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

ABSTRACT

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 (H_o) and expected (H_e) 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 (1985, 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 ℃ in dark conditions in the summer.

Table 1. Garlic accessions used in the present study

No.	Geographical region	Managing number or name	Collection country or site	Accession information	Remarks
1	Japan - Group A (30°N–40°N)	5	Japan	Etoh 1985	‘‘Howaito-Roppen’’
2		6	Japan	Etoh 1985	‘‘Niigata-Sado’’
3		8	Japan	Etoh 1985	‘‘Ibaraki’’
4		9	Japan	Etoh 1985	‘‘Chiba-A’’
5		15	Japan	Etoh 1985	‘‘Hamamatsu’’
6		16	Japan	Etoh 1985	‘‘Wakayama-Roppen’’
7		37	Japan	Etoh 1985	‘‘Okute-B’’
8		40	Japan	Etoh 1985	‘‘Kokotsu’’
9		56	Japan	Etoh 1985	‘‘California Early’’
10		63	Japan	Etoh 1985	‘‘Saga-zairai’’
11		75	Japan	Etoh 1985	‘‘Kushikino-wase’’
12		100	Japan	Etoh 1985	‘‘Takasaki-C’’
13		124	Japan	Etoh 1985	‘‘Kanchi-Howaito’’
14		230	Japan	–	‘‘Kawanabe-zairai’’
15		360	Japan	–	‘‘Hiru’’
16		Hagi	Japan	–	‘‘Hagi’’
17		Hirado	Japan	–	‘‘Hirado’’
18		Chugokukei ninniku	Japan	–	–
19		Chiba-shoukyu	Japan	Saga University, Japan	–
20		Enhei	Japan	Saga University, Japan	–
21		Enshuu-gokuwase	Japan	Saga University, Japan	–
22		Kagoshima	Japan	Saga University, Japan	–
23		Kashu-wase	Japan	Saga University, Japan	–
24		Kashu-okute	Japan	Saga University, Japan	–
25		Katei-shu(Touhiru)	Japan	Saga University, Japan	–
26		Kouchi-shoukyuu	Japan	Saga University, Japan	–
27		Nagano	Japan	Saga University, Japan	–
28		Saga-ooninniku	Japan	Saga University, Japan	–
29		S62-tashirouetuke	Japan	Saga University, Japan	–
30		Setouchi	Japan	Saga University, Japan	–
31		Wase-ninniku	Japan	Saga University, Japan	–
32	Japan - Group B (20°N–30°N)	32	Japan	Etoh 1985	‘‘Iki-No. 1’’
33		65	Japan	Etoh 1985	‘‘Iki-shu’’
34		67	Japan	Etoh 1985	‘‘Amami-A’’
35		68	Japan	Etoh 1985	‘‘Amami-B’’
36		112	Japan	Etoh 1985	‘‘Ishu-wase (Sakata)’’
37		129	Japan	Etoh 1985	‘‘Iriomote’’
38		501	Japan	–	‘‘Tarama’’
39		540	Japan	–
40		Okinawa(Naha)	Japan	–	‘‘Naha’’
41		Okinawa(Tamagusuku)	Japan	–	‘‘Tamagusuku’’
42		Okinoerabu	Japan	Saga University, Japan	–
43		Kikai(Onodu)	Japan	Saga University, Japan	–
44		Kikai(Oodama)	Japan	Saga University, Japan	–
45		Kikai(Ikumi)	Japan	Saga University, Japan	–
46		Iki-ooninnniku	Japan	Saga University, Japan	–
47		Iki-wase	Japan	Saga University, Japan	–
48		Okinawa-nanbu	Japan	Saga University, Japan	–
49		Taishu-san	Japan	Saga University, Japan	–
50	China (30°N–40°N)	54	China	Etoh 1985	‘‘Fukushu (Foochow, China)’’
51		64	China	Etoh 1985	‘‘Shanhai-wase’’
52		291	China	–	‘‘Kunming’’
53		362	China	Hong and Etoh 1996	‘‘Urumchi’’
54		397	China	Hong and Etoh 1996	‘‘Kashgar’’
55		524	China	–	‘‘Guizhou-D’’
56		534	China	–	‘‘Guizhou’’
57		Kankousan	China	Saga University, Japan	–
58		Hong Kong-wase	China	Saga University, Japan	–
59		Shanghai	China	Saga University, Japan	–
60	Southeast Asia (15°N–20°N)	39	Taiwan	–	‘‘Seira’’
61		44	Taiwan	Etoh 1985	‘‘Taiwan-daikyu-pinku’’
62		45	Taiwan	Etoh 1985	‘‘Taiwan-shokyu-pinku’’
63		180	Taiwan	–	‘‘Taipei’’
64		509	Thailand	–	‘‘Chiang Mai’’
65		Mai Dinh	Vietnam	–	‘‘Mai Dinh’’
66		IIT	India	–	–
67		Chang Mai small	Thailand	–	–
68		Chang Mai large	Thailand	–	–
69		67-4	Thailand	Saga University, Japan	–
70		151-1	Thailand	Saga University, Japan	–
71		73-4	Thailand	Saga University, Japan	–
72		16-5	Thailand	Saga University, Japan	–
73		174-1	Thailand	Saga University, Japan	–
74		210-3	Thailand	Saga University, Japan	–
75		202-1	Thailand	Saga University, Japan	–
76	Central Asia (40°N–45°N)	199	Frunze	Etoh 1986	‘‘Frunze-2’’
77		211	Moscow	Etoh 1986	‘‘Moscow-5’’
78		369	Kazakhstan	Hong and Etoh 1996	‘‘Almaty’’
79		F17	Central Asia	–	–
80		F30	Central Asia	–	–
81		F31	Central Asia	–	–
82		F112	Central Asia	Hong et al. 2000	–
83		F115	Central Asia	Hong et al. 2000	–
84		F117	Central Asia	Hong et al. 2000	–
85		F138	Central Asia	Hong et al. 2000	–
86		F146	Central Asia	Hong et al. 2000	–
87		F147	Central Asia	Hong et al. 2000	–
88		F189	Central Asia	Hong et al. 2000	–
89		F215	Central Asia	Hong et al. 2000	–
90		F227	Central Asia	Hong et al. 2000	–
91		F424	Central Asia	Hong et al. 2000	–
92		F436	Central Asia	Hong et al. 2000	–
93		F1-200-23	Central Asia	Hong et al. 2000	–
94		F1-200-34	Central Asia	Hong et al. 2000	–
95		F1-200-92	Central Asia	Hong et al. 2000	–
96		Fs405	Central Asia	Hong et al. 2000	–
97		Fs407	Central Asia	Hong et al. 2000	–
98		Fs410	Central Asia	Hong et al. 2000	–
99		Fs412	Central Asia	Hong et al. 2000	–
100		Fs414	Central Asia	Hong et al. 2000	–
101		Fs422	Central Asia	Hong et al. 2000	–
102		Fs423	Central Asia	Hong et al. 2000	–
103		Fs424	Central Asia	Hong et al. 2000	–
104		Kazakhstan	Central Asia		‘‘Chimkent’’
105	Northern Mediterranean (40°N–45°N)	225	Spain	–	‘‘Spain-1’’
106		307	Greece	–	‘‘Thessaloniki market-1’’
107		434	Spain	Etoh et al. 2001	‘‘Spanish Gene Bank’’
108		445	Spain	Etoh et al. 2001	‘‘Spanish Gene Bank’’
109		454	Spain	Etoh et al. 2001	‘‘Spanish Gene Bank’’
110		462	Portugal	Etoh et al. 2001	‘‘Portuguese Gene Bank’’
111		465	Portugal	–	‘‘Braga Gene Bank’’
112		469	Portugal	–	‘‘Braga Gene Bank’’
113		552	Germany	Germany IPK collection All 130	–
114		553	Germany	Germany IPK collection All 146	–
115		556	Germany	Germany IPK collection All 1035	–
116		557	Germany	Germany IPK collection All 1038	–
117		560	Germany	Germany IPK collection All 1473	–
118	Southeastern Mediterranean (30°N–40°N)	55	Egypt	Etoh 1985	‘‘Egypt’’
119		144	Algeria	Etoh 1985	‘‘Kabyle’’
120		489	Egypt	–	‘‘Egypt-2’’
121		490	Egypt	–	‘‘Egypt-3’’
122		491	Jordan	–	‘‘Jordan-1’’
123		493	Syria	–	‘‘Syria-1’’
124		542	Turkey	–	–
125		Egypt	Egypt	–	‘‘Aswan’’
126		Syria-1	Syria	–	–
127		Syria-2	Syria	–	–
128		Syria-3	Syria	–	–
129		Syria-4	Syria	–	–
130		Syria-5	Syria	–	–
131		Gatur	Turkey	–	–
132	The New World (20°N-30°S)	60	Chile	Etoh 1985	‘‘Chile’’
133		69	Colombia	–	–
134		137	Peru	Etoh 1985	‘‘Peru’’
135		222	Mexico	–	–
136		Chile	Chile	Saga University, Japan	–
137		Colombia	Colombia	Saga University, Japan	–
138	Unknown	94or378	unknown	–	–
139		523	unknown	–	–
140		539	unknown	–	–

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.

Isozyme analysis

In April and May, 2011, phenotypes of two enzymes, leucine aminopeptidase (LAP; E.C. 3.4.11.1) and phosphoglucoisomerase (PGI; E.C. 5.3.1.9), in the 140 garlic accessions were analyzed using polyacrylamide gel electrophoresis. Newly expanding leaves, 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. (1995, 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. 1.

Various morphological traits of garlic accessions observed in this study. (A) Bulb structure and cloves in accession No. 230 (Japan). (B) Bulb structure and cloves in accession No. 490 (Egypt). (C) Bulbils produced in accession No. 45 (Taiwan). (D) Bulbils produced in accession No. F147 (Central Asia). (E) Incomplete bolting in accession No. 39 (Taiwan). Scale bars, 15 mm.

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

Group		Scape length (cm)					Number of bulbils					Bulb weight (g)					Bulb diameter (cm)					Number of cloves					Clove weight (g)					Bolting period (days)					Bolting type
		N	Mean	±	SE		N	Mean	±	SE		N	Mean	±	SE		N	Mean	±	SE		N	Mean	±	SE		N	Mean	±	SE		N	Mean	±	SE		CBa	IB	NB
Central Asia		27	81.7	±	3.3	b	25	66.9	±	5.8	b	27	9.6	±	1.0	a	27	2.8	±	0.1	ab	27	6.9	±	0.3	a	27	1.4	±	0.1	a	25	191.3	±	1.3	b	25	0	0
Northern Mediterranean		12	52.4	±	7.5	ab	9	57.5	±	13.9	ab	11	12.6	±	2.6	a	11	3.0	±	0.3	a	9	7.4	±	0.9	a	9	1.9	±	0.2	a	7	189.9	±	2.6	ab	10	0	2
Southeastern Mediterranean		7	63.5	±	6.8	ab	12	34.2	±	12.5	a	14	25.0	±	3.7	b	14	4.4	±	0.3	b	14	19.3	±	3.7	b	14	1.7	±	0.3	a	12	185.5	±	1.4	ab	3	9	2
China		6	60.9	±	10.2	ab	5	54.8	±	14.9	b	6	14.4	±	3.0	ab	6	3.4	±	0.3	ab	6	7.8	±	0.5	a	6	1.8	±	0.3	a	6	181.3	±	3.2	b	6	0	0
Japan	Group A	16	35.6	±	4.2	a	14	10.9	±	2.7	a	16	8.4	±	1.1	a	16	2.8	±	0.2	ab	16	6.2	±	0.4	a	16	1.4	±	0.2	a	14	181.9	±	1.4	b	13	3	1
	Group B	8	39.7	±	9.1	a	7	17.1	±	3.7	a	8	11.8	±	1.8	ab	8	3.3	±	0.2	ab	8	9.0	±	2.4	ab	8	1.9	±	0.6	a	8	181.3	±	1.1	b	5	3	0
Southeast Asia		6	40.0	±	11.2	a	6	18.1	±	8.4	a	9	9.1	±	2.2	a	9	2.9	±	0.3	ab	9	5.8	±	1.1	a	9	1.7	±	0.4	a	5	179.2	±	2.8	a	2	4	3
The New World		2	83.3				2	19.8				3	15.8	±	5.8	ab	3	3.7	±	0.7	ab	3	6.5	±	0.1	a	3	2.4	±	0.9	a	2	188.5				2	0	1

Means followed by different letters differ at the 5% significance level by Tukey’s test.

^a  CB, Complete Bolting; IB, Incomplete Bolting; NB, Non-Bolting.

Isozyme variation

Multiple loci in one enzyme system were numbered starting from the cathode and progressing to the anode (Fig. 2). These alleles were labeled alphabetically, starting with the slowest-migrating band. LAP and PGI putative genotypes at Lap-1, Lap-2 or Pgi-1 showed clear polymorphism. Lap-1 was detected as a single band (genotypes ‘aa’ and ‘bb’) or as two bands (genotype ‘ab’) because LAP is a monomeric enzyme (Maass and Klaas, 1995). In this study, the ‘ab’ bands did not resolve clearly for technical reasons, such as non-optimal sample volume or polyacrylamide gel concentration. Lap-2 was detected clearly in several banding patterns (genotypes ‘ac,’ ‘bb,’ ‘bc,’ ‘bd,’ ‘cc’ and ‘cd’). For PGI, single or triple bands were detected, indicating that Pgi was composed of either (1) a single gene or (2) two genes. In the former case, there are two bands of homodimers with heterodimers. In the latter, there are two Pgi genes that form an intergenic heterodimer. Some reports have indicated that in several plants, such 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.

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.

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

Isozyme genotype	Genotype			Central Asia (n = 29)	Northern Mediterranean (n = 13)	Southeastern Mediterranean (n = 14)	China (n = 10)	Japan		Southeast Asia (n = 16)	The New World (n = 6)	Unknown (n = 3)
	Lap-1	Lap-2	Pgi-1					Group A (n = 31)	Group B (n = 18)
1	aa	ac	bb	1	2	0	0	0	0	0	0	0
2	aa	bb	bb	2	1	0	0	0	0	0	0	0
3	aa	bc	bb	5	1	0	0	0	0	0	0	0
4	aa	cc	bb	2	0	0	0	0	0	0	0	0
5	ab	bb	ab	0	0	0	0	2	0	0	0	0
6	ab	bb	bb	1	3	2	2	4	2	2	2	2
7	ab	bc	bb	6	4	0	0	0	0	0	0	0
8	ab	bd	bb	1	1	0	2	0	0	0	0	0
9	ab	cc	bb	4	1	9	0	0	0	0	0	1
10	ab	cd	bb	1	0	0	0	0	0	0	0	0
11	bb	cd	bb	1	0	0	0	0	0	0	0	0
12	bb	bb	ab	0	0	0	1	3	5	10	0	0
13	bb	bb	bb	3	0	1	5	22	9	2	4	0
14	bb	bc	bb	1	0	0	0	0	0	0	0	0
15	bb	cc	bb	1	0	2	0	0	2	2	0	0

Group A; collected from Honshu, Group B; collected from islands in Western Japan.

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 frequency (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).

Fig. 3.

Allelic frequencies in Lap-1, Lap-2 and Pgi-1 isozyme loci in various regions. Numbers in parentheses represent the number of accessions.

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 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 Southeast 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 chi-square 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, respectively. The overall G_st value was 0.259. This score indicates that about 26% of the total genetic variation was derived from genetic differentiation.

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

Isozyme locus	Group	Whole	Central Asia (n = 29)	Northern Mediterranean (n = 13)	Southeastern Mediterranean (n = 14)	China (n = 10)	Japan Group A (n = 31)	Japan Group B (n = 18)	Southeast Asia (n = 16)	The New World (n = 6)
Lap-1	Na	2.0	2.0	2.0	2.0	2.0	2.0	2.0	2.0	2.0
	Ho	0.386	0.448	0.692	0.786	0.400	0.194	0.111	0.125	0.333
	He	0.310	0.552	0.453	0.477	0.320	0.175	0.105	0.117	0.278
	dHWE		ns	ns	*	ns	ns	ns	ns	ns
Lap-2	Na	2.3	4.0	4.0	2.0	2.0	1.0	2.0	2.0	1.0
	Ho	0.171	0.552	0.615	0.000	0.200	0.000	0.000	0.000	0.000
	He	0.260	0.561	0.583	0.337	0.180	0.000	0.198	0.219	0.000
	dHWE		ns	ns	***	ns	Monomorphic	***	***	Monomorphic
Pgi-1	Na	1.5	1.0	1.0	1.0	2.0	2.0	2.0	2.0	1.0
	Ho	0.146	0.000	0.000	0.000	0.100	0.161	0.278	0.625	0.000
	He	0.115	0.000	0.000	0.000	0.105	0.148	0.239	0.430	0.000
	dHWE		Monomorphic	Monomorphic	Monomorphic	ns	ns	ns	ns	Monomorphic
Mean Ho over loci		0.234	0.333	0.436	0.262	0.233	0.118	0.130	0.250	0.111

N_a, number of alleles; H_o, observed heterozygosity; H_e, expected heterozygosity; dHWE, deviations from Hardy-Weinberg equilibrium (ns = not significant, * = P < 0.05, *** = P < 0.001).

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).

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 Table 3.

DISCUSSION

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.

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 non-bolting 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.

ACKNOWLEDGMENTS

The authors are grateful to Professor Emeritus Takeomi Etoh (Kagoshima University, Japan) for providing materials collected worldwide, including Central Asian regions. Furthermore, we are grateful to Professor Emeritus Yosuke Tashiro (Saga University, Japan) for providing materials collected from a wide area in Japan.

REFERENCES

•  Buso,  G. S.,  Paiva,  M. R.,  Torres,  A. C.,  Resende,  F. V.,  Ferreira,  M. A.,  Buso,  J. A., and  Dusi,  A. N. (2008) Genetic diversity studies of Brazilian garlic cultivars and quality control of garlic-clover production. Genet. Mol. Res. 7, 534–541.
•  Chen,  S.,  Zhou,  J.,  Chen,  Q.,  Chang,  Y.,  Du,  J., and  Meng,  H. (2013) Analysis of the genetic diversity of garlic (Allium sativum L.) germplasm by SRAP. Biochem. Syst. Ecol. 50, 139–146.
•  Etoh,  T. (1985) Studies on the sterility in Garlic, Allium sativum L. Mem. Fac. Agric. Kagoshima Univ. 21, 77–132.
•  Etoh,  T. (1986) Fertility of the garlic clones collected in Soviet Central Asia. J. Jpn. Soc. Hortic. Sci. 55, 312–319.
•  Etoh,  T., and  Simon,  P. W. (2002) Diversity, fertility and seed production of garlic. In: Allium Crop Science - Recent Advances, (eds.: Rabinowitch, H. D., and Currah, L.), pp. 101–117. CABI Publishing, Wallingford, UK.
•  Etoh,  T.,  Watanabe,  H., and  Iwai,  S. (2001) RAPD variation of garlic clones in the center of origin and the westernmost area of distribution. Mem. Fac. Agr. Kagoshima Univ. 37, 21–27.
• FAO (2012) FAOSTAT: FAO Statistical Database. Food and Agriculture Organization, New York. http://faostat.fao.org. Accessed May 26, 2015.
•  Figliuolo,  G.,  Candido,  V.,  Logozzo,  G.,  Miccolis, V., and  Spagnoletti Zeuli,  P. L. (2001) Genetic evaluation of cultivated garlic germplasm (Allium sativum L. and A. ampeloprasum L.). Euphytica 121, 325–334.
•  Guri,  A.,  Levi,  A., and  Sink,  K. C. (1988) Morphological and molecular characterization of somatic hybrid plants between Lycopersicon esculentum and Solanum nigrum. Mol. Gen. Genet. 212, 191–198.
•  Hartl,  D., and  Clark,  A. G. (1988) Principles of Population Genetics, Second edition. Sinauer Associates, Sunderland, MA.
•  Hirata,  S.,  Abdelrahman,  M.,  Yamauchi,  N., and  Shigyo,  M. (2015) Characteristics of chemical components in genetic resources of garlic Allium sativum collected from all over the world. Genet. Resour. Crop Evol. DOI 10.1007/s10722-015-0233-7.
•  Hong,  C. J., and  Etoh,  T. (1996) Fertile clones of garlic (Allium sativum L.) abundant around the Tien Shan Mountains. Breed. Sci. 46, 349–353.
•  Hong,  C. J.,  Etoh,  T., and  Iwai,  S. (2000) An attempt of crossbreeding in garlic. Mem. Fac. Agr. Kogoshima Univ. 36, 17–28.
•  Hornickova,  J.,  Velisek,  J.,  Ovesna,  J., and  Stavelikova,  H. (2009) Distribution of S-alk(en)yl-L-cysteine sulfoxides in garlic (Allium sativum L.). Czech J. Food Sci. 27 (special issue 1), S232–S235.
•  Jabbes,  N.,  Arnault,  I.,  Auger,  J.,  Al Mohandes Dridi,  B., and  Hannachi,  C. (2012) Agro-morphological markers and organo-sulphur compounds to assess diversity in Tunisian garlic landraces. Sci. Hortic. 148, 47–54.
•  Jo,  M. H.,  Ham,  I. K.,  Moe,  K. T.,  Kwon,  S. W.,  Lu,  F. H.,  Park,  Y. J.,  Kim,  W. S.,  Won,  M. K.,  Kim,  T. I., and  Lee,  E. M. (2012) Classification of genetic variation in garlic (Allium sativum L.) using SSR markers. Aust. J. Crop Sci. 6, 625–631.
•  Kamenetsky,  R.,  London Shafir,  I.,  Khassanov,  F.,  Kik,  C.,  Van Heusden,  A. W.,  Vrielink-Van Ginkel,  M.,  Burger-Meijer,  K.,  Auger,  J.,  Arnault,  I., and  Rabinowitch,  H. D. (2005) Diversity in fertility potential and organo-sulphur compounds among garlics from Central Asia. Biodivers. Conserv. 14, 281–295.
•  Kazakova,  A. A. (1971) The most widely distributed species of onion, their origin and intraspecific classification. Trudy Prikl. Bot. Genet. Selek. 45, 19–41.
•  Keller,  E. R. J. (2002) Cryopreservation of Allium sativum L. (Garlic). In: Cryopreservation of Plant Germplasm. vol. 2, (eds.: Towill, L. E., and Bajaj, Y. P. S.), pp. 37–47. Springer-Verlag, Berlin Heidelberg, Germany.
•  Kik,  C.,  Kahane,  R., and  Gebhardt,  R. (2001) Garlic and Health. Nutr. Metab. Cardiovasc. Dis. 11, 57–65.
•  Lallemand,  J.,  Messian,  C. M.,  Briand,  F., and  Etoh,  T. (1997) Delimitation of varietal groups in garlic (Allium Sativum L.) by morphologigal, physiological and biochemical characters. Acta Hortic. 433, 123–132.
•  Lu,  X.,  Ross,  C. F.,  Powers,  J. R.,  Aston,  D. E., and  Rasco,  B. A. (2011) Determination of total phenolic content and antioxidant activity of garlic (Allium sativum) and elephant garlic (Allium ampeloprasum) by attenuated total reflectance- fourier transformed infrared spectroscopy. J. Agric. Food Chem. 59, 5215–5221.
•  Ma,  K. H.,  Gwag,  J. G.,  Zhao,  W. G.,  Dixit,  A.,  Lee,  G. A.,  Kim,  H. H.,  Chung,  I. M.,  Kim,  N. S.,  Lee,  J. S.,  Ji,  J. J., et al. (2009) Isolation and characteristics of eight novel polymorphic microsatellite loci from the genome of garlic (Allium sativum L.). Sci. Hort. 122, 355–361.
•  Maass,  H. I., and  Klaas,  M. (1995) Infraspecific differentiation of garlic (Allium sativum L.) by isozyme and RAPD markers. Theor. Appl. Genet. 91, 89–97.
•  Mario,  P. C.,  Viviana,  B. V., and  María,  I. G. A. (2008) Low genetic diversity among garlic accessions detected using RAPD. Chilean J. Agric. Res. 68, 3–12.
•  Micales,  J. A., and  Bonde,  M. R. (1995) Isozymes: methods and applications. In: Molecular Methods in Plant Pathology, (eds.: Singh, R. P., and Singh, U. S.), pp. 115–130. CRC Press, London.
•  Nei,  M. (1973) Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. USA 70, 3321–3323.
•  Ovesna,  J.,  Kucera,  L.,  Hornickova,  J.,  Svobodova,  L.,  Stavelikova,  H.,  Velisek,  J., and  Milella,  L. (2011) Diversity of S-alk(en)yl cysteine sulphoxide content within a collection of garlic (Allium sativum L.) and its association with the morphological and genetic background assessed by AFLP. Sci. Hort. 129, 541–547.
•  Peakall,  R., and  Smouse,  P. E. (2012) GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics 28, 2537–2539.
•  Pooler,  M. R., and  Simon,  P. W. (1993) Characterization and classification of isozyme and morphological variation in a diverse collection of garlic clones. Euphytica 68, 121–130.
•  Shaaf,  S.,  Sharma,  R.,  Kilian,  B.,  Walther,  A.,  Ozkan,  H.,  Karami,  E., and  Mohammadi,  B. (2014) Genetic structure and eco-geographical adaptation of garlic landraces (Allium sativum L.) in Iran. Genet. Resour. Crop Evol. 61, 1565–1580.
•  Shigyo,  M.,  Tashiro,  Y.,  Isshiki,  S., and  Miyazaki,  S. (1995) Chromosomal locations of five isozyme gene loci (Lap-1, Got-1, 6-Pgdh-2, Adh-1 and Gdh-1) in shallot (Allium cepa L. Aggregatum group). Jpn. J. Genet. 70, 399–407.
•  Shigyo,  M.,  Tashiro,  Y.,  Isshiki,  S., and  Miyazaki,  S. (1996) Genetic analysis of phosphoglucoisomerase isozymes in cultivated and wild species of section Cepa in Allium. Euphytica 90, 195–200.
•  Tanksley,  S. D., and  Jones,  R. A. (1981) Effects of O₂ stress on tomato alcohol dehydrogenase activity: description of a second ADH coding gene. Biochem. Genet. 19, 397–409.
•  Weeden,  N. F., and  Gottlieb,  L. D. (1979) Distinguishing allozymes and isozymes of phosphogluco-isomerases by electrophoretic comparisons of pollen and somatic tissues. Biochem. Genet. 17, 287–296.
•  Wendel,  J. F. (1983) Electrophoretic analysis of genetic variation in wild and cultivated Camellia japonica L. Ph. D. Thesis, Univ. North Carolina, Chapel Hill, NC.
•  Zhao,  W. G.,  Chung,  J. W.,  Lee,  G. A.,  Ma,  K. H.,  Kim,  H. H.,  Kim,  K. T.,  Chung,  I. M.,  Lee,  J. K.,  Kim,  N. S.,  Kim,  S. M., and  Park,  Y. J. (2011) Molecular genetic diversity and population structure of a selected core set in garlic and its relatives using novel SSR markers. Plant Breed. 130, 46–54.