Diversity evaluation based on morphological, physiological and isozyme variation in genetic resources of garlic (Allium sativum L.) collected worldwide

The aim of this study was to obtain primary information about the global diversity of garlic (Allium sativum L.) by evaluating morphological, physiological and isozyme variation. A total of 107 garlic accessions collected worldwide were grown in Yamaguchi, Japan. Five morphological traits (bulb weight, bulb diameter, number of cloves per bulb, number of bulbils and scape length) and one physiological trait (bolting period) of the collected garlic showed wide variation. Meanwhile, a total of 140 garlic accessions, including the 107 mentioned above, were characterized by leucine aminopeptidase (LAP) and phosphoglucoisomerase (PGI) isozyme analyses; they clearly showed polymorphisms in putative isozyme loci (Lap-1, Lap-2 and Pgi-1). Allelic frequencies were estimated in each group of accessions categorized by their geographical origin, and the observed (Ho) and expected (He) heterozygosities were calculated. The allelic frequencies differed between groups. A principal component analysis based on morpho-physiological data indicated a grouping of the garlic accessions into Central Asian and Northern Mediterranean groups as well as others. We discuss the roles of artificial and natural selection that may have caused differentiation in these traits, on the assumption that ancestral domesticated garlic populations have adapted in various regions using standing variation or mutations that accumulated during expansion, and have evolved along with human-preferred traits over a long history of cultivation.


INTRODUCTION
Edible Allium species are major crops cultivated worldwide.Next to the onion, garlic (Allium sativum L.) is the most important cultivated Allium species.Garlic is grown in many countries at a wide range of latitudes; China, India, the Republic of Korea and Egypt are its principal producers (FAO, 2012).Today, large quantities of garlic bulbs are consumed worldwide as a functional food or for pharmaceutical purposes (Kik et al., 2001).Garlic is completely sterile; therefore, it has a long history of vegetative propagation.Garlic was certainly cultivated in ancient Egypt; its cultivation history dates back to approximately 3000 BC, and it has been propagated, probably by bulbs or bulbils, since then (Etoh, 1985;Figliuolo et al., 2001).Its center of origin is presumed to be the northwestern side of the Tien Shan Mountains, Central Asia, because fertile garlic, which is a primitive form, has been found in this area (Etoh and Simon, 2002).It is uncertain whether garlic became sterile after the beginning of its cultivation, but sterility in garlic is undoubtedly a consequence or a product of the species' evolution, including domestication (Etoh, 1985).
For centuries, this plant has been propagated clonally, which has, perhaps, resulted in a bottleneck effect for genetic variation (Ma et al., 2009).However, cultivated garlic or clonal lineages exhibit remarkably wide morphological variation, such as in leaf number, bulb size and structure (such as arrangement, number and size of the cloves), floral scape length and inflorescences (Pooler and Simon, 1993;Keller, 2002;Kamenetsky et al., 2005;Buso et al., 2008).Garlic has specific adaptations to different agroclimatic regions (Figliuolo et al., 2001;Mario et al., 2008).It is likely that in ancient times, ancestral garlic populations would have had some standing variation.When they dispersed widely from their own growing fields to different agroclimatic regions, only adaptable clones survived.Alternatively, after the start of domestication, and distinct from the variation resulting from sexual reproduction, the variation in domesticated garlic seen today may be attributable to mutations that have accumulated through the history of cultivation (Shaaf et al., 2014).Thus, it is possible that garlic has specifically adapted and spread to various agroclimatic environments through the process of domestication.As a result, garlic varied morphologically in various regions.
Characterization of garlic germplasm has been based largely on phenotypic characteristics, but these can vary under different agroclimatic conditions (Jo et al., 2012).This situation complicates the characterization of garlic clones (Mario et al., 2008).Many researchers have studied morphological traits and molecular markers such as isozymes and DNA to evaluate the diversity of garlic (Pooler and Simon, 1993;Maass and Klaas, 1995;Etoh et al., 2001;Zhao et al., 2011;Jo et al., 2012).Isozyme analysis has long been used to evaluate genetic diversity in animals, fungi and higher plants (Micales and Bonde, 1995).Lallemand et al. (1997) stated that the isozyme types of Central Asian garlic clones were different from those of the Western world, and Asian clones are distinguished from those in other parts of the world in terms of isozyme types.Maass and Klaas (1995) categorized garlic species into four subspecies based on morphological and isozyme variation: the longicuspis group, including most garlic clones from Central Asia; the subtropical group, which developed in the climatic conditions of Southeast and East Asia; the ophioscorodon group, which is derived from Eastern Europe; and the sativum group, which is from the Mediterranean.However, reports evaluating morphological characteristics and isozyme polymorphisms of garlic, including these subspecies, have been limited.
In this study, we investigate morphological and physiological traits and two isozymes of garlic accessions collected worldwide in order to evaluate garlic's diversity.

MATERIALS AND METHODS
Plant materials Bulbs of 107 garlic accessions collected from around the world since the 1970s have been managed at Yamaguchi University, Japan (34.14°N, 131.47°E).In addition, bulbs of 33 garlic accessions were managed at Saga University, Japan (33.24°N, 130.29°E) until 2012, when management of both collections was taken over by Yamaguchi University.The following eight groups were categorized based on their origins: 31 accessions from Honshu, Japan (Group A), 18 accessions from islands in Western Japan (Group B), 10 accessions from China, 16 accessions from Southeast Asia, 29 accessions from Central Asia, 13 accessions from the Northern Mediterranean, 14 accessions from the Southeastern Mediterranean and six accessions from the New World.Thus, a total of 140 accessions (including three of unknown origin) were used in this study (Table 1).These bulbs were obtained from local markets or national institutions in each country.Detailed information regarding some accessions was reported by Etoh (1985Etoh ( , 1986)), Hong et al. (2000) and Etoh et al. (2001).These accessions have no clear information as to which subspecies they belong to; however, they include representatives of all four subspecies.The bulbs were stored at 4 °C in dark conditions in the summer.
The 140 garlic accessions were planted in an experimental field at Yamaguchi University at the end of October, 2011 and 2012.A compound fertilizer was applied before planting.Total amounts of three major nutrients in a basal dressing were 100 N (as ammonium sulfate), 120 P (as calcium superphosphate), and 100 K (as potassium chloride) kg/ha.During the growing season, eight cloves of uniform size per accession were randomly selected from the bulbs and were grown in rows 10 cm apart and in columns 20 cm apart.

Morphological and physiological observations
In 2011, morphological and physiological traits of the 107 Yamaguchi accessions were examined for eight plants of each accession.Bolting types of the accessions were identified as follows: complete bolting-plant always bolts, its scape elongates high above the ground, and the inflorescence comes out of the leaf sheath; incomplete bolting-plant produces a thin, short scape and bears only a few bulbils in its leaf sheath; non-bolting-plant neither bolts nor develops flower buds.Identification was carried out in accordance with the guidelines of Kamenetsky et al. (2005), with minor modifications.The bolting period was recorded as a physiological trait by counting the days until the accession's spathes differentiated.In incomplete-bolting accessions, the number of days to the development of bulbils in the leaf sheath was recorded.Just before harvest season, accessions that did not develop scapes or inflorescences could be identified as the non-bolting type.These accessions were removed from the survey of the bolting period.All accessions were harvested and cured (completely dried of umbels, scapes, leaves, leaf sheaths and roots) in a vented greenhouse.About one month later, the scape length, number of bulbils, and bulb-related traits (bulb weight, bulb diameter and number of cloves) for each accession were recorded with mature plants.The number of bulbils per accession was determined by counting all bulbils removed from umbels and dividing by the number of umbels examined.approximately 5 cm long from base to tip, were collected from all accessions to be analyzed for enzymes.Enzyme extraction, electrophoresis, and staining were carried out following the method of Shigyo et al. (1995Shigyo et al. ( , 1996)).Minor modifications were applied to the extraction buffer  (Wendel, 1983) for enzyme extraction.The polyacrylamide gel was composed of Tris-HCl running gel (7% acrylamide, pH 8.9) and Tris-HCl stacking gel (4.2% acrylamide, pH 8.9).The crude enzymes extracted from the leaves were loaded into the wells of the gel at volumes of 20 μl and 10 μl each for LAP and PGI samples, respectively.The number of alleles and allelic frequencies at each isozyme gene were evaluated for each group of accessions, defined by their origin.
Data analysis All obtained morphological (scape length, number of bulbils per accession and bulb-related traits) and physiological (bolting period) data from the groups' accessions by their origins were subjected to various statistical tests.A principal component analysis (PCA) was completed using SPSS 22.0 statistical software (SPSS Japan, Tokyo, Japan) to clarify the relationship between the morpho-physiological data and the collection site.In each group, we examined whether the allele frequencies were the same as the average of frequencies across all samples using a chi-square test.Genetic diversity can be measured by heterozygosity (Nei, 1973).Thus, to evaluate genetic diversity at the isozyme loci, the observed (H o ) and expected (H e ) heterozygosity in each group were calculated.In addition, deviations from Hardy-Weinberg equilibrium (HWE) in each locus were evaluated by a chi-square test.G st values were calculated using GenAlEx ver.6.5 software for Windows (Peakall and Smouse, 2012) to show the level of geographical differentiation.MANOVA (multivariate analysis of variance) was carried out on the obtained data to clarify the relationships between morpho-physiological characteristics, isozyme genotypes and the group of origin of the accessions.

RESULTS
Morphological and physiological variation Garlic accessions showed various types of morphological variation when grown in the experimental field at Yamaguchi University.The number of cloves per plant varied among the accessions from two to 44.Almost all garlic accessions developed several cloves, ranging from two to 20 among the groups (Fig. 1A), except for the Southeastern Mediterranean group.Accessions from this group developed many cloves, ranging from seven to 44 (Fig. 1B).All accessions of the Central Asia group developed complete bolting.These accessions produced many more small bulbils (less than 5 mm in size) with florets than did those from other groups (Fig. 1, C and D).Incomplete bolting was also observed in some accessions (Fig. 1E).These accessions mainly belonged to the Southeast Asia and Southeastern Mediterranean groups.Morphological and physiological traits were compared among groups of accessions classified by their origins (Table 2).The scape length and number of bulbils per plant in the Central Asia group were significantly greater than those in Japan Groups A and B and the Southeast Asia group; however, the China group had traits similar to those of Central Asia in scape length and the number of bulbils.In bulb weight, the Central Asia group, Japan Group A and the Southeast Asia group showed similar values.Southeastern Mediterranean accessions produced significantly heavier bulbs (25.0 ± 3.7 g) than did those of other groups.While there was no significant difference in clove weight among the regional groups, the number of cloves in Southeastern Mediterranean accessions was greater than that in accessions of the other groups.Bolting periods in Southeast Asia accessions were significantly shorter (179.2 ± 2.8 days) than those in other groups.Regarding bolting types, all Central Asia and China accessions bolted completely, while accessions from the other groups bolted incompletely or did not bolt.In particular, Southeastern Mediterranean and Southeast Asia accessions had high ratios of these bolting types.
Fig. 2. Isozyme zymograms and genotypes of the Lap-1, Lap-2 and Pgi-1 enzyme systems.Numbers in parentheses represent the number of accessions.In Pgi, two cases were presumed: case (1), in which there is single gene in garlic, and case (2), in which there are two monomorphic genes and they form heterodimers.

Lap-1 Lap-2 Pgi
as rice and tomato, a heterodimer of the products of two isozyme genes is formed (Weeden et al., 1979;Tanksley et al., 1981;Guri et al., 1988).Shigyo et al. (1996) reported Pgi isozyme banding patterns using various Allium species and hybrid plants; the banding pattern in this study was very similar to their hybrid banding pattern.Therefore, in this paper, we tentatively regarded as case (1) the Pgi bands shown in Pgi-1 genotype 'bb' or 'ab.'The triple-band pattern indicates two homodimer bands and one heterodimer, as PGI is a dimeric enzyme.The homodimer 'aa' in Pgi-1 was not observed in any accession.In addition, accessions with heterodimers were observed only in Southeast and East Asia accessions.These three isozyme loci provided variable band patterns, namely, three Lap-1 patterns, six Lap-2 patterns and two Pgi-1 patterns.From these combinations, there are, theoretically, 36 isozyme genotypes possible; however, only 15 genotypes were observed in this study.The numbers of genotypes found in each regional group are shown in Table 3. Accessions possessing an 'aa' band in Lap-1 were seen in the Central Asia and Northern Mediterranean groups, and they did not have an 'ab' band in Pgi-1.

Allelic frequencies and heterozygosity
Allelic frequencies showed different tendencies among groups (Fig. 3).Lap-1a appeared mainly in the Central Asia (58.6%) and Northern Mediterranean (65.4%) groups, while in the China, Japan and Southeast Asia groups its frequency was low.Japan Group A showed an especially low fre-quency (9.7%), although it is located at the same latitude as the Central Asia group (approximately 40°N).Thus, high Lap-1a frequency was specific to the Central Asia and Northern Mediterranean groups.In other groups, Lap-1b frequency was high (more than 40%).Regarding Lap-2, four alleles (-2a, -2b, -2c and -2d) were observed in the Central Asia and Northern Mediterranean groups, while one or two alleles (-2b and -2c) were observed in other groups.Groups from Japan, China, Southeast Asia (the eastern side of Central Asia) and the New World showed mainly -2b.Moreover, the frequency of Lap-2c in the Central Asia and Northern and Southeastern Mediterranean groups was high, and its frequency tended to decrease toward the east.Allele -2a was seen in the Northern Mediterranean and Central Asia groups, and allele -2d was shared across groups from the Northern Mediterranean to China.In Pgi-1, almost all groups showed -1b.Allele -1a was only seen in the China, Japan, and (especially) Southeast Asia groups.In each group, the allelic frequencies of isozyme loci were compared to the average of the whole by chi-square testing to detect their deviation from the average of the whole.There were no significant deviations in the frequencies of Lap-1 and -2 from the average as a whole.However, in Pgi-1, there were significant deviations in the Central Asia, New World, and Northern and Southeastern Mediterranean groups (Supplementary Table S1).
The observed (H o ) and expected (H e ) heterozygosity in each locus ranged from 0.111 to 0.786 and 0.105 to 0.585 Group A; collected from Honshu, Group B; collected from islands in Western Japan.Diversity evaluation of garlic collected worldwide in Lap-1, from 0.000 to 0.615 and 0.000 to 0.583 in Lap-2, and from 0.000 to 0.625 and 0.000 to 0.430 in Pgi-1, respectively (Table 4).The means over loci in each group ranged from 0.111 to 0.436, and the average was 0.234.In Lap-1, the Central Asia and Northern and Southeastern Mediterranean groups had higher levels of heterozygosity than did other groups.The Lap-2 tendency was similar to that of Lap-1, but the heterozygosity was zero in the Southeastern Mediterranean group.In Pgi-1, the South-east Asia group showed a high level of heterozygosity.This demonstrated that the Central Asia, Southeast Asia, and Northern and Southeastern Mediterranean groups had large amounts of genetic diversity.According to chisquare testing, some loci deviated significantly (P < 0.05) from HWE (Lap-1 and Lap-2 loci in the Southeastern Mediterranean group, and a Lap-1 locus in the Southeast Asia group and Japan Group B).The G st values for Lap-1, Lap-2 and Pgi-1 were 0.222, 0.336 and 0.133, respec-

Lap-1 Lap-2
Southeastern Mediterranean (n=14) tively.The overall G st value was 0.259.This score indicates that about 26% of the total genetic variation was derived from genetic differentiation.The obtained data for morphological and physiological traits of garlic were subjected to PCA to examine their relationship to geographical origin.Three PCs were obtained, accounting for 84.2% of the total variance.PC1 represented 43.1% and was strongly related to bulb weight, bulb diameter, number of cloves and clove weight; PC2 represented 27.5% and was positively related to scape length, number of bulbils and bolting period; PC3 represented 13.6% and was strongly related to the number of cloves (Supplementary Table S2).Moreover, scatter plots were made from the obtained scores, and all plots were given isozyme groups as shown in Table 3 to evaluate their association with several garlic traits.A 2D scatter plot of the PCA scores for PC1 and PC2 principal components is shown in Fig. 4. Of the 107 accessions, only 66 are plotted in Fig. 4 because no data were available for some accessions (especially accessions from the Southeastern Mediterranean and Southeast Asia groups, which possess incomplete bolting or non-bolting types).However, PCA could divide garlic accessions into several groups.Central Asia and Northern Mediterranean accessions were separated from East and Southeast Asia accessions.Accessions from Central Asia and the Northern Mediterranean, located in high-latitude regions (40°N-45°N), had elongated scapes, produced many bulbils, and matured late.However, accessions from middle-to low-latitude regions (15°N-30°N) developed short scapes, produced small bulbils and heavy bulbs, and matured early.Meanwhile, isozyme genotypes 3, 4, 7 and 8, which possessed homozygous 'aa' in Lap-1 or were heterozygous in Lap-2, were found in the Central Asia and Northern Mediterranean groups, while genotypes 6, 9, 12, 13 and 15, which possessed homozygous 'bb' in Lap-1 and 'bb' or 'cc' in Lap-2, were found in other groups (Table 3).Moreover, based on MANOVA tests, there were several significant differences in garlic traits between isozyme genotypes and the groups of origin (Supplementary Table S3).Maass and Klaas (1995) classified 300 garlic accessions from the Old World into four subgroups using isozymes and RAPD, but they did not fully evaluate the morphological characteristics.In this study, we could evaluate the morphological and physiological traits in Japanese climate conditions as well as the isozyme variation of garlic accessions collected worldwide.

DISCUSSION
Groups of garlic accessions showed various types of morphological and physiological variation based on their origins.There are two possible reasons for these differences: (1) mutations have accumulated through the domestication process in each region; or (2) mutations had already occurred in the ancestral population.There have been reports of intraspecific variation among cultivated garlic landraces in Iran (Shaaf et al., 2014), Tunisia (Jabbes et al., 2012), Brazil (Buso et al., 2008) and China (Chen et al., 2013).The variation found in this study probably will have been derived from standing variation or mutations caused by adaptation, hitchhiking or genetic drift in the process of garlic's domestication before its cultivation.Etoh (1985) proposed the hypothesis that garlic has evolved from fertility to sterility and from a complete bolting type to a non-bolting type through an incomplete bolting type.Developing bulb-related traits in the Southeastern Mediterranean group were superior to those in the other groups, although the bolting period of that group was not significantly different from that of other groups.However, many Southeastern Mediterranean accessions bolted incompletely or did not bolt.Garlic is frequently called a medium-temperature plant because it grows well in medium temperatures (Etoh, 1985).Etoh (1985) also reported that the Mediterranean climate (cold in the winter, hot and dry in the summer) is suitable for growing garlic.In this plant, bulbil formation at the top of the scape causes a decrease in bulb yield due to competition with the bulbs for nutrients (Etoh, 1985;Hong and Etoh, 1996).
Additionally, geographical conditions accelerated garlic selection.Etoh (1985) suggested that garlic collected from areas with harsh, cold winters with heavy snow, such as Northern Europe, Northern America, and Northern Japan (high latitude areas), have evolved to be nonbolting because of the severe agroclimatic conditions.Both non-and incomplete-bolting traits are presumably the products of adaptation to unfavorable climatic conditions (Etoh, 1985).Specifically, garlic clones might have ceased bolting due to farmers' efforts to avoid decreased bulb yields.In a tropical area, however, other traits were required.Etoh and Simon (2002) reported that many tropical garlic cultivars develop only light bulbs because the differentiation of axillary buds and their development into cloves require low temperatures.In South Asia, garlic leaves are consumed as a green vegetable, and special clones have been selected for leaf production.Selection for leaf-producing rather than bulb-producing plants may have occurred in warm or hot regions (Etoh and Simon, 2002).
To discuss the sources of the present variation of garlic, we assume the following two hypotheses: (1) domestication with some artificial selection occurred in Central Asia (these populations may have standing variation) and spread widely to other regions; or (2) domesticated garlic expanded to other regions of the world with accumulating mutations.In these cases, there are two possibilities: (1) the sources of local adaptation and artificial selection are derived from standing variation; or (2) the sources of local adaptation and artificial selection are derived from mutations accumulated during the expansion.Therefore, it has been assumed that ancestral domesticated garlic populations have adapted in various regions using standing variation or mutations accumulated during the expansion, evolving with human-preferred traits over a long history of cultivation.
Isozyme loci showed that polymorphisms and allelic frequencies were different among the regional groups of accessions.In this study, only 15 isozyme genotypes were observed (Table 3).Central Asia has all genotypes except genotypes 5 and 12 (containing the 'ab' banding pattern in Pgi-1), while other groups have only a few specific genotypes.This is probably due to linkage disequilibrium caused by regional differentiation or other factors.Central Asia and Northern and Southeastern Mediterranean accessions showed high heterozygosity.On the other hand, accessions from Japan, China, Southeast Asia and the New World showed low levels of heterozygosity.Notably, the lowest H o among groups was seen in the New World group.This is probably due to small population size or other factors, such as selective sweeps at those loci and the founder effect.The Northern and Southeastern Mediterranean groups showed tendencies different from those of the Asian groups.This result is in agreement with previous reports (Etoh, 1985;Pooler and Simon, 1993;Maass and Klaas, 1995).
Chi-square testing showed no significant differences in allelic frequencies as compared to the average of the whole, except for Pgi-1.However, from the G st scores, genetic differentiation between regions was expected to be high.Additionally, some loci deviated significantly (P < 0.05) from the HWE (Lap-1 and Lap-2 loci in the Southeastern Mediterranean and the Lap-1 locus from Japan Group B and Southeast Asia) (Table 4).Some factors are considered to disturb HWE (e.g., genetic drift, migration, natural or artificial selection, and non-random mating).In these groups, there is a possibility that these factors affect the allelic frequencies.Kazakova (1971) reported that the Mediterranean region (from the west side of the Tien Shan Mountains to the Caucasus) contains a mix of fertile and sterile garlic.This region is regarded as garlic's secondary center of origin.In other words, ancestral garlic was widespread in this area, and domestication was begun.It is believed that a random-mating population then ceased random mating and started clonal reproduction.Thus, garlic from the Southeastern Mediterranean may have been affected by selective pressure for human needs (such as superior bulb formation) during a long cultivation history.
Japan Group B (collected from islands in Western Japan) also has a history of varietal establishment.For example, accession 33 "Iki-shu" was originally introduced from Jeju Island in Korea to Iki Island in Japan.Thus, it probably originated from a local clone in Jeju Island (Etoh, 1985).Information about the origins of accessions in this region is limited and complicated.It is likely that many kinds of garlic clones from surrounding countries were introduced to this region.Alternatively, accessions in this region may have been strongly affected by selection for human-preferred traits (such as high adaptability to agroclimatic conditions).
In Southeast Asia, it is estimated that garlic was introduced from the Mediterranean to India more than 5,000 years ago and then spread to this region (Etoh and Simon, 2002).Maass and Klaas (1995) inferred that garlic in Southeast Asia might have originated from A. longicuspis a long time ago through India, after acquiring special adaptations for various climatic stressors (such as heat, desert, strong sunshine and disease) necessary for its spread through the tropics.It is also possible that these stressors resulted in natural selection.Meanwhile, the Pgi-1 genotype 'aa' was not observed in any accessions, not even in the Central Asia group.On the other hand, Pgi-1 'ab' was observed mainly in the Southeast Asia group.It is possible that garlic possessing an 'aa' genotype in Pgi-1 exists in the region from India to Southeast Asia.Alternatively, some garlic from the ancestral population in India obtained genes that form intergenic heterodimers and expanded to Southeast Asia.Further investigation is needed to confirm genetic variation in India and the surrounding areas.
PCA showed relationships between morpho-physiological traits and isozyme genotypes (Fig. 4).According to MANOVA tests, there were significant differences in some traits among isozyme genotypes; however, the tests suggested that geographical factors also have significance (Supplementary Table S3).Thus, it was expected that the significant associations of alleles with traits were caused by regional differences.To reveal the relationships between genetic structures and particular traits, further analyses with many loci, such as those using microsatellites or others, are necessary.
Genetic changes or different combinations of genes after garlic's exposure to different agroclimatic conditions would result in different phenotypes.Geographical (latitude) information could explain the selection of garlic based on bolting traits.Therefore, adaptation and selection in garlic seem to depend on various environmental conditions and human preferences.In this study, garlic accessions showed great diversity of morpho-physiological traits and isozymes.Other diversity studies have been carried out regarding the variability of chemical production in a set of garlic collections, such as organosulfur compounds (Kamenetsky et al., 2005;Hornickova et al., 2009;Ovesna et al., 2011;Jabbes et al., 2012) or phenolic compounds (Lu et al., 2011), which have benefits for human health.In our previous report, we demonstrated the association between bio-morphological traits (bolting types and chemical production levels mentioned above) and geographical distribution (Hirata et al., 2015).It is possible that our materials are diverse not only in their visible traits but also in their DNA or other chemical production levels.Moreover, Kamenetsky et al. (2005) concluded that garlic from the place of origin possesses superior traits, such as tolerance to disease and pests and better adaptation to biotic or abiotic stress, to those that are seen in current cultivars.Further research on the genetic structure of garlic populations is necessary to utilize new breeding materials for future marker-assisted garlic breeding programs.

Table 3 .
Determination of 15 groups based on the observation of isozyme genotype and number of groups in each origin of

Fig. 4 .
Fig. 4. Plot of the first and second principal components obtained from garlic morphological and physiological data.PC1 and PC2 accounted for 43.1 and 27.5% of the total variation, respectively.The numbers next to the data points denote isozyme groups determined in Table3.

Table 2 .
Comparison of morphological and physiological traits of garlic accessions collected from various areas

Table 4 .
Genetic variability at three isozyme loci in each origin of accessions