﻿Allium cytogenetics: a critical review on the Indian taxa

﻿Abstract The genus Allium Linnaeus, 1753 (tribe Allieae) contains about 800 species worldwide of which almost 38 species are reported in India, including the globally important crops (onion, garlic, leek, shallot) and many wild species. A satisfactory chromosomal catalogue of Allium species is missing which has been considered in the review for the species occurring in India. The most prominent base number is x=8, with few records of x=7, 10, 11. The genome size has sufficient clues for divergence, ranging from 7.8 pg/1C to 30.0 pg/1C in diploid and 15.16 pg/1C to 41.78 pg/1C in polyploid species. Although the karyotypes are seemingly dominated by metacentrics, substantial variation in nucleolus organizing regions (NORs) is noteworthy. The chromosomal rearrangement between A.cepa Linnaeus, 1753 and its allied species has paved way to appreciate genomic evolution within Allium. The presence of a unique telomere sequence and its conservation in Allium sets this genus apart from all other Amaryllids and supports monophyletic origin. Any cytogenetic investigation regarding NOR variability, telomere sequence and genome size in the Indian species becomes the most promising field to decipher chromosome evolution against the background of species diversity and evolution, especially in the Indian subcontinent.


Introduction
The genus Allium Linnaeus, 1753 is considered a wonder crop of global importance, catering to the agriculture, condiment, pharmaceutical, nutraceutical and cosmetic sectors of economy owing to the presence of numerous species with tremendous significance. Among several herb species, an onion (A. cepa Linnaeus, 1753) that is valued throughout the continent attracts a lot of attention of the economic sectors mentioned above, followed by garlics, leeks and shallots having limited uses. Onion is the second of the five main world vegetables species (after tomato) whose worldwide production accounted for 9% of the total (42-45%) increase in production of vegetables between 2000-2019 (https://www.fao.org/3/cb4477en/online/cb4477en.html#chapter-2_1).
Allium, previously referred to Liliaceae, is now a member of Amaryllidaceae sensu Angiosperm Phylogeny Group or APG III (Haston et al. 2009). This large genus (about 800 species, Costa et al. 2020) was divided into 15 subgenera and 56 sections (Friesen et al. 2006). At present, Allium has its primary evolution centre across the Irano-Turanian phytochorion while secondary centres of diversity include Mediterranean basin and western North America (Friesen et al. 2006). The taxonomy and evolution of this diverse genus has been accepted as difficult.
Cytogenetics, being the only elementary discipline of genetics, focuses on genome structure, function and evolution. The evolutionary history of organisms is inscribed in the chromosomes, the physically visible form of genome. The very fundamental parameters such as chromosome count reports, when combined with molecular cytogenetic and phylogenetic data (Islam-Faridi et al. 2020;Senderowicz et al. 2021), or genome size estimates, can elucidate trends of evolution in context of ploidy changes. Molecular cytogenetic approaches, in line with the parameters mentioned already, can accelerate the understanding of the evolutionary questions (Borowska-Zuchowska et al. 2022;Nath et al. 2022). A general correlation between evolutionary trends and chromosomal features has been shown in many plant families ( Van-Lume et al. 2017;Carta et al. 2020;Bhowmick and Jha 2022;Nath et al. 2022). Recently, a broad concurrence between karyology and geographical distribution has been shown in three Allioidae tribes, with respect to the diversification of Allieae to Northern Hemisphere from the Indian tectonic plate around 30 million years ago (Costa et al. 2020).
India is the world's second-largest producer of onion after China, with a production rate of 16360 kg/ Ha (2020-2021) (https://eands.dacnet.nic.in/). After onion, A. sativum Linnaeus, 1753 (garlic) is the second largest species of Allium contributing significantly to agro-economical development of the country (https://eands.dacnet.nic.in/). Among the other species, A. schoenoprasum Linnaeus, 1753 and A. roylei Stearn, 1947 exhibited resistance qualities (Nanda et al. 2016) and promise adoption of advanced breeding. Keeping in mind the significance of Allium and the complications in taxonomy and evolution, a comprehensive summary of cytogenetic characters has been presented for Indian species of Allium.

Chromosome counts
The chromosome counts and karyotype details are known perhaps in 33 and 25 species, respectively (Table 1, Fig. 1). The prominent base number (x) is 8, irrespective of the subgenera, sections or the distribution pattern. Some western Himalayan species which are still not assigned to any of the subgenera (e.g. A. atropurpureum, A. caesioides, A. consanguineum, A. ascalonicum Linnaeus, 1756, A. blandum Wall., 1832, A. hypsistum Stearn, 1960 and endemic A. stracheyi have x=8. Divergent numbers such as x=7, 10 and 11 are found in the Indian species of the subgenus Amerallium (Table 1) which also justifiy their inclusion in a separate subgenus (Peruzzi et al. 2017). Chromosome number has not been studied in the newly discovered A. negianum of Rhizirideum, sect. Eduardia (Pandey et al. 2021), which together with its close relative A. przewalskianum of sect. Caespitosoprason (Pandey et al. 2021) not studied from the territory of India, needs to be investigated. Similarly, A. loratum, A. auriculatum, A. rhabdotum and an endemic A. gilgiticum still are not assigned to any of the subgenera, and any cytological information is also missing. The meiotic studies in some species have shown various configurations like multivalents or univalents and occasional irregularities as in A. chinense G. Don, 1827 Koul 1973, 1981), A. hookeri , A. roylei Gohil 2003, 2011a;Kohli and Gohil 2011), A. rubellum M. Bieb., 1808(Khoshoo and Sharma 1959A. tuberosum (Gohil andGohil 2004, 2013a, b). In case of tetraploid A. ampeloprasum Linnaeus, 1753 (as A. porrum Linnaeus, 1753 in many studies), 16 bivalents were recorded regularly with complete absence of any multivalent (Koul and Gohil 1970b;Ved Brat and Dhingra 1973;Gohil and Koul 1977;Pandita and Mehra 1981a;Stack and Roelofs 1996). In this species, some peculiar features like appearance of bivalents in metaphase I instead of quadrivalents, localized chiasmata at pericentromeric regions have been reported (Levan 1940;Koul and Gohil 1970b;Stack and Roelofs 1996). Considering the incidence of vivipary and hybridization in A. cepa (Singh et al. 1967;Langer and Koul 1983;Puizina and Papea 1996), thorough meiotic analysis of the agriculturally important species (A. cepa, A. sativum, etc.) would be a significant aspect of future revision.

A. blandum
Wall. Among the diploid species, the range of genome size (  Wang et Tang, 1937) to 40.5-41.78 pg/1C in A. victorialis. Thus, the lowest values of genome size for the entire array of Allium species in India is represented by diploid and polyploid species of A. schoenoprasum (subgenus Cepa).
The genome size evolution of Allium species has been envisaged in relation to growth pattern (dormancy), habitat preference and evolutionary history of the subgenera and sections (Ohri et al. 1998). The authors suggested an overall lack of correlation between genome size and chromosome numbers, although continuity in variation was particularly evident in few species. The present review has showed a 2.25-fold (diploid) or 2.43-fold (tetraploid) difference in genome size in the species occurring in India, although the base number (x) is predominantly 8.
Nucleolus organizer regions or NORs are significant markers for chromosome identification. Among the species considered presently, NORs/ satellite-bearing chromosomes often show infra-specific or cultivar-specific differences particularly in A. cepa, A. sativum and A. tuberosum (Table 2).
In case of subgenus Allium, eight active NORs have been shown in A. ampeloprasum by C-banding, CMA3 + /DAPIbanding, AgNOR staining and FISH (Table 2). In A. sativum secondary constrictions were observed in two to even six chromosomes by C and N banding (Ghosh and Roy 1977;Roy 1978;Cortes et al. 1983), in addition to showing population specific differences (Roy 1978). NORs were also confirmed in four chromosomes by N banding (Cortes and Escalza 1986;Wajahatullah and Vahidy 1990). Recently, two pairs of chromosomes with secondary constrictions were reported in some Brazilian accessions of A. sativum of which one pair was suggested to contain intercalary NOR (Bacelar et al. 2021). CMA banding method was used to show the infraspecific heterochromatin variability of nucleolar (proximal) and non-nucleolar (distal and proximal) CMA bands in the Brazilian garlic accessions for their identification. This study remains to be done in case of Indian cultivars.
Allium cepa varieties with different ploidy levels (e.g. A. cepa var. viviparum, then supposed to be a hybrid between A. cepa and A. fistulosum Linnaeus, 1753) (Singh et al. 1967;Langer and Koul 1983;Puizina and Papea 1996) show variable number of satellited chromosomes (Bozzini 1964;Singh et al. 1967;Langer and Koul 1983;Puizina and Papea 1996). Many of the conventional staining and C-banding studies showed the presence of two satellite chromosomes in A. cepa (Ved Brat and Dhingra 1973;Fiskesjo 1975;Bhattacharyya 1976;Talukder and Sen 2000). Application of differential staining with sequence specific fluorochromes elucidated two NORs in A. cepa (Kim et al. 2002). However, reports claiming variable numbers of NORs (Battaglia 1957;Sato 1981;Puizina and Papea 1996) could not be ruled out. With the application of silver staining, 1-4 active NORs in the satellite region were observed (Sato 1981) while variable number of NORs (2-5) was elucidated by 45S-rDNA hybridization ( Table 2). The 45S rDNA sites are distally located and found to co-occur with telomeric tandem repeats (18S). The 5S rDNA loci are reported to range from 2-4 and do not co-occur with the 45S rDNA site.
One interesting feature is that satellites occur mostly in the short arms except for some cases in the subgenera Allium and Amerallium (Peruzzi et al. 2017). The same phenomenon has been found to exist in case of A. cepa, A. sativum and A. ampeloprasum (Kim et al. 2002;Maragheh et al. 2019;Bacelar et al. 2021). However, the localization of satellites in the species of Amerallium and other subgenera of Indian occurrence opens interesting scope of future study. The major difference between subgenera Allium and Cepa lie in the localization of the NORs rather than numbers of rDNA loci. The NORs are interstitial in Allium and distal in Cepa (Fig. 2) as confirmed by heterochromatic CMA banding, Ag-NOR staining as well as rDNA FISH (Kim et al. 2002;Fajkus et al. 2016;Maragheh et al. 2019;Bacelar et al. 2021).

Chromosome specialization in A. cepa
Telomeres and rDNA loci are the two especially variable features of A. cepa chromosomes. Many authors have previously argued that genomic rearrangements are responsible for positional variations of 45S rDNA loci in A. cepa (Ricroch et al. 1992;Do et al. 2001;Mancia et al. 2015). The rDNA sequences have been found to contain Copia-like retroelements in A. cernuum Roth, 1798 that were dispersed via homogenization mechanisms (Fajkus et al. 2016). The rDNA loci in A. cepa have been observed to co-occur with telomeric repeats although telomeres evolved independently of rDNA sequences (Fajkus et al. 2016).
The plant telomere was once thought to be composed of Arabidopsis Heynhold, 1842 prototype TTTAGGG repeats (Richards and Ausubel 1988). Exception to this was observed in Asparagales, where an 80 million years old mutation gave rise to human type (TTAGGG) repeat in the family Iridaceae (Adams et al. 2001;Weiss and Scherthan 2002;Sýkorová et al. 2003) and subfamily Allioideae (Sýkorová et al. 2006). The genus Allium is different from all other subfamilies of Amaryllidaceae and also other plant groups in terms of the unique telomere sequence. The telomeric sequence (TTATGGGCTCGG) n surfaced long back (Fuchs et al. 1995) and is neither Arabidopsis nor human type. The sequence has been found to be conserved in Allium, probing for monophyletic origin of this genus (Fajkus et al. 2016). The telomeres of land plants, including the unique ones like that of Amaryllids, have received less attention (Peska and Garcia 2020). For example, telomeric repeat in Arabidopsis thaliana (Linnaeus, 1753) Heynhold, 1842 is a Pol III transcribed lncRNA (Fajkus et al. 2019). Hence, the Allium and non-Allium taxa of Amaryllidaceae provide excellent scope for studying telomere evolution in eukaryotes.

Recent updates on cytogenetic relationships
A robust phylogenetic analysis supported by genome size and karyotype parameters was found to elucidate the evolution of Gilliesieae of Allioideae (Pellicer et al. 2017). The phylogenetic background of the genus Allium has paved way for refinement of classification, inter-species relationships and cytogeographic evolution (Friesen et al. 2006;Gurushidze et al. 2007Gurushidze et al. , 2008Fritsch et al. 2010;Li et al. 2010Li et al. , 2017Abugalieva et al. 2017;Herden et al. 2016;Huo et al. 2019;Costa et al. 2020). Global sampling of 207 species of Allium (Allieae) highlighted the ancestral number (x=8) and the reasons behind symmetric karyotype evolution (Peruzzi et al. 2017).
The utility of cytogenetic mapping remains unparallel to investigate synteny comparison between phylogenetically related species that has been employed to interpret chromosome evolution in Allium crop species from Russia (Khrustaleva et al. 2019). The presence of flavonoids and sulphur-containing compounds are responsible for the onion's characteristic flavour and the enzyme alliinase is part of the biosynthesis (Lancaster and Collin 1981). Recent techniques like ultra-sensitive tyramide-FISH (tyr-FISH) and SteamDrop protocol have facilitated the physical detection of the alliinase as well as chalcone synthase genes along with expressed sequence tag (EST) markers. The bulb alliinase gene was located on the long arm of chromosome 4 in A. cepa and A. schoenoprasum while the same gene was found in the short arm of chromosome 4 in the related (A. fistulosum, A. altaicum Pallas, 1773, A. oschaninii O. Fedtschenko, 1906, and A. pskemense B. Fedtschenko, 1905 and phylogenetically distant species (A. roylei and A. nutans Linnaeus, 1753) (Khrustaleva et al. 2019). Khrustaleva et al. (2019) proposed a pericentric inversion model for rearrangements in chromosome 4 in line with divergence of A. cepa and A. fistulosum, responsible for breaking collinearity of the genes controlling flavour and bulb colour. This particular report focussed on genomic kinship and genomic rearrangement among the closely related Allium species. Also, the practical benefit of molecular cytogenetic mapping becomes apparent in terms of suitably utilizing the genomic resources for onion breeding. These studies would also help to address genomic relationships among A. cepa, A. schoenoprasum and A. roylei, occurring in India.

Summary and future prospects
Considering the impact of cytogenetic investigation in Allium phylogeny at a global scale, it is unfortunate to notice the lack of attention in an Indian context in spite of species abundance. Although A. cepa has often been regarded as the common material for cytogenetic analysis and the popular 'Allium cepa test' (Pathiratne et al. 2015;Bonciu et al. 2018), systematic chromosome analysis is still missing in Indian A. cepa as well as other species. The present dataset and existing references are not exhaustive but furnish the prerequisite to search for further chromosomal landmarks (NORs, genome size etc) and complement future phylogenetic studies or cyto-geographical evolution of Allium, involving the unexplored wild and endemic species in the subcontinent. The crops, onion and garlic, have been admired from ancient time in global cuisines and Indian culinary practices (c.a. 5000 years ago) and continue to be tremendously important in agriculture and pharmaceutical industries (Rana et al. 2011;Nile and Park 2013). The cultivation of onions is challenged by a number of biotic threats which are the direct or indirect manifestation of the current climatic adversity (Le et al. 2021). Identification of wild relatives of the crop having high resistance is germane to address available genomic sources (Dempewolf et al. 2014), which is necessary for Allium crop species of India (Gedam et al. 2021). Interesting discoveries on the 'neodomesticate' western Himalaya taxon A. negianum (Pandey et al. 2021) along with other endemic less-known species (A. stracheyi, A. roylei, A. wallichii and A. przewaliskianum) are assets of Indian repository in line with global assemblages. The genomic attributes of Indian Allium germplasm as outlined in this review, could help strategic upgradation of cultivation practices.