Keywords

8.1 Introduction

Garlic, Allium sativum L., is an economically important diploid species of the genus Allium belonging to the Alliaceae family. This crop is considered to be one of the oldest horticultural crops. The earliest known documents indicated that garlic and onion (A. cepa) formed an essential part of the daily diet of several Egyptian working classes involved in the building of the pyramids, presumably to maintain and increase their strength, thereby enabling them to work harder (Moyers 1996; Mostafa et al. 2013; Abdelrahman et al. 2016, 2019). Besides this, garlic was exploited as an antiseptic to avoid gangrene during the First World War (Hedrick 1972). It is evident from the world production scenario of the last 30 years that world garlic production has increased from 5.78 to 28.16 million tons (Fig. 8.1), which is more than five times (FAOSTAT 2012; Wu et al. 2015). However, the cultivation area increased only twofold in the last 30 years (FAOSTAT 2012; Wu et al. 2015). Currently, more than 90% of garlic is produced by the Asian countries especially China and India. This crop is an asexually propagated plant with less multiplication rate, and hence having less genetic diversity. Owing to this, the development of genetically improved cultivars or creation of genetic variations through conventional breeding methods is cumbersome. Even though very few garlic genotypes flower at specific geographical regions, these genotypes exhibited sterility and location specificity which led to limit the development of new genetically improved cultivars. In the modern molecular breeding and genomic era, very less genomic studies have been conducted in garlic compared with other vegetable crops. The genome size of garlic is 15.9 GB which is marginally smaller than the onion genome (Arumuganathan and Earle 1991; Jones et al. 2004; Abdelrahman et al. 2017). Nevertheless, for the improvement of garlic, meristem culture, genetic transformation, and molecular breeding have been embarked on, but more precise research is required to develop smart climate traits in the garlic. Till date, most of the garlic cultivars released by public sector are susceptible to several viruses like onion yellow dwarf virus, leek yellow stripe virus, garlic common latent virus, shallot latent virus, and others (Sako et al. 1991; Conci et al. 1992).

Like other crops, garlic is also affected by various biotic and abiotic stresses. The changing climatic scenario may affect garlic production, but no documentary proof is there. According to Reddy et al. (2000), crop production is expected to decrease year to year even under controlled conditions due to climatic changes. Yield component of garlic is susceptible to environmental conditions (Panse et al. 2013). Thus, it needs the attention of the breeders to develop climate-smart garlic cultivars for better adaptation concerning climate changes by utilizing available genetic resources and modern biotechnological tools to ensure production sustainability. This chapter will focus on the use of breeding and genomic approaches for the climate-resilient sustenance of garlic.

Fig. 8.1
figure 1

(FAO http://www.fao.org)

World garlic production (million tons) and harvested area from 1987 to 2017 according to  Food and Agriculture Organization

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The reproduction of garlic is done entirely by using its underground parts called as clove or by inflorescence vegetative top sets which are usually sterile but have high diversity morphologically (Bradley et al. 1996; Wu et al. 2016). The asexual reproduction in garlic for many generations led to chromosomal aberrations in the form of aneuploidy and translocations/inversions which considerably limit the incidence of balanced gametes. Thus, the source of genetic variations in garlic remains the mutations (induced or random), somaclonal variations, and genetic transformation (Jones and Mann 1963; Novak 1990; Burba et al. 1993; Rubatzky and Yamaguchi 1997; Robinson 2007; Sandhu et al. 2015). The dearth of flowering and sexual reproduction in garlic limits the increase of variability that is useful for breeding for economically important traits, such as tolerance to biotic and abiotic stresses and higher yield (Kamenetsky 2007). Use of modern biotechnological tools such as molecular markers is limited and challenging due to the bigger size and complex nature of garlic genome in addition to vegetative nature of reproduction (Egea et al. 2017). Asexual reproduction could lead to narrow genomic diversity, subsequently in the clonal production since no meiosis is involved (Kamenetsky et al. 2015).

8.1.1 Climate-Smart Agronomic Trait Improvement

Development of new cultivars is dependent not only on clonal selection but also can be achieved through introduction from other garlic growing regions or environments (Jones and Mann 1963; Rubatzky and Yamaguchi 1997). Cultivation location has a significant effect on the characteristics of cultivar. Several reports revealed that changing climate could have an immense influence on flower stalk formation, taste; and a soft neck variety at a particular location might produce flower stalk when cultivated at another location (Kamenetsky et al. 2004; Kamenetsky 2007) (Table 8.1).

Table 8.1 Interaction effects of temperature and photoperiod treatments on garlic bulb characteristics in 2012
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8.1.2 Diversity Evaluation Study and Potential for Breeding Materials

Garlic is one of the most widely used cultivated Allium species and is grown in many countries at a wide range of latitudes. For centuries, this plant has been propagated clonally in various countries. It has, perhaps, caused a bottleneck effect for genetic variation (Ma et al. 2009). However, cultivated garlic or clonal lineages exhibit remarkably wide range of morphological variation in leaf number, bulb size, and structure (such as arrangement, number, and size of the cloves), floral scape length, and inflorescences (Pooler and Simon 1993a; Keller 2002; Kamenetsky et al. 2005a; Buso et al. 2008). The center of origin of garlic is opined as Central Asia region because fertile garlic was found in Kyrgyzstan, Kazakhstan, and Uzbekistan. Many researchers have studied morphological traits and molecular markers such as isozymes and DNA markers to evaluate the diversity of garlic (Pooler and Simon 1993a; Maaß and Klaas 1995; Etoh et al. 2001; Lampasona et al. 2003; Zhao et al. 2011; Jo et al. 2012; Hirata et al. 2016b). Etoh (1985) collected various garlic germplasms from worldwide including Central Asia and hypothesized that garlic evolved from fertility to sterility and from a complete bolting type to a non-bolting type through an incomplete bolting type. Moreover, Hirata et al. (2016a) demonstrated that garlic has acquired high environmental adaptability by changing the chemical composition in the bulb. Today, the evolution of garlic seems to be continuing. Other diversity studies have been carried out regarding the production level of chemicals in a set of garlic collections such as organosulfur compounds (Kamenetsky et al. 2005b; Hornickova et al. 2009; Ovesna et al. 2011; Hirata et al. 2016a) or phenolic compounds (Lu et al. 2011), which have benefits for human health. Kamenetsky et al. stated that garlic from the place of origin possesses superior traits, such as tolerance to disease and pests and better adaptation to biotic or abiotic stresses, than are seen in current cultivars. This research field could be even more important for garlic in the future.

8.1.3 Genetic Resources for Climate-Smart Genes

Albeit being utmost important bulb vegetable crops, substantial attention has not been paid to the Allium species for their germplasm collection and conservation since long which have led to the shortage of enough germplasm (Kamenetsky 2007). In case of garlic, not much efforts have been devoted to collect and preserve its crop wild relatives and landraces systematically which are potential source for further genetic improvement. (Rabinowitch and Zeltzer 1984; Kamenetsky 1993; Baitulin et al. 2000; Fritsch 2001; Keller and Senula 2001). The precious local gene pool is currently under severe threat of extinction, due to the rapid replacement of traditional landraces with modern cultivars (Kamenetsky et al. 2005b; Ovesna et al. 2011). Internationally, construction of an information structure for genetic resources of garlic should be imperative in near future.

8.1.4 Abiotic Stress Tolerance

8.1.4.1 Water Stress Tolerance

Apart from genetic potential of any crop, growth and yield also depend on prevailing environmental conditions during crop development which is highly stage specific. Among all environmental aspects, water stress in the form of excess or deficiency is a challenging factor for crop production especially for vegetable crop production since these crops are of short duration requiring sufficient moisture content for their growth and development. With the continuing changes in the climate, both excess and deficit of water are the major limiting factors for vegetable production. Garlic being a shallow rooted plant exhibits significant reduction in anthocyanin, chlorophylls (a, b, and total), carotenoids, growth parameters like fresh weight of plant and root, bulb yield, quality and elevated allicin content, and increase in ion leakage under drought conditions (Bideshki et al. 2013; Diriba-Shiferaw 2016). Heavy rainfall and waterlogging conditions are also damaging to the plant growth and bulb formation (Diriba-Shiferaw 2016). In Romania, Csiszár et al. (2007) observed activities of antioxidant enzyme in three Allium species under drought conditions and found that after 1 week there were manipulations in the activities of enzymes related to glutathione (GR, GST) and POD in shoots linked with relative water content of leaves. Furthermore, they revealed that inducible antioxidants played great role against drought in Allium ancient populations. This investigation could be immensely useful for the development of new climate-smart cultivars of garlic. In Egypt, Badran (2015) conducted comparative analysis by taking four commercial garlic varieties, namely, Egaseed 2, Balady, Egaseed 1, and Sids 40 under drought conditions. On the basis of drought tolerance index, superiority measure, yield injury %, and relative performance, Egaseed 1 was found highly tolerant while Balady was found the highly sensitive variety. Further, he used five inter-simple sequence repeat (ISSR) primers and observed 50.83% of mean polymorphism and only three primers (HB08, HB11, and 44B) showed unique bands. The ISSR marker analysis could be exploited to distinguish garlic cultivars across any breeding program.

8.1.4.2 Salinity Tolerance

Salinity is an important stress which affects the crop yield worldwide. Not much research work has been done on this area in garlic. With the changing climatic scenario, knowledge about the salt stress levels of garlic cultivars will be a viable option to work toward identification and development of salt-tolerant varieties. Silenzi et al. (1985) suggested that salinity (0.96–5.40 dS m−1) delays sprouting but has no effect on the final amount of sprouting. Mangal et al. (1990) estimated that in garlic, 50% yield reduction occurs at 5.60–7.80 dS m−1, depending upon the genotype. They also estimated that if soil salinity exceeds 1.70 dS m−1, the mean garlic yield declined by 1.68% per unit increase in soil salinity. Francois (1994) indicated a tolerance threshold of 3.9 dS m−1 and a yield decline of 14.3% for each unit increase in salinity above the threshold. Although salt tolerance threshold of garlic was slightly higher than most vegetable crops, yields drop rapidly once the soil salinity values exceed the threshold (Maas and Hoffman 1977).

8.1.4.3 Thermal Stress and Photoperiod

Thermal stress is one of the major abiotic stresses which restricts germination, plant growth, metabolism, and productivity worldwide. The processes starting from seed germination to senescence of plant include several biochemical reactions and enzyme activities that are highly sensitive to temperature. Response of crop plants to temperature depends upon the duration and the degree of the temperature. Temperature stress is now a foremost apprehension for the crop breeders for sustaining crop productivity.

The documented studies revealed that environmental factors such as temperature, photoperiod, etc. play immense role in Allium vegetative and reproductive growth and development (Takagi 1990; Pooler and Simon 1993b; Brewster 1994; Kamenetsky and Rabinowitch 2002; Etoh and Simon 2002; Kamenetsky et al. 2004). In garlic, the transition of the apical meristem from a vegetative to a reproductive state occurs during the active growing phase (Kamenetsky and Rabinowitch 2001). Low temperatures promote floral development, and long photoperiod is essential for floral scape elongation (Takagi 1990). Kamenetsky et al. (2004) observed that high temperature with long photoperiod enhanced the translocation of reserves to the cloves, and the degeneration of the developing inflorescence. It was further concluded that in bolting garlic genotypes, manipulation of the environment, both before and after planting, can regulate the development of flowers and regain fertility. Recently, Wu et al. (2016) concluded that higher endogenous phytohormone (especially GA) and MeJA levels are beneficial for garlic bolting and bulbing which varied with various treatment combinations of photoperiod and temperature. Son et al. (2012) studied response of garlic to cold stress and isolated 15 upregulated and 4 downregulated cold-responsive genes. These cold-responsive (CR) genes can be manipulated to overcome frost damage in garlic during its hibernation in the field conditions.

8.1.5 Biotic Stress Tolerance

8.1.5.1 Insect-Pest and Disease Resistance

Under changing climate scenario, there are many documented reports of damage caused by the abrupt spread of insect-pests and diseases in field and horticultural crops. It is a strong indication of climate change that is manipulating the intensity, distribution, and incidence of crop pests and diseases (Lamichhane et al. 2015). Garlic is prone to many diseases such as basal rot (Fusarium culmorum) (Mishra et al. 2014), white rot (Sclerotium cepivorum) (Zewde et al. 2007), downy mildew (Peronospora destructor) (Schwartz 2004), Botrytis rot (Botrytis porri) (Wu et al. 2012), Penicillium decay (Penicillium hirsutum) (Dugan 2007), and nematodes (Insunza and Valenzuela 1995). Most of the major garlic diseases are soil-borne, so proper site assessment and yearly rotations are crucial in maintaining a healthy garden of garlic.

8.1.5.2 White Rot

This disease is caused by fungus, Sclerotium cepivorum, which is one of the devastating global garlic diseases (Schwartz and Mohan 1995; Nabulsi et al. 2001). In Syria, Al-Safadi et al. (2000) started mutation breeding of garlic to get mutants resistant to white rot using gamma radiation and successfully achieved resistant mutants. Furthermore, Nabulsi et al. (2001) used random amplified polymorphic DNA (RAPD) analysis to elucidate molecular diversity among eight mutants of garlic through 13 random primers. Twelve primers showed polymorphism in amplification products and further highly resistant mutants were quite distant from the control with low correlation coefficients. The pattern of bands displayed by primer OPB-15 (GGAGGGTGTT) with highly resistant mutant could be exploited as genetic marker for further garlic breeding program

8.1.5.3 Blue Mold Disease

This garlic disease is caused by many Penicillium species and has been attributed to significant annual crop losses. Symptoms include stunted and chlorotic plants with withered leaves and reduced bulb size (Valdez et al. 2006). In Argentina, Cavagnaro et al. (2005a, b) evaluated garlic accessions against Penicillium hirsutum and found significant differences in the accessions. Accessions Castano and Morado were most resistant and it was further observed that there was a low correlation (r = 0.17) between allicin content and tolerance against this disease, indicating that allicin is not the main factor involved in the resistance against P. hirsutum.

8.1.5.4 Purple Blotch and Stemphylium Blight

The causal organism of purple blotch infection is Alternaria porri while Stemphylium blight is caused by Stemphylium vesicarium. Often both the diseases appear together and exhibit a complex of symptoms. There are less sources of available host plants that exhibit resistance against purple blotch naturally. Genetic engineering or transgenic could be an alternate toward purple blotch resistance (Eady et al. 2003) but consumer non-preference due to ethical/biosafety issues have not allowed the transgenics to grow on commercial level. In this situation, host resistance breeding could be the most efficient way to control purple blotch disease (Nanda et al. 2016). Rout et al. (2016) isolated and characterized a PR5 gene, designated as AsPR5, induced in response to Fusarium oxysporum f. sp. cepae (FOC) infection in garlic. Their results suggest that, besides antifungal activities, AsPR5 also plays a significant role in activating multiple defense pathways for enhancing stress resistance.

8.2 Genomic Approaches for Climate-Smart Garlic

Changing climate is a global phenomenon, and it is a continuous process since long with a long-term effect on agriculture productivity and food security. Thus, managing such changes is now demanding the attention of the agri-scientists and policymakers (Raza et al. 2019). Furthermore, it is predicted that future agriculture evolution will be designed by its response to climate change (Zilberman et al. 2018). For the adoption and development of climate-smart garlic cultivars, subsequent strategies are needed to combat environmental stresses.

8.2.1 Interspecific Hybridization

With the inception of agriculture, the genus Allium has played a significant role as vegetable and spice crop with a long history of cultivation of various Allium species either in cultivated or semi-cultivated form. This genus has immense genetic diversity in the form of bulb species, leaf shape, sexual or asexual reproduction, and tolerance to various biotic and abiotic stresses. To impart resistance or tolerance against various abiotic and biotic stresses, interspecific hybridization plays an important role to introgress alien genes into domesticated cultivated species. Among the Allium species, several attempts have been made for the improvement of onion (Allium cepa) (Keller et al. 1996; Peffley and Hou 2000) but in case of garlic not much efforts have been put due to its sterile nature and asexual mode of reproduction. There are very few scientific reports on interspecific hybridization between leek (Allium ampeloprasum L.) and garlic to introduce fertility and disease resistance into garlic (Sugimoto et al. 1991; Yanagino et al. 2003). The Allium species A. ampeloprasum is mainly used as a leafy vegetable, propagated through seeds and has the possibility to develop bulb in the summer season; on the other side, garlic is used as bulb spice crop and vegetatively propagated. Nevertheless, taxonomically garlic and leek are narrowly associated and both are grouped into the subgenus Allium. Porter and Jones (1932) documented that leek has some disease resistance genes while garlic has not. To create genetic variation and new Allium crops, interspecific hybridization between A. sativum and A. ampeloprasum could be the alternative option for the climate-smart cultivar development in garlic crop. In Japan, Yanagino et al. (2003) produced interspecific hybrids between leek and garlic successfully through ovary culture. They used some identified fertile clones of garlic as male parent through ovary culture. The hybridity of the interspecific cross was validated through morphological observations cytologically (2n = 3x = 24) and molecular analysis using RAPD markers. Further, their results revealed that the hybrid exhibited intermediate characteristics between the parental species such as growth, foliage, and bigger bulb size and garlic odor. Success and results of this interspecific hybrid indicated that this could have the potential to be a new crop having diverse genetic makeup and wide adaptability (Table 8.2).

Table 8.2 Foliage and bulb characteristics of leek, garlic and the interspecific hybrid ± indicates standard deviation
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8.3 Biotechnological Approaches

Being a vegetatively propagated crop, selection of diverse clones is one of the conventional approaches for garlic improvement. These diverse clones are developed through natural mutations or occasional production of sexual progenies (Etoh and Simon 2002; Havey and Ahn 2016). Landraces and local farmer developed cultivars are the significant source of various climate-smart genes for the various stresses. With the advent of biotechnological approaches, improvement of garlic can be initiated by utilizing a plethora of non-conventional approaches.

8.3.1 Genetics and Genomics Strategies In Vitro Culture Based Methods

8.3.1.1 Genetic Transformation

Foreign gene transfer to plants is becoming a routine technique for many important crop species. The presence of efficient methods of genetic transformation—Agrobacterium-mediated transformation or direct gene transfer by particle bombardment (Songstad et al. 1995)—is of considerable importance for the improvement of modern crops. Agrobacterium tumefaciens is routinely utilized in gene transfer in case of dicotyledonous plants. Monocotyledonous plants were thought to be recalcitrant to this technology as they were outside the host range of the bacterium. However, recently transgenic plants have been obtained in some monocotyledonous spp. using specific Agrobacterium strains (Arencibia et al. 1998; Liu et al. 1998; Khanna and Raina 1999). Therefore, the monocotyledonous nature of species no longer prevents the application of Agrobacterium-mediated techniques for the transfer of genes to these species as soon as the methodological parameters are optimized (Hiei et al. 1997).

Genetic transformation in garlic is of utmost importance because of its sexual sterility. Due to difficulties of inducing flowering, breeding programs have been limited to clonal selection and production of virus-free stocks via meristem culture. Although, tissue culture is a useful technique for producing virus-free garlic seedlings, the propagation rate of virus-free plantlets is very low. And the process is laborious and time-consuming. Since no other methods of gene transfer exist, the genetic transformation may be a promising tool. Transformation also holds the key for the improvement of garlic toward biotic and abiotic stresses. It is possible to use the genetic transformation for the production of transgenic garlic with the desired characteristics. Unfortunately, both onion and garlic have proved to be recalcitrant to genetic transformation and plant regeneration (Eady et al. 1996).

Introduction of alien DNA into plant cells can be achieved by using the bacterium A. tumefaciens (indirect method) or biolistic method (direct method) as a vehicle. In biolistic approaches, Barandiaran et al. (1998) were first to attempt garlic transformation using biolistic approach to transfer and detect the transient expression of uidA gene into different garlic tissues, including regenerable calli using nuclease inhibitor aurintricarboxylic acid. Later, Ferrer et al. (2000) introduced, by biolistic method, reporter gene uidA and selection gene bar in leaf tissue, basal plate disc, and embryogenic calli and reported maximum expression of uidA gene in calli and leaves. Sawahel (2002) showed that biolistic transformation could lead to the expression and stable integration of a DNA fragment into immature cloves, whereas Park et al. (2002) established an effective biolistic transformation procedure for obtaining chlorsulfuron-resistant transgenic plants by incorporating ALS gene coding for acetolactate synthase. Later Robledo-Paz et al. (2004) were able to introduce DNA into embryogenic garlic callus and produce stably transformed garlic plants.

Use of Agrobacterium-mediated transformation was initiated by Kondo et al. (2000) who were able to develop a stable transformation system of garlic using highly regenerative calli. Zheng et al. (2004) developed a reliable transformation system to produce garlic plants containing Bt resistance genes which conferred resistance to beet armyworm (Spodoptera exigua). Khar et al. (2005) studied the transitory expression of the reporter gene gusA in two garlic cultivars after infecting them with A. tumefaciens, whereas Eady et al. (2005) recovered transgenic garlic plants from immature embryos using A. tumefaciens containing the vector pBIN mgfp-ER which includes the modified gfp reporter gene and the nptII selectable marker gene. Later, Kenel et al. (2010) developed a method for garlic transformation from immature leaves containing the mgfp-ER reporter gene and hpt selectable gene. Regenerated transgenic plants survived in the glasshouse and matured into healthy plants. Ahn et al. (2013) were successful in increasing the stable transformation efficiency (up to 10.6%) by using a two-step selection involving hygromycin resistance and green fluorescent protein (GFP) expression. Transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene, and they demonstrated that transgenic plants conferred herbicide resistance, while nontransgenic plants and weeds died. Quality and yield of garlic are diminished due to white rot disease (Sclerotium cepivorum Berk). Fortiz et al. (2013) developed a transformation protocol to introduce tobacco chitinase and glucanase genes into garlic embryogenic calli using A. tumefaciens and were able to develop transformed plants which were not completely resistant but exhibited a delay in fungal infection.

8.3.1.2 Meristem Tip Culture

Vegetative propagation leads to accumulation of viruses, and it is well established that garlic is susceptible to accumulation of a complex of viruses, notably members of the genera Potyvirus, Carlavirus, Allexivirus, and Potexvirus (King et al. 2012). Losses in yield and deterioration in quality are the well-established problems associated with virus infections. Control of these viruses is problematic and involves the production of virus-free plants by meristem tip culture and subsequent multiplication of plants under aphid-free conditions. Production of virus-free garlic plants has been attained through shoot tip culture (Peña-Iglesias and Ayuso 1982), scape tip culture (Ma et al. 1994), small inflorescence bulbils culture (Ebi et al. 2000), “stem disc dome culture” (Ayabe and Sumi 2001), and meristem tip culture (Wei and We 1992). Attempts to obtain virus-free garlic through thermotherapy (Conci and Nome 1991; Ucman et al. 1998), a combination of meristem tip culture and thermotherapy (Robert et al. 1998) and use of chemotherapy (Ramírez-Malagón et al. 2006) have been reported. It has also been concluded that virus-free garlic yields better and has better quality than the virus-infected plants (Ramírez-Malagón et al. 2006). New methods of development of virus-free garlic through cryotherapy of shoot tips (Vieira et al. 2015) and root tip culture (Haque and Hattori 2017) have been reported. Although many papers on development of virus-free garlic through various methods have been documented, field performance and a protocol for production of these virus-free garlic plants for commercial production are still lacking.

8.3.1.3 Somaclonal Variations

Since all commercially grown garlic cultivars are sterile, they can only be vegetatively propagated; this habit of garlic has restricted the development of new, improved cultivars through the utilization of plant breeding approaches. To create genetic variation and new forms of the crop, somaclonal variants produced during long-term tissue culture could be a potential option for garlic (Al-Zahim et al. 1999). In vitro regeneration of plants through callus culture has been documented since long (Kehr and Schäffer 1976; Abo El-Nil 1977; Xue et al. 1991) but achievement in this aspect is limited. Novák (1980) observed such variations phenotypically and cytologically. Furthermore, higher bulb weight was noticed in some somaclones compared to the parental ones (Vidal et al. 1993).

8.3.2 Molecular Breeding

8.3.2.1 Molecular Markers

Molecular markers are being used extensively for determination of genetic diversity because of their neutral nature, reproducibility of results across labs, and no environmental effect on their expression. Genetic diversity of garlic has been assessed by isozymes (Pooler and Simon 1993a), random amplified polymorphic DNA (RAPD) markers (Ipek et al. 2003; Khar et al. 2008; Maaß and Klaas 1995), inter-simple sequence repeat (ISSR) markers (Jabbes et al. 2011), combination of RAPD and ISSR markers (Shaaf et al. 2014), sequence-related amplified polymorphism (SRAP) markers (Chen et al. 2013), amplified fragment length polymorphism (AFLP) markers (Volk et al. 2004; García-Lampasona et al. 2012), and locus-specific markers (Ipek et al. 2008). Estimation of garlic diversity using microsatellite markers was first reported by Ma et al. (2009) wherein they were able to develop a simple sequence repeat (SSR) enriched library and finally reported eight SSRs for diversity estimation. The same eight SSRs were used by Zhao et al. (2011) for molecular genetic diversity studies, population structure analysis, and core collection estimation followed by Jo et al. (2012) who classified genetic variation in 120 accessions from five different countries using the seven primers out of the same eight SSRs reported earlier. Cunha et al. (2012) reported a new set of 16 SSR markers using (CT)8- and (GT)8-enriched library and found 10 markers to be polymorphic, whereas Chen et al. (2014) used the same set of markers and found eight to be polymorphic. Khar (2012) used 99 SSRs and reported 18 polymorphic SSR for estimation of genetic diversity in garlic. Recently, Cunha et al. (2014) were able to assess the genetic diversity and population structure of Brazilian accessions using 17 SSR markers developed by Ma et al. (2009) and Cunha et al. (2012) (Fig. 8.2).

Fig. 8.2
figure 2

Source Cunha et al. (2014)

UPGMA dendrogram based on Rogers-W genetic distance for 73 garlic accessions with a cophenetic correlation coefficient of 0.92. The major clusters are highlighted with the corresponding color of subgroups identified by the model-based clustering technique: SA and SB (K = 2); and S1 (green), S2 (yellow), S3 (blue), and S4 (red) (K = 4).

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8.3.2.2 Genetic Linkage Maps

Genetic linkage maps are powerful tools for localization of genes, understanding the genetic basis of complex traits, marker-assisted breeding, and map-based cloning of important genes. Development of a genetic linkage map will enhance garlic improvement by allowing marker-assisted selection (MAS) and identification of genes that control economically important traits. The first genetic map of garlic (Zewdie et al. 2005) was developed using 37 markers forming nine linkage groups, and a male fertility locus was placed on the map. This was followed by the development of the first low-density genetic map (Fig. 8.3) based on AFLP markers (Ipek et al. 2005).

Fig. 8.3
figure 3

Source Ipek et al. (2005)

Linkage map in the genetic map of Allium sativum, based on the MP1 family. Map distance is in centiMorgans. Numbers in boldface are common markers segregating in both MP1 and MP2.

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8.3.3 Genomics-Aided Breeding

For crop improvement, omics approaches offer potential resources to study biological functions of any genetic information (Stinchcombe and Hoekstra 2008) which also help to unravel meaningful biological regulatory networks (Keurentjes et al. 2008). Almost all important crop improvement breeding programs include genomics approaches combined with conventional breeding to shorten the time and to evaluate the elite germplasm (Bevan and Waugh 2007). Such modern biotechnological tools assist considerably to develop climate-smart crops with higher yield potential under climate change scenario (Roy et al. 2011).

8.3.3.1 Genomics-Based Markers

It could be said that molecular plant breeding is a critical and efficient approach to augmenting crop production under several abiotic and biotic stresses (Wani et al. 2018). Garlic is a diploid (2n = 2x = 16) Allium species having 16.2 GB genome per 1C nucleus size (Bennett and Leitch 2012; Havey and Ahn 2016). With such a large genome size, sequencing of expressed regions is an effective method to detect various genes and escaping of repetitive DNA issues (Newman et al.1994; Kuhl et al. 2004; Gore et al. 2009; Havey and Ahn 2016). Genetic diversity and polymorphism in garlic have been studied through various molecular markers such as RAPD, SSR, AFLP, and insertions–deletions (Maaß and Klaas 1995; Ipek et al. 2005; Ovesná et al. 2014; DaCunha et al. 2014; Ipek et al. 2015; Wang et al. 2016). However, genetic diversity studies in garlic are still challenging (Kim et al. 2009). Till 2009, there was no available tool or integrated database for garlic which could give the information about annotations and expressed sequence tags (ESTs). Later on, Kim et al. (2009) developed the GarlicESTdb using a pipeline system which offers access to all garlic EST resources and comprehensive database containing information about the cluster, annotation, protein domain, pathway, tandem repeat, single nucleotide polymorphism (SNP), etc. To carry forward this genomics research in garlic, polymorphism among garlic germplasm has been revealed through transcriptome sequencing in expressed regions which is helpful for diversity analysis and genetic map development (Kuhl et al. 2004; Martin et al. 2005; Gore et al. 2009; Duangjit et al. 2013; Ipek et al. 2015). Use of molecular markers like AFLPs, SSRs, and SNPs have been identified (Ipek et al. 2005, 2015; Ma et al. 2009; Zewdie et al. 2005; Zhao et al. 2011). Until now, similar appearance and phenotypic plasticity of garlic varieties hinder their morphological classification. Molecular studies are challenging, due to the large and expected complex genome of this species, with asexual reproduction. Classical molecular markers, like isozymes, RAPD, SSR, or AFLP, are not convenient to generate germplasm core collections for this species. The recent emergence of high-throughput genotyping-by-sequencing (GBS) approaches, DArTseq technology, allows to overcome such limitations to characterize and protect genetic diversity. Therefore, such technology can be used in garlic to: (i) assess genetic diversity and structure of a large garlic germplasm bank; (ii) create a core collection; (iii) relate genotype to agronomical features; and (iv) describe a cost-effective method to manage genetic diversity in garlic germplasm banks.

8.3.3.2 Genome-Wide Association Studies (GWAS) for Stress Tolerance

To understand the full set of genetic variants in crop cultivars and to identify allelic variants linked with any specific trait, genome-wide association studies (GWAS) is one of the powerful genomic technologies (Manolio 2010). GWAS has been conducted to reveal the genetic background responsible for the resistance at the genetic level under climate change (Mousavi-Derazmahalleh et al. 2018). In plants, GWAS has widespread applications related to biotic and abiotic stresses (Lafarge et al. 2017; Thoen et al. 2017). The first work of high-throughput garlic genotyping was done by Egea et al. (2017). They reduced significantly  the garlic germplasm bank size, identifying redundant accessions and thus generating a unique (non-redundant) core collection, with the consequent reduction in space and maintenance expenses. They further suggested that DArTseq analysis is a cheaper method to perform genotyping-by-sequencing and genetic diversity analyses of garlic having gigantic, complex, and without a reference genome, and gave reliable results, according to genotype and their geographical origin. With this study, it would be easy for the breeders to select genotype from characterized core collection for better adaptability against various biotic and abiotic stresses under changing climate and global warming.

8.3.3.3 Next-Generation Sequencing

Recently, transcriptome profile of garlic buds, using Illumina sequencing technology, has been achieved. A total of 127,933 unigenes have been generated, annotated functionally, and analyzed about their gene ontology and metabolic pathways. Genes encoding enzymes involved in sulfur assimilation pathway were discovered which will provide the foundation for the research on gene expression, genomics, and functional genomics in Allium sativum and closely related species (Sun et al. 2012). Sun et al. (2013) studied the transcriptional profiling between the dormant and sprouting garlic shoot apex and observed that the expression of 22,836 unigenes was increased by more than two fold in sprouting garlic shoot apex as compared with dormant shoot apex. Based on the findings, they postulated that differential expression of genes, such as ENHYDROUS, DAG1, DAM, DTH8, play a critical role in shoot apex sprouting and may serve as candidate genes for sprouting regulation in Allium species. Studies on the molecular characterization of nuclear binding site encoding resistance genes and induction analysis of a putative candidate gene linked to Fusarium basal rot resistance in Allium sativum (Rout et al. 2014) led to identification of 28 AsRGA (A. sativum resistance gene analogs) sequences from a resistant garlic genotype CBT-As153 that can form the basis towards Fusarium basal rot resistance. The identified AsRGAs can act as a valuable resource toward the development of resistance gene analog-based molecular markers for genetic mapping in garlic that will pave the way toward cloning of novel R-gene against F. oxysporum f. sp. cepae.

8.4 Future Perspectives

Garlic is an essential horticultural crop, because of its nutritional and medicinal properties. In addition to fresh garlic consumption, the production of processed and dried garlic products for use as dietary health-food supplements and food processing is an important industry. Garlic breeding has been constrained by the absence of adequate methods to generate variation in the existing germplasm due to sexual sterility of garlic. In addition, flower development and fertility of garlic plants are strongly regulated by environmental conditions, and therefore the garlic seed production in various climatic zones is challenging and needs further studies. Currently, most progress in achieving genetic improvement in garlic has been through clonal selection, but standardization of biotechnological methods to induce variations still needed further efforts. Restoring fertility in garlic provides new genetic possibilities for breeding purposes. Garlic breeding improvement through using modern techniques to increase variation like mutagenesis, sexual hybridization, genetic transformation, and the current developments in florogenesis can be successfully implemented, which might help to increase the genetic variability, opening new avenues for the breeding of this important crop. For instance, biolistic and Agrobacterium gene transfer systems were improved in the last years and the first transgenic garlic lines have already been produced. Moreover, garlic embryogenic cell suspensions have been described. Although large steps forward have been made at the fundamental level (e.g., garlic genome organization, genetic transformation, florogenesis, and embryogenic cell suspension development), it is also clear that there are still large gaps present in our knowledge. Few accessions have been developed to carry out interspecific gene introgression, and genetic linkage maps have begun to be developed with few significant loci placed on approximate locations of the genome. In addition, molecular markers like RAPD, SSR have been developed for diversity studies. All these developments in garlic breeding system innovation show that there are good opportunities for the production of improved garlic cultivars. If results of these researches are systematically interpreted and applied in garlic breeding, production, and storage, garlic can become highly remunerative.