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INTRODUCTION
The understanding and conservation of plant diversity provides benefits for humans, such as in prospecting for natural chemical compounds helpful in medicine (Tharanathan & Mahadevamma 2003; Al-Islam et al. 2012) or in use of phytodiversity for improvement of cultivated plants to meet challenges of future nutritional demands (Govindaraj et al. 2015). In this context it is especially important to evaluate genetic diversity in crops of agronomic importance.
Many cultivated crops belong to the legume family (Fabaceae), which consists of about 800 genera and 20000 species (Lewis et al. 2005). According to Stagnari et al. (2017), the benefits offered by legumes can be classified at three levels: good nutrition (Tharanathan & Mahadevamma 2003; Al-Islam et al. 2012), high productivity (Liting et al. 2019), and a stable system of cultivation (Pacajes et al. 2002; Zander et al. 2016), all of which provide high significance for many zones of the world. Among the legumes, there exists a subgroup called the dry legumes (pulses), which are harvested for their dry seeds.
An important dry leguminous crop world-wide is the lentil (Lens culinaris Medik.). This annual, herbaceous, and autogamous species originated in the near East, and it was one of the crops from which agriculture developed in the Old World (Zohary 1999). In 2018, the lentil was the fifth most productive dry leguminous crop in the world with six million tons harvested (FAOSTAT 2020). In this same year, the largest producers were Canada, India, USA and Turkey, in that order, with Canada producing two million tons and 13953 hg ha-1 (FAOSTAT 2020).
The lentil was introduced to Chile in the 17th century with the arrival of the Spanish, and it became an important domesticated crop within the country. In 1979 Chile produced up to 31690 tons, but 2018/2019 production dropped significantly to only 1400 tons and 7642 hg ha-1 (FAOSTAT 2020). Lentils are now being grown by small farmers only in Maule, Ñuble, La Araucanía, and Biobío regions, the latter region showing the highest yields (ODEPA 2019). Due to low domestic production, Chile now imports a large portion (97%; 14726 t) of its crop from Canada (ODEPA 2019).
Diversity within cultivated L. culinaris is classified principally on the basis of the diameter of its seeds, these being the microspermas (2-6 mm) and macrospermas (6-9 mm) (Barulina 1930), with the latter plants also being taller and having heavier seeds (Sharma et al. 1995). The macrospermas also flower and fruit over a longer period of days.
Recent studies with molecular markers on germplasm of L. culinaris throughout the world have revealed three agro-ecological zones, the northern temperate region, the Mediterranean, and southern Asia (Khazaei et al. 2016). Studies on genetic diversity of L. culinaris in Chile are few, and hence there is no clear picture of the status of this crop within the country. The Instituto de Investigaciones Agropecuarias (INIA) has made an effort to generate cultivars that possess seeds of high caliber, e.g., Calpun-INIA and Super Araucana- INIA (Tay et al. 2001; Peñaloza et al. 2007) and resistant to “la roya” (a devastating leaf rust, Uromyces fabae (Pers.) de Bary). Rodríguez et al. (1999) completed an isozyme study that revealed low genetic distance among samples of Chilean germplasm, with 87.2% of Chilean lentils being incorporated in the same phenetic grouping. These results suggest that low genetic diversity exists within the naturalized Chilean lentils.
Another approach to recognize patterns of genetic variation within diverse crop plants is use of karyological data. These methods permit evaluation of differences in the number, form, and size of chromosomes, band polymorphisms, and architectural alterations in the presence of secondary restrictions or nuclear organizing regions (NORs; Guerra 2008). General cytotaxonomic studies on L. culinaris have revealed a chromosome number of 2n = 14 (Bhattacharjee 1953; Sinha & Acharia 1972; Balyan et al. 2002; Jha et al. 2015). The morphology of the chromosomes varies from metacentric, submetacentric, to subtelocentric or acrocentric (Sinha & Acharia 1972; Manara & Manara 1979; Ladizinsky & Muehlbauer 1993; Rehman & Altaf 1994; Balyan et al. 2002; Gaffarzadeh-Namazi et al. 2007; Jha et al. 2015). Size of the individual chromosomes varies from 3.0 to 12.5 μm, depending upon which cultivar is examined, whereas the total length of the chromosomes (TLC) ranges from 28 to 121 μm (Sinha & Acharia 1972; Naithani & Sarbhoy 1973; Manara & Manara 1979; Rehman & Altaf 1994; Gaffardazeh-Namazi et al. 2007; Jha et al. 2015). Secondary constrictions range from 0 to 3 (Battacharjee 1953; Sinha & Acharia 1972; Naithani & Sarbhoy 1973; Kumar et al. 2001; Galasso et al. 2001; Balyan et al. 2002; Gaffarzadeh-Namazi et al. 2007; Jha et al. 2015).
In the present study, we contribute to a better understanding of Chilean lentils by characterizing cytotaxo- nomically selected Chilean cultivars and accessions and comparing these with accessions from Canada. Specific data involve chromosome formula, indexes of chromosomal asymmetry, total length of chromosomes, and a detailed examination of the architecture of each of the chromosomes.
MATERIALS AND METHODS
Plant material
Five accessions and two cultivars of Lens culinaris Medik. were utilized from Chile, plus three cultivars from Canada (Líder, Martini, Campo Lindo) obtained from Chilean supermarkets. For the Chilean accessions, these were obtained from small farmers in distinct localities of the Ñuble (Yumbel, Ninhue) and Biobío (Collanco, Hualqui, Los Ángeles) Regions. Finally, the Chilean cultivars, Calpun-INIA and Super Araucana- INIA, were obtained from the Instituto de Investigaciones Agropecuarias (INIA).
Preparation of plant material
Seeds of L. culinaris were thoroughly hydrated in Petri dishes at room temperature (18-22 °C), germinating after 4 d. This was followed by an antimitotic treatment of 8-hydroxyquinoline in the darkness at 4 °C for 24 h. Later, the germinated seeds were fixed in ethanol/acetic acid (ratio 3:1) and kept in the darkness at 4 °C. The root tips were subsequently excised from the seeds and washed with distilled water, followed by maceration with HCl 0.5 N at 45 °C for 17 min. Immediately the root tips were washed in distilled water and placed on a microscope slide to which was added a drop of distilled water to titrate the tissue. The root tips were then squashed in a drop of 1% aceto-orcein for observation under the microscope.
Seven metaphase plates were observed for each cultivar and accession. Data obtained were: long arm/short arm ratio of the chromosomes following the modified method of Levan et al. (1964); and indices of the interchromosomal (coefficient of variation of chromosome length; CVCL) and intrachromosomal asymmetry (mean centromeric asymmetry; MCA) of the karyotypes (Peruzzi & Eroglu 2013). For measuring dimensions of the chromosomes, the software Micromeasure (Reeves 2001) was used. Statistical analyses were done with the software Past 3.26b (Hammer et al. 2001).
RESULTS AND DISCUSSION
Uniformity of chromosomal formula
As can be observed in the metaphase plates (Fig. 1), all samples of L. culinaris analyzed in this study showed 2n = 14 chromosomes, as has been reported previously (Bhattacharjee 1953; Sinha & Acharia 1972; Balyan et al. 2002; Jha et al. 2015). The chromosomal formula was mostly 4m+3sm (Fig. 2) with no differences being detected between Chilean and Canadian cultivars and accessions, probably due to the frequent introduction of Canadian lentils to Chile. There were two exceptions: Collanco and Campo Lindo, which had 3m+4sm. Chromosome 1 in these two samples can be defined as submetacentric (following Levan et al. 1964), although the standard deviation might permit one alternatively to consider it as metacentric. The important point is that these samples were effectively distinct from the others.
In studies in other regions of the world, uniformity of the chromosome formula of 4m+3sm has also been documented, e.g., in cultivars from Iran (Gaffarzadeh-Namazi et al. 2007) and also from India, but here with a slight difference of 4m+2sm+1st (Jha et al. 2015).
Variation in number of secondary constrictions
Among the cultivars and accessions from Canada and Chile, there is variation in number of secondary constrictions, 4 to 5 from those in Canada and 4 to 6 from those in Chile (Fig. 3). In Chile this variation is due principally to the six secondary constrictions from Yumbel, which has a distinctive secondary subtelomeric constriction on chromosome 2 (Fig. 3). This constriction is related to the ribosomal gene 5S, which is positioned in a subtelomeric zone of chromosome 2 (Balyan et al. 2002) or 1 (Galasso et al. 2001). Three nuclear organizing regions (NORs) have been reported previously (Galasso et al. 2001; Balyan et al. 2002), one of 5S rDNA in a subtelomeric zone on chromosome 1 or 2, which correlates with the secondary subtelomeric constriction observed in Yumbel, and another for the 18S-5.8S-25S rDNA in an easily observable centromeric zone on chromosome 3 or 4 (Fig. 2). In addition, we also observed a secondary subcentromeric constriction on chromosome 6 that is related to the position of 5S rDNA on the same chromosome (also reported by Balyan et al. 2002 and Galasso et al. 2001). Another secondary constriction that has not been reported previously is a centromeric type found on chromosome 1 (Fig. 1). Given these variations in the presence of secondary constrictions, it would be important in the future to obtain more information, especially from fluorescence in situ hybridization (FISH).
Total length of chromosomes (TLC)
A broader variation in total length of chromosomes (TLC) exists among Chilean cultivars and accessions than among those from Canada, with 150.86-234.28 μm in the former, and 176.11-190.92 μm in the latter (Fig. 4). Hualqui possesses TLC of 150.86 μm, the lowest among samples examined, and significantly differentiated from the highest TLCs in Yumbel and Collanco (p = 0.04 and p = 5.5 × 10-5, respectively) (Table 2). Collanco, with the largest TLC of 234.28 μm, is differentiated significantly from Ninhue, Los Ángeles, and Hualqui (p = 3.1 × 10-4, p = 6.0 × 10-3 and p = 5.5 × 10-5, respectively) (Table 2). Other studies have reported TLC for cultivars of L. culinaris in different countries that range from 28 to 121 μm (Sinha & Acharia 1972; Naithani & Sarbhoy 1973; Manara & Manara 1979; Rehman & Altaf 1994; Gaffarzadeh-Namazi et al. 2007; Jha et al. 2015), revealing that this character is quite variable. The results obtained in the present study indicate TLC even higher than those reported previously.
Interchromosomal asymmetry
Differences in the asymmetric karyotypes among Canadian and Chilean cultivars and accessions reveal clusters that are somewhat distinct from each other between the two regions (Fig. 5). Separating these data into cultivars and accessions, however, shows that only Hualqui (Fig. 6) is separated from all other samples. With exclusion of these data, the cultivars and accessions from Chile and Canada are completely overlapping. The interchromosomal asymmetry (CVCL) of the Hualqui accession was an average 31.25, significantly differentiated from all the rest of the cultivars or accessions (Table 1).
FIGURE 1 Metaphase plates from each cultivar and accession of Lens culinaris, with chromosomes numbered: Calpún-INIA (A), Super Araucana-INIA (B), Yumbel (C), Ninhue (D), Collanco (E), Hualqui (F), Los Ángeles (G), Campo Lindo (H), Líder (I), Martini (J). Black arrows indicate the most representative secondary constrictions. The blue arrow indicates the single subtelomeric secondary constriction. / Placas metafásicas de cada cultivar y procedencia de Lens culinaris, con cromosomas numerados: Calpún-INIA (A), Super Araucana-INIA (B), Yumbel (C), Ninhue (D), Collanco (E), Hualqui (F), Los Ángeles (G), Campo Lindo (H), Líder (I), Martini (J). Las flechas negras indican las constricciones secundarias más significativas. La flecha azul indica la única constricción secundaria subtelomérica.
FIGURE 2 Idiograms of the cultivars and accessions of Lens culinaris evaluated in this study. The chromosomes are ordered according to their length; whiskers indicate standard deviations. The black areas represent secondary constrictions observed in metaphase plates. / Ideogramas de los cultivares y procedencias de Lens culinaris evaluados en este estudio. Los cromosomas están ordenados de acuerdo a su longitud, cada uno lleva su desviación estándar. Las áreas negras representan las constricciones secundarias observadas en las placas metafásicas.
TABLE 1 Tukey test for comparisons of the coefficient of variation of chromosome length (CVCL) index of the Hualqui accession with all other cultivars and accessions. / Test de Tukey para la comparación del índice de coeficiente de variación de la longitud del cromosoma (CVCL) de la procedencia de Hualqui con respecto a los demás cultivares y procedencias.
| Comparisons with Hualqui | CVcl -p value |
|---|---|
| Calpún-INIA | 1.80 x 10-11 |
| Super Araucana-INIA | 1.81 x 10-11 |
| Ninhue | 1.80 x 10-11 |
| Collanco | 1.81 x 10-11 |
| Yumbel | 1.79 x 10-11 |
| Los Ángeles | 1.79 x 10-11 |
| Campo Lindo | 1.79 x 10-11 |
| Líder | 1.80 x 10-11 |
| Martini | 1.79 x 10-11 |
TABLE 2 Significant differences in Dunn test comparisons for total chromosome length (TLC). All p-values are adjusted by Bonferroni correction. / Diferencias significativas en comparaciones del test de Dunn para la longitud total del cromosoma (TLC). Todos los valores p están ajustados a la corrección de Bonferroni.
| Comparisons | TLC -p value |
|---|---|
| Hualqui - Yumbel | 0.04307 |
| Hualqui - Collanco | 0.00006 |
| Collanco - Los Ángeles | 0.00602 |
| Collanco - Ninhue | 0.00031 |
FIGURE 3 Karyotypes from the accessions of Collanco (A), Hualqui (B), and Yumbel (C). Scales = 10 μm. / Cariotipos de las procedencias de Collanco (A), Hualqui (B), y Yumbel (C). Escala = 10 μm.
FIGURE 4 Total chromosome length (TLC) for each cultivar and accession evaluated in this study. / Longitud total del cromosoma (TLC) para cada cultivar y procedencia evaluada en este estudio.
FIGURE 5 Karyotypic asymmetry of the coefficient of variation of chromosome length (CVCL) and mean centromeric asymmetry (MCA) for the Chilean and Canadian cultivars and accessions of Lens culinaris. / Asimetría cariotípica del coeficiente de variación de la longitud del cromosoma (CVCL) y asimetría centromérica media (MCA) para los cultivares y procedencias de Lens culinaris chilenos y canadienses.
FIGURE 6 Asymmetric karyotype of the indices coefficient of variation of chromosome length (CVCL) and mean centromeric asymmetry (MCA) for each cultivar and accession of Lens culinaris evaluated in this study. The blue ellipse includes values from plants of Hualqui. / Asimetría cariotípica de los índices coeficiente de variación de la longitud del cromosoma (CVCL) y asimetría centromérica media (MCA) para cada procedencia y cultivar de Lens culinaris evaluado en este estudio. La elipse azul incluye valores de Hualqui.
Differences in intrachromosomal asymmetry (MCA) also exist between the Chilean ‘Calpún-INIA’ and ‘Super Araucana-INIA’ (Fig. 6; Pino-Palma & Baeza-Perry 2019). One possible explanation for the interchromosomal asymmetric karyotype in Hualqui might be chromosomal rearrangements, possibly deletions, given that the lowest TLC occurs in this accession (Fig. 4). Vu et al. (2015) indicated that repair of a rupture of the DNA double helix can generate insertions or deletions, which can determine the size of the genome as consequence of the net value between insertions and deletions. Polymorphisms in some chromosomal bands have been found in cultivars of L. culinaris from Iran (Gaffarzadeh- Namazi et al. 2007), which also might reflect processes such as chromosomal rearrangements, such as have been documented in cultivars of wheat (Schneider et al. 2003). In studies of other Chilean species, such as Alstroemeria ligtu L., differences in asymmetrical karyotypes have been suggested to correlate with environmental factors, such as type of soil, habitat, and distribution (Baeza et al. 2016). In addition, in two subspecies of Alstroemeria presliana Herb., karyotypic asymmetry appears to correlate with phenotypic variations in intensity of color of tepals and anthers (Baeza et al. 2015).
CONCLUSIONS
In this karyological study of Lens culinaris, we have shown the broadest variation of total length of chromosomes (TLC) to occur within Chile. We found some Chilean accessions with distinct cytotaxonomic characteristics, such as Hualqui with marked interchromosomal asymmetry. Yumbel possesses a secondary subtelomeric constriction on chromosome 2, and Collanco has the highest TLC. It is not known that the physiological implications might be from these variations, but further study and understanding of L. culinaris in Chile is certainly warranted. With further sampling, it might be possible to detect consistent cytotaxonomic signatures for each of these regions, distinct from those in Canada, which would permit identification of origin for any marketable product. The agronomic importance of this species in Chile recommends protection of these cytotaxonomic variants as a national genetic resource.
