Arka Bheem has been released. One of the parents used in synthetic development may be male sterile. Except for two (GJWO-11 and Bhima Shweta), all S-cytoplasm-containing varieties belong to the red bulb category. This implies that various institutes are actively engaged in breeding efforts to introduce S cytoplasm into Indian varieties where these cultivars have been released. However, the absence of the use of molecular markers might have led to oversight, potentially causing breeders to miss identifying the sterility status. Khar and Saini (2016) reported that 31 (93.9%) of the Indian OPVs had N cytoplasm and only 2 (5.8%) had S cytoplasm. Similarly, Khar et al. (2022) reported that 91.3% of Indian OPVs had N cytoplasm whereas 8.7% had S cytoplasm. These reports were based on one plant per OPV whereas we used 24 plants per OPV and the results are a true indicator of the sterility status in Indian onion.

Genotyping of the Ms locus

In general, aberrant mitochondrial genes induce male sterility in the higher plants which can be reverted by the interaction of nuclear gene(s) known as fertility restorer genes (Rf). In onion, one dominant Rf gene (Ms locus) was reported to govern the restoration of fertility in the S-cytoplasm (Jones and Clarke 1943). However, three independent loci were observed to be involved in the restoration of T-cytoplasm fertility status (Schweisguth 1973).

In this study, four PCR-based molecular markers (AcPMS1, AcSKP1, jnurf13 and OPT) were exploited for genotyping the Ms locus in diverse Indian onion populations. Among 840 individual plants, AcPMS1, AcSKP1, jnurf13, and the OPT primer predicted 98.33% (826/840), 99.52% (836/840), 99.40% (835 /840) and 38.81% (326/840) homozygous recessive genotypes (msms), respectively. Whereas, these markers predicted 0.12% (1/840), 0.0% (0/840), 0.12% (1/840) and 21.67% (182/840) homozygous dominant alleles (MsMs), respectively. The frequencies of observed heterozygous dominant (Msms) allele were 1.55% (13), 0.48% (4), 0.48% (4) and 39.52% (332), respectively (Table 2).

Table 2 Frequencies of N-, S- type cytoplasm and the Ms locus in the Indian short day onion population
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For the Ms locus, the AcPMS1 marker identified 13 heterozygous (Msms) plants from 840 plants including 4 each from Early Grano (Fig. 3) and Bhima Safed, 3 from Pusa Red and one from Pusa White Round and Bhima Dark Red.

Fig. 3
figure 3

Amplification of the Ms locus in the Early Grano population (n = 24) using AcPMS1 marker (MsMs 242 bp; msms 276 bp)

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Except for AcSKP1, all the other three Ms specific markers predicted homozygous dominant alleles in the Indian short-day onion population. Maximum homozygous recessive alleles were observed in all the plants of all the accessions. The marker jnurf13 identified 99.40% of the plants with the msms genotype, and AcSKP1 predicted 98.33% of the plants with homozygous recessive Ms alleles. The marker OPT showed considerably unpredictable results compared to the other markers (Fig. 4).

Fig. 4
figure 4

Amplification of the Ms locus in Bhima Safed population (n = 24) using the OPT1 marker (MsMs 242 bp; msms 276 bp)

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This marker detected 332 plants out of 840 (39.52%) with heterozygous (Msms) and 21.67% homozygous dominant (MsMs) alleles. However, the AcSKP1 marker exhibited no dominant homozygous genotype. A total of 836 out of the 840 plants possessed a recessive homozygous Ms locus (msms) according to the AcSKP1 marker. One plant had a homozygous dominant genotype at the Ms locus (MsMs) according to AcPMS1 and jnurf13 markers (Table 3).

Table 3 Genotyping of the Ms locus of individual twenty-four plants of 35 commercial varieties
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Khar and Saini (2016) predicted that the primer AcPMS1 gave more accurate results than AcSKP1 based on visual observations of 25 commercially released varieties of Indian onion and some exotic lines grown under short-day conditions. The findings of this study suggest that all the primers (except OPT) predicted OPVs with similar frequencies. The efficacy of the use of the AcPMS1 and jnurf13 markers for correctly classifying onion populations was contested by Khar and Saini (2016) as they did not always predict the correct type of Ms genotype in the Indian population. However, the same markers were used on the North American onion population (Havey and Von Kohn 2017). AcPMS1 was recommended as a better marker as it displayed no recombination events with Ms locus whereas jnurf13 showed 3 events of recombination. The frequency of the Ms locus predicted by the OPT marker is completely different from that predicted by other three markers and needs further study. The results obtained from PCR markers need to be correlated with parents and their selfed progenies to arrive at a meaningful conclusion. Till then, we can conclude that the PCR marker AcPMS1 can be used for Ms locus determination in OPVs. Based on our findings, the homozygous recessive (msms) allele was the predominant Ms locus type with N cytoplasm, which contradicts the findings of previous studies (Pike 1986; Pathak 1997) in which the frequency of Nmsms plants in a population was found to be less than 5%. This identification of A, B and R/C (restorer line) lines from OPVs will aid in the development for high-yielding F1 hybrids with the low production cost of hybrid seed.