**7. Karyotyping Mexican garlic genotypes**

Karyotypes of C-CN-95/2, C-37 1/8, C-3 1/25 (all these 'Perla' type genotype), 'Chino', 'Coreano' y 'Criollo' were obtained from root tips. Cloves of these genotypes were placed inside petri dishes containing wet cotton wool in order to induce roots 1-2 cm long. Roots were removed and soaked with 0.05% w/v colchicine and placed in the darkness for 3:30 h at 25°C. These roots were fixed with Farmer´s solution (ethanol and glacial acetic acid, 3:1 v/v); then, they were hydrolyzed with 1N HCl at 60 C for 10 min. Feulgen stain was applied to fixed roots before maceration with an enzymatic solution (2% pectinase, 5% celulase and citrate buffer pH 4.5) for 30 to 60 min. Roots tips were placed on microscope glass slides with a drop of 2% propionic orcein, and sandwiched with a cover glass. The slides were heated for few seconds with an alcohol burner with a very soft press so that single cells could be freed from the tissue. Then the cover glasses were gently tapped with a pencil in order to squeeze single cells for releasing and spreading the chromosomes. Observation of microphotography 100x25" allowed the following counts and measurements: chromosome number, short arms (p), long arms (q) total length, relative size of the chromosome and arm relationship (García, 1990). All of these observations were useful to classify each garlic genotype according to karyotype nomenclature and formule from Levan et al., (1964).

According to the observations, all of the genotypes tested have a chromosome number 2*n*=8x=16 (Fig. 4) in agreement with other reports (Battaglia, 1963; Verma & Mital, 1978; Koul et al., 1979). The karyotype characteristics found for centromer position were as follows: 'P-CN-95/2' (1M+4m+3sm), 'P-37-1/8' (1M+6m+1st), 'P-C-3-1/25' (7m+1sm), 'Chino' (1M+3m+3sm+1st), 'Coreano' (6m+2sm) and 'Criollo' (5m+3sm); Code: M or m=metacentric, sm= submetacentric and st=subtelocentric (Table 3). The nuclear content (2C value), which is considered one of the highest among the cultivated plants (Ipek et al*.,* 2005), is 32.7 pg. Additionally, garlic has a high karyotype variability that may be attributed to repetitive ADN (Kirk et al., 1970).

#### **8. Genetic profile of Mexican garlic**

Genetic markers are efficient tools for genetic analysis of populations and individuals. According to this concept, molecular characterization of garlic around the world has been performed either through RAPDs (Bradley & Collins, 1996; Eom & Lee, 1999; Shasany et al.,

Fig. 3. Protoplast isolation from P-C-3 1/25 genotype: A,B) Bulb ('Perla' type), C) *In vitro* 

Karyotypes of C-CN-95/2, C-37 1/8, C-3 1/25 (all these 'Perla' type genotype), 'Chino', 'Coreano' y 'Criollo' were obtained from root tips. Cloves of these genotypes were placed inside petri dishes containing wet cotton wool in order to induce roots 1-2 cm long. Roots were removed and soaked with 0.05% w/v colchicine and placed in the darkness for 3:30 h at 25°C. These roots were fixed with Farmer´s solution (ethanol and glacial acetic acid, 3:1 v/v); then, they were hydrolyzed with 1N HCl at 60 C for 10 min. Feulgen stain was applied to fixed roots before maceration with an enzymatic solution (2% pectinase, 5% celulase and citrate buffer pH 4.5) for 30 to 60 min. Roots tips were placed on microscope glass slides with a drop of 2% propionic orcein, and sandwiched with a cover glass. The slides were heated for few seconds with an alcohol burner with a very soft press so that single cells could be freed from the tissue. Then the cover glasses were gently tapped with a pencil in order to squeeze single cells for releasing and spreading the chromosomes. Observation of microphotography 100x25" allowed the following counts and measurements: chromosome number, short arms (p), long arms (q) total length, relative size of the chromosome and arm relationship (García, 1990). All of these observations were useful to classify each garlic genotype according to karyotype nomenclature and formule

According to the observations, all of the genotypes tested have a chromosome number 2*n*=8x=16 (Fig. 4) in agreement with other reports (Battaglia, 1963; Verma & Mital, 1978; Koul et al., 1979). The karyotype characteristics found for centromer position were as follows: 'P-CN-95/2' (1M+4m+3sm), 'P-37-1/8' (1M+6m+1st), 'P-C-3-1/25' (7m+1sm), 'Chino' (1M+3m+3sm+1st), 'Coreano' (6m+2sm) and 'Criollo' (5m+3sm); Code: M or m=metacentric, sm= submetacentric and st=subtelocentric (Table 3). The nuclear content (2C value), which is considered one of the highest among the cultivated plants (Ipek et al*.,* 2005), is 32.7 pg. Additionally, garlic has a high karyotype variability that may be attributed to

Genetic markers are efficient tools for genetic analysis of populations and individuals. According to this concept, molecular characterization of garlic around the world has been performed either through RAPDs (Bradley & Collins, 1996; Eom & Lee, 1999; Shasany et al.,

callus culture and D) Protoplasts at 40x magnification.

**7. Karyotyping Mexican garlic genotypes** 

from Levan et al., (1964).

repetitive ADN (Kirk et al., 1970).

**8. Genetic profile of Mexican garlic** 

Fig. 4. Chromosomes from somatic cells (*n*=8x=16) from garlic genotypes: 'C-CN-95/2', 'C-37- 1/8', C-3 1/25, 'Chino', 'Coreano' and 'Criollo'.

2000; Peiwen et al., 2001; Ipek et al., 2003; Paredes et al., 2008; Pardo et al., 2009) or through AFLP (Rosales & Molina, 2007). Our version of this kind of analysis with 20 Mexican genotypes was as follows: Twenty garlic genotypes were subjected to RAPDs in order to construct a distance tree using clustering with the Unweighted Pair Group Method with Erithmetic Mean (UPGMA). DNA was extracted according to Doyle and Doyle (1990); DNA samples were run on agarose gel 0.8% and DNA concentration was measured with a spectrometer (model GBC Cintra 10e UV-visible). RAPD reactions were performed in a 25 ml volume, consisting of 10x buffer solution [10 mM Tris-HCl buffer (pH 8.0), 50 mM KCl2], 2.5 mM MgCl2, 2.5 units of Taq DNA polymerase (Promega), 100 µM dNTP, 50 ng genomic DNA and 0.4 µM OPB series (OPB-8, OPB-9, OPB-10, OPB-11, OPB-15 and OPB 17) primer (Operon Technologies, Alameda, CA, USA). A total of 20 µl of mineral oil was placed over the reaction mixture. Amplifications were carried out in a DNA thermocycler (Model FPR0G02Y Techne Progene, England), under the following conditions: an initial denaturalization step of 2 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 35 °C, and 2 min at 72 °C, with a final extension step of 7 min at 72 °C. Amplification products were analyzed by electrophoresis in a 1.2% agarose gel. It was run at 100 V for 4 h, and detected by staining the gel with ethidium bromide (10 ng/100 ml of agarose solution in TBE). All visible and unambiguous fragments amplified by the chosen primers were entered under the heading of total visible fragments. Fragment data were entered on a spreadsheet to form a binary matrix, where (1) represented fragment presence and (0) fragment absence for each fragment accession combination. Cluster analysis was conducted by converting the data matrix into a similarity matrix using a simple matching coefficient. This coefficient was calculated by dividing the number of matches (0-0 and 1-1) by the total number of comparisons (Nei & Li, 1979). A cluster analysis was then conducted using the unweighted pair group method, with arithmetical averages (UPGMA) process using the S–Professional Plus 2000 program. The results obtained were compared with others studies realized by different authors, and were discusses as following: Six decamer OPB primers showing


Table 3. Chromosome morphological description from Mexican garlic genotypes. LBL=Long arm, LBC=Short arm, LT=Total length, LR=Relative length, r=Arm relationship and N=Centromere nomenclature.

**'P-CN-95/2' Chino'**  1 6.75 2.5 9.25 0.12 2.7 sm 1 6.75 4 10.75 0.13 1.7 sm 2 6.5 2.5 9 0.11 2.6 sm 2 6.5 6.25 12.75 0.16 1.04 m 3 6 6 12 0.15 1 M 3 6.5 2 8.5 0.10 3.25 st 4 6 5.5 11.5 0.15 1.09 m 4 6 6 12 0.15 1 M 5 6 5 11 0.14 1.2 m 5 6 5.5 11.5 0.14 1.09 m 6 5.5 4 9.5 0.12 1.3 m 6 5.5 4.75 10.25 0.13 1.2 m 7 5 4.5 9.5 0.12 1.1 m 7 5.5 3.5 9 0.11 1.6 sm 8 4.5 2.5 7 0.09 1.8 sm 8 4.5 2.5 7 0.08 1.8 sm

**'P-37-1/8' 'Coreano'**  1 6.75 2 8.75 0.14 3.4 st 1 7 5.5 12.5 0.15 1.2 m 2 5 4.5 9.5 0.15 1.1 m 2 7.5 4 11.5 0.14 1.8 sm 3 5 4 9 0.14 1.2 m 3 7 2.5 9.5 0.11 2.8 sm 4 4 4 8 0.13 1 M 4 6.5 6 12.5 0.15 1.08 m 5 4.5 2.75 7.25 0.12 1.6 m 5 6.5 5 11.5 0.14 1.3 m 6 4 3.25 7.25 0.12 1.6 m 6 5 4.5 9.5 0.11 1.1 m 7 4 3 7 0.11 1.3 m 7 5 4.5 9.5 0.11 1.1 m 8 3.5 2.25 5.75 0.09 1.5 m 8 4.5 3 7.5 0.09 1.5 m

**'P-C-3-1/25' 'Criollo'**  1 6.75 4 10.7 0.14 1.7 m 1 7 5.5 12.5 0.15 1.2 m 2 6.5 5.5 12 0.15 1.2 m 2 7.5 4 11.5 0.13 1.8 sm 3 6 5 11 0.14 1.2 m 3 6.5 6 12.5 0.15 1.0 m 4 5.75 5.5 11.25 0.15 1.04 m 4 6.5 6 12.5 0.15 1.0 m 5 5.75 2.75 8.5 0.11 2.0 sm 5 6.5 4.5 11 0.13 1.4 m 6 5 4 9 0.12 1.3 m 6 6.5 2.5 9 0.10 2.6 sm 7 5 3.5 8.5 0.11 1.4 m 7 5.5 3.25 8.75 0.10 1.7 sm 8 4 2.5 6.5 0.08 1.6 m 8 4.5 3.5 8 0.09 1.3 m

Table 3. Chromosome morphological description from Mexican garlic genotypes. LBL=Long

arm, LBC=Short arm, LT=Total length, LR=Relative length, r=Arm relationship and

N=Centromere nomenclature.

*no.*

Formula: 1M+4m+3sm Formula: 1M+3m+3sm+1st

Formula: 1M+6m+1st Formula: 6m+2sm

Formula: 7m+1sm Formula: 5m+3sm

*LBL LBC LT LR r N* 

*Chrom. no. LBL LBC LT LR r N Chrom.*

distinct polymorphic fingerprint were selected to reveal the genetic variation among the garlic samples. In almost all varieties, it was possible to identify around 10 bands. A dendrogram was generated from the binary matrix of measured data (Fig. 5), and two groups were identified. The first group was formed by eight varieties ('Durango', 'Nicaragua', 'Cortazar', 'Hermosillo', 'Massone', 'Pepita', 'Sonora' and 'Napuri') that are characterized by a lower production (smaller clove weight and/or greater number of cloves: Fig. 6), required more days to dormancy (6 months) and need fewer days (150) to harvest (data not shown). The second group was constituted by white, colored and marbled garlic ('Coreano', 'Positas', and 'Perla' cultivars, 'Criollo Aguascalientes', 'Español', 'Chileno', 'Ixmiquilpan', 'California', 'Chino', 'Pata de Perro' and 'Guatemala'). These are characterized by better bulb and clove weights, lower clove numbers/bulb, fewer days to dormancy (5-6 months), and between 180-210 days to harvest. In general, garlic varieties were clustered according to yield level, clove and bulb weights, number of cloves/bulb and dormancy period (Table 1). These results agree with those of García et al., (2003) using the AFLP technique. The most productive variety ('California') has the inconvenient of having a larger number of cloves/bulb and requires lower temperatures to achieve complete bulb formation. Dissimilarity among the two groups was 0.33. The lowest dissimilarity (0.0) corresponded to the most related varieties ('Sonora'-'Napuri', 'Criollo Aguascalientes'- 'Español', 'California'-'Chino' and 'Pata de Perro'-'Guatemala'). The highest dissimilarity (0.70) was between the variety 'Cortazar' and the varieties 'Ixmiquilpan', 'Pata de Perro', and 'Guatemala'. (Choi et al., 2003) reported a dissimilarity of 0.4 between two large groups from a total of 75 garlic varieties using the "M" or affinity coefficient. Using the Jaccard coefficient, Ipek et al*.,* (2003) obtained a lowest dissimilarity of 0.0 and a highest dissimilarity of 0.75 between two large groups of garlic. These results are similar to those of Al-Zaim et

Fig. 5. RAPD from Mexican genotypes. Lanes: 1: C-3-1/25, 2: C-37-1/8, 3: 'Coreano', 4: 'California', 5: 'Chino', 6: 'Criollo Aguascalientes', 7: 'Español', 8: 'Cortazar', 9: 'Positas', 10: 'Pepita', 11: 'Massone', 11: 'Durango', 13: 'Chileno', 14: 'Hermosillo', 15: 'Sonora', 16: 'Nápuri', 17: 'Nicaragua', 18: 'Ixmiquilpan', 19: 'Pata de perro', 20: 'Guatemala', (+): positive control, (-): negative control and M: Molecular weight marker.

al*.*, (1997). Evaluating diversity and genetic relationships among the progenitor *A. longicuspis* and 27 garlic varieties collected from different regions of the world, these authors found a dissimilarity of 0 between two samples, and a highest dissimilarity of 0.82 between two large groups. Through the RAPD technique used in this work, the two 'Perla' cultivars were grouped with the best production varieties, where the two selections presented a dissimilarity of 0.1. However, the 'Perla' C-3-1/25 cultivar showed a band of 2100 bp, and could thus be identified as a possible molecular marker. Our results allowed to identify highly related garlic varieties ('Sonora'-'Napuri', 'Criollo Aguascalientes'-'Español', 'California'-'Chileno' and 'Pata de Perro'-'Guatemala'), and separate them from varieties that are characterized by a lower yield (i.e., 'Pata de Perro' and 'Napuri'), and from mixed garlic that has been generated from introduced commercial varieties ('Criollo

Fig. 6. Dendrogram obtained by the RAPD technique and general description according to physiological, morphological and genetic characteristics in garlic (*Allium sativum* L.) varieties cultivated in the Central Region of Mexico. Arms or branches of the dendrogram: 1. 'Perla' C-3-1/25, 2. 'Perla' C-37-1/8, 3. 'Coreano', 4. 'California', 5. 'Chino', 6. 'Criollo Aguascalientes', 7. 'Español', 8. 'Cortazar', 9. 'Positas', 10. 'Pepita', 11. 'Massone', 12. 'Durango', 13. 'Chileno', 14. 'Hermosillo', 15. 'Sonora', 16. 'Napuri', 17. 'Nicaragua', 18. 'Ixmiquilpan', 19. 'Pata de Perro' 20. 'Guatemala'.

Aguascalientes'); these have lost their potential yield because they have not been subjected to an appropriate selection process. Because we do not know the source of most garlic varieties and cultivars used in this work, we cannot establish relationships among their origins.
