Validation of morphological and molecular descriptors in the identification of chickpea cultivars for cultivar registration

RESUMO:

O cultivo de grão-de-bico (Cicer arietinum) tem se expandido no Brasil, o que demanda a oferta de cultivares registradas, adaptadas às diferentes regiões. Assim, são necessários descritores seguros visando à distinção de cultivares para a habilitação destas para a produção, beneficiamento e comercialização de sementes. Objetivou-se avaliar descritores morfológicos e moleculares de proteínas e DNA para a identificação de 10 cultivares de grão-de-bico. Os descritores morfológicos, baseados nas diretrizes da UPOV, foram avaliados em plantas cultivadas em casa de vegetação, em parcelas contendo plantas com diferentes níveis de misturas destas cultivares, em diferentes estádios de desenvolvimento. Nesta avaliação, foram calculadas as porcentagens de acerto e erro de quatro especialistas da área de sementes que desconheciam a porcentagem de misturas das cultivares. Também foram avaliadas expressões de proteínas de armazenamento, resistentes ao calor e de enzimas, em sementes das diferentes cultivares, as quais foram produzidas sob dois níveis de nitrogênio (50 e 200 kg.ha⁻¹). A caracterização das cultivares também foi realizada por meio de marcadores SSR, simples sequencia repetida. Conclui-se que que a combinação de descritores morfológicos, proteicos e moleculares é importante para a diferenciação das cultivares de grão de bico. Para os marcadores SSR, foi possível discriminar o maior número de cultivares por meio do iniciador TA-203. A expressão das proteínas não foi influenciada pelos níveis de nitrogênio, e o estádio ideal para a avaliação da pureza genética variou entre as cultivares.

Termos para indexação:
Cicer arietinum; caracterização genética; pureza genética; distinção genotípica; identificação varietal

ABSTRACT:

Chickpea (Cicer arietinum) cultivation has been expanding in Brazil, which demands the availability of registered cultivars adapted to different regions. Therefore, reliable descriptors are needed to distinguish cultivars for their certification in seed production, processing, and commercialization. In this study, morphological and molecular descriptors based on proteins and DNA were evaluated to identify 10 chickpea cultivars. Morphological descriptors, based on UPOV guidelines, were assessed in plants grown in a greenhouse, in plots containing plants with different levels of cultivar mixtures, at various developmental stages. In this evaluation, accuracy and error rates were calculated for four seed specialists who were unaware of the mixture percentages of the cultivars. Additionally, the expressions of storage proteins, heat-resistant proteins, and enzymes were evaluated in seeds from the different cultivars, which were produced under two nitrogen levels (50 and 200 kg.ha⁻¹). The characterization of cultivars was also performed using SSR (simple sequence repeat) markers. It is concluded that the combination of morphological, protein, and molecular descriptors is important for differentiating chickpea cultivars. For the SSR markers, the highest number of cultivars was discriminated using the TA-203 primer. Protein expression was not influenced by nitrogen levels, and the optimal developmental stage for evaluating genetic purity varied among the cultivars.

Index terms:
Cicer arietinum; genetic characterization; genetic purity; genotypic distinction; varietal identification

INTRODUCTION

Chickpea (Cicer arietinum L.) is an important species belonging to the Fabaceae family and is among the most important food grain legumes in the world in terms of consumption, occupying the third position among the most produced legumes (Artiaga et al., 2015; Nascimento, 2016; FAOSTAT, 2025). In Brazil, the implementation of new cultivation areas has grown quite significantly, and in the Cerrado, the yields obtained already exceed the national and world averages (Avelar et al., 2018; Pegoraro et al., 2018). Thus, it is essential to develop cultivars more adapted to local climatic conditions and seed production technologies to meet the demands of producers, farmers and consumers.

For a new cultivar to be released, it is essential to comply with the entire legal framework. In this context, as recommended by Law No. 10,711 of August 5, 2003, the cultivars developed for cultivation in the country must be certified for seed production and commercialization. The law determines that the production, processing and commercialization of seeds and seedlings are subject to prior registration of the respective cultivars in the National Register of Cultivars (Registro Nacional de Cultivares - RNC). This registration aims, above all, to ensure the identity and quality of the propagative material for the entire market (Brasil, 2003).

In addition, Decree No. 10,586 and MAPA Ordinance No. 502 of 2022 recommend that, for registration purposes, morphological, physiological, biochemical, or molecular descriptors, which are genetically inherited, can be used for the identification and certification of cultivars with the RNC. In addition, it should be noted that descriptors can only be used to distinguish cultivars when homogeneous and stable (Brasil, 2022). For chickpea cultivars that are registered in the RNC of MAPA, it is verified that the official report of descriptors is not available. Thus, conducting studies and research aimed at validating descriptors to identify and characterize the cultivars developed and registered in the country is extremely relevant.

Descriptors of different natures have been used for the characterization and identification of chickpea cultivars. Upadhyaya (2003) used morphological markers to evaluate chickpea accessions from Africa, South and Southeast Asia, the Americas, Europe, Western Asia and the Mediterranean region. Morphological markers have also been employed by Joshi et al. (2018) and Biswal et al. (2021) in Indian genotypes. As for molecular characterization, Hameed et al. (2009) analyzed proteins in genotypes from India and Pakistan, while Kumar et al. (2011) and Kaur et al. (2012) performed proteomic analyses in Indian materials. Regarding DNA markers, the studies conducted by Mir et al. (2022) with genotypes from Turkey and India, by Afzal et al. (2018) with materials from Saudi Arabia, and by Ghaffari et al. (2014) with genotypes from Iran stand out.

Thus, in this study, morphological and molecular descriptors of proteins and DNA were evaluated for the distinction of chickpea cultivars, aiming at the National Registry of Cultivars.

MATERIAL AND METHODS

The study was conducted in the experimental area and at the Central Seed Research Laboratory (LCPS) of the Department of Agriculture (DAG) of the Universidade Federal de Lavras (UFLA), in the municipality of Lavras, located in the southern region of Minas Gerais, Brazil.

Experiment I: Evaluation of morphological descriptors

Seeds of 10 chickpea cultivars from Embrapa Vegetables (BRS Kalifa, BRS Cícero, BRS Toro, BRS Aleppo, BRS Cristalino, 10108, BRS Hari, 10209, UPL 05 and UPL 06) were produced under the same edaphoclimatic conditions in the experimental area of the Department of Agriculture. In the subsequent season, these seeds were sown in plastic pots under the same climatic conditions (Figure 1) in a greenhouse, and the treatments were defined by combining the values from 0 to 75%, with four replications, of contamination with a second cultivar. Each plot was composed of six pots, containing two plants per pot, and the level of contamination and the contaminating cultivar were randomly defined within the plots. Identification of the cultivars was carried out by four evaluators who were unaware of the percentage of contamination in each plot.

Figure 1
Climatic conditions: relative humidity (%) and maximum, minimum and average temperatures (°C) inside the greenhouse, between the months of June and October 2023. UFLA, Lavras, MG, Brazil.

The contamination was aimed at assessing the ability of the evaluators to differentiate the cultivars based on morphological descriptors. Concomitantly, under the same conditions, plants of all cultivars were cultivated and served as a reference for the evaluators.

During the evaluations, the morphological descriptors recommended by the International Union for the Protection of New Varieties of Plants (UPOV) for chickpea crop were considered: growth habit, ramification, presence of anthocyanin in the stem, intensity of green color in the leaves, type of leaf, flower color, seed color, intensity of seed color, seed shape and ribbing, time of flowering, seed weight and time of maturity.

The evaluations were carried out at 40, 100 and 140 days after sowing, representing the vegetative, reproductive/flowering and pre-harvest stages, respectively. Accuracy and error rates were calculated. Accuracy rate was calculated by the quotient between the observed frequency and the expected frequency, multiplied by one hundred.

The experimental design used was completely randomized. The standard deviation of the mean was calculated according to Pimentel-Gomes (1987). Significance of the deviations between the results obtained by the evaluators was assessed using the chi-square test (χ²). In this test, the deviations were transformed into a single value of χ², representing the standardized measure of the magnitude of the deviations (Ramalho et al., 2012). The value of χ² was estimated by the following expression: $\mathrm{\chi }2\mathrm{}=\mathrm{}\sum \mathrm{}(\mathrm{F}\mathrm{o}\mathrm{}-\mathrm{}\mathrm{F}\mathrm{e})²\mathrm{}/\mathrm{}\mathrm{F}\mathrm{e}$, where Fo = observed frequency of contaminating plants; and Fe = expected frequency of contaminating plants.

Experiment II: Evaluation of molecular descriptors

For the molecular analyses, 50 seeds of the same cultivars evaluated in Experiment I were sampled, and the samples were prepared according to Konarev and Gavriljuk (1988).

Electrophoretic analysis of isoenzymes was carried out using the Nativa PAGE method, following the protocol proposed by Alfenas (2006) for their extraction. The electrophoretic run was performed in a system of 7.5% (separator gel) and 4.5% (concentrator gel) polyacrylamide gels. The gels were revealed for the systems alcohol dehydrogenase (ADH), catalase (CAT), esterase (EST), malate dehydrogenase (MDH), according to methodologies described by Alfenas (2006), with three biological replications, each composed of 50 seeds, and two technical duplicates for analysis.

Electrophoretic analysis of heat-resistant proteins was carried out according to José et al. (2005), and electrophoretic analysis of storage proteins, which are the proteins that act as a nutrient reserve and that, in the case of legumes, have globulins as their largest fraction, was performed using the SDS-PAGE method, according to the protocol of Vieira (2009). Electrophoresis and gel development were performed according to Alfenas (2006).

In order to verify the influence of environmental factors on protein expression, seeds of the cultivars BRS Aleppo, BRS Kalifa and BRS Toro were multiplied, under two levels of nitrogen fertilization, in a split-plot scheme, according to planting recommendations by Nascimento (2016). Plots contained the cultivars and subplots contained two nitrogen levels, 50 kg.ha^-1 and 200 kg.ha^-1 according to Barbosa et al. (2022), applied as topdressing, 30 days after seedling emergence. The analytical procedures, as well as the isoenzymes and proteins evaluated, were the same as those previously described.

DNA extraction was performed on leaves of the 10 cultivars, collected 30 days after sowing and stored at -80 °C, using the CTAB method according to Castro et al. (2011) for C. arietinum. The DNA obtained was quantified in a NanoDrop ND-100 spectrophotometer and in 0.8% agarose gel.

For the evaluation of the microsatellite markers (SSR), 50 pairs of species-specific primers, described as polymorphic for chickpea crop (Castro et al., 2011; Sefera et al., 2011; Getahun, et al., 2021), were tested.

The amplification products, from DNA and protein analyses, were used to generate a genetic similarity matrix, by recording the presence (1) and absence (0) of bands for each cultivar. The estimate of genetic similarity (Sgij) between each pair of genotypes was calculated by Jaccard’s coefficient. The genotypes were clustered by the UPGMA (Unweighted Pair-Group Method), using the NTSYS program version 2.11.

RESULTS AND DISCUSSION

In the evaluation of the morphological descriptors (Table 1), the cultivars BRS Kalifa, BRS Cícero, BRS Toro, BRS Aleppo, BRS Cristalino, 10108 and 10209 showed an erect growth habit and the cultivars BRS Hari, UPL 05 and UPL 06 showed an erect to semi-erect growth habit. Plant ramification varied among the cultivars and, for this trait, there were plants with strong ramification (BRS Kalifa and BRS Cristalino), medium ramification (BRS Aleppo, BRS Hari,) medium to weak ramification (BRS Toro) and weak ramification (BRS Cícero, 10108, 10209, UPL 05 and UPL 06).

Table 1
Characterization of 10 chickpea cultivars based on UPOV descriptors.

	BRS Kalifa	BRS Cícero	BRS Toro	BRS Aleppo	BRS Cristalino	10108	BRS Hari	10209	UPL 05	UPL 06
Growth habit	Erect	Erect	Erect	Erect	Erect	Erect	Erect to semi-erect	Erect	Erect to semi-erect	Erect to semi-erect
Ramification	Strong	Weak	Weak to medium	Medium	Strong	Weak	Medium	Weak	Weak	Weak
Anthocyanin coloration in stem	Absent	Absent	Absent	Absent	Absent	Present	Present	Present	Present	Present
Leaflet size	Small to medium	Large	Small to medium	Small to medium	Small to medium	Small to medium	Small to medium	Small to medium	Small to medium	Small to medium
Intensity of green color of leaves	Medium	Light to medium	Light to medium	Light to medium	Light to medium	Light to medium	Light to medium	Light to medium	Medium	Medium
Type of leaf	Pinnate	Pinnate	Pinnate	Pinnate	Pinnate	Pinnate	Pinnate	Pinnate	Pinnate	Pinnate
Time of flowering	Early	Early	Intermediate	Intermediate	Late	Intermediate	Intermediate	Intermediate	Early	Intermediate
Flower color	White	White	White	White	White	Purplish pink	Purplish pink	Purplish pink	Purplish pink	Purplish pink
Pod size	Medium	Large	Large	Medium to large	Medium to large	Small to medium	Small	Very small to small	Small	Small
Intensity of green color of pods	Medium	Light to medium	Medium	Medium	Medium	Light to medium	Medium	Light to medium	Light to medium	Light to medium
Seed color	Greyed brown	Greyed brown	Greyed brown	Greyed brown	Greyed brown	Reddish brown	Brown	Brown	Brown	Brown
Intensity of color of seeds	Light	Light	Light	Light	Light	Medium	Dark	Dark	Dark	Dark
Seed shape	Round to angular	Round to angular	Round to angular	Round to angular	Round to angular	Round	Round	Angular	Angular	Angular
Seed ribbing	Weak	Medium	Strong	Strong	Weak	Medium	Absent or very weak	Very strong	Strong	Strong
Seed weight	High	Very high	Medium	Medium	Medium	Very low	Medium	Medium	Low	Medium
Time of seed maturity	Late	Early	Intermediate	Early	Early	Intermediate	Early	Intermediate	Early	Early

It was possible to observe the presence of anthocyanin in all cultivars of the desi group. In the vegetative stage, the presence of anthocyanin was observed only in the stem, at ground level, and in petioles; in the reproductive stage, it was observed in petioles, stems and pedicels (Figure 2). In a study carried out with cultivars of the genus Cicer, it was found that the genes for anthocyanin expression in the corolla, for example, have pleiotropic effects on pigmentation in other parts of the plant, with evidence that multiple alleles may be involved in its expression (Singh et al., 2006).

Figure 2
Anthocyanin pigmentation in chickpea cultivars of the desi group.

The cultivar BRS Cícero differed from the others as it had larger leaflets and morphological differences in the leaf apexes. These differences were reported by Puntel et al. (2023), who used mathematical models to estimate the leaf area of chickpea cultivars and found that the same mathematical model used for the cultivars BRS Aleppo, BRS Kalifa, Jamu and BRS Toro could not be applied to BRS Cícero due to the specific leaf characteristics of this cultivar. All cultivars used in this study have pinnate leaves.

For the time of flowering and time of maturity, the cultivars could be classified as early, intermediate and late. BRS Kalifa was the earliest in flowering (42 days), but the latest in terms of maturity (145 days). The second earliest was BRS Cícero, which reached flowering at 45 days after sowing and was also the earliest for maturity (100 days), whereas BRS Cristalino had the latest flowering, 73 days for 50% of the plants to reach full flowering.

Daba et al. (2016) reported that the genes that control the flowering period of chickpeas are sensitive to temperature and latitude changes and that chickpea plants flowered earlier on long days and under higher temperatures than on short days and under lower temperatures.

It was possible to distinguish all cultivars that belong to the kabuli group and the desi group based on flower color. For chickpeas, this trait is monogenic and has complete dominance (Atanasova and Mihov, 2006; Hasan and Deb, 2013; Burse et al., 2017). These characteristics are important for the characterization of cultivars because they are not fundamentally influenced by the environment to the point of promoting changes in phenotypic expression.

In the evaluation of 100-seed weight, the cultivars were classified into groups of very low weight seeds (< 20 g) for 10209, low weight seeds (20-25g) for 10108, medium weight seeds (26-35 g) for UPL 05, UPL 06, BRS Hari, BRS Aleppo, BRS Toro and BRS Cristalino, high weight seeds (36-45 g) for BRS Kalifa and very high weight seeds (> 45 g) for BRS Cícero. Seed weight is a complex trait because it involves a greater number of genes. In general, quantitative traits are more sensitive to environmental variations than qualitative traits.

Regarding seed color and color intensity, according to the classifications available in the UPOV descriptors, the five cultivars of the kabuli group were classified as grayish brown and with light color intensity. Seeds of the cultivars of the desi group were classified as reddish brown and brown, with medium to dark color intensity. Regarding the shape of the seeds, those of the kabuli group are round to angular and those of the desi group are round or angular. Regarding seed ribbing, there was a diverse variation within the two major groups. For the cultivars of the kabuli group, classification was weak for BRS Kalifa and BRS Cristalino, medium for BRS Cícero, and strong for BRS Toro and BRS Aleppo. As for the ribbing in seeds of the desi group, the classification was absent or very weak for the cultivar BRS Hari, medium for 10108, strong for UPL 05 and UPL 06, and very strong for 10209.

Regarding the evaluation carried out by the specialists (Table 2) in the vegetative stage, there was an accuracy rate ≥ 50% for six cultivars. The highest accuracy rate was 96.1% for the cultivar BRS Cícero, with distinct leaf size and morphology from the other cultivars. The lowest accuracy rate occurred for the cultivar 10209, 29.8%, which did not have any characteristics that would allow it to be distinguished from the others.

Table 2
Results of identification (% of accuracy and % of error) of plants of different chickpea cultivars, in three evaluation periods.

Cultivar	Evaluation periods
		Vegetative stage	Flowering	Pre-harvest
BRS Kalifa	% accuracy	52.7 ± 20.2*	56.9 ± 20.2	38.8 ± 17.2
	% error	29.1 ± 4.66	22.4 ± 7.0	38.5 ± 4.3
BRS Cícero	% accuracy	96.1 ± 6.8	98.6 ± 2.3	52.6 ± 11.2
	% error	3.6 ± 3.7	1.6 ± 1.7	26.0 ± 6.2
BRS Toro	% accuracy	68.3 ± 7.3	46.4 ± 11.8	37.5 ± 7.8
	% error	31.2 ± 9.0	36.4 ± 3.5	35.4 ± 12.6
BRS Aleppo	% accuracy	63.3 ± 7.5	52.0 ± 9.1	43.7 ± 3.6
	% error	22.3 ± 5.8	23.4 ± 6.8	30.7 ± 7.0
BRS Cristalino	% accuracy	38.8 ± 7.4	50 ± 9.4	36.2 ± 11.9
	% error	34.4 ± 3.5	28.6 ± 3.1	38.0 ± 2.7
10108	% accuracy	46 ± 11.5	43.4 ± 6.9	45.6 ± 13.9
	% error	39.5 ± 3.3	35.9 ± 3.4	39.5 ± 2.9
BRS Hari	% accuracy	41.7 ± 8.5	65.7 ± 16.9	46.0 ± 15.5
	% error	36.9 ± 6.3	28.1 ± 3.5	39.5 ± 8.7
10209	% accuracy	29.8 ± 9.7	48.8 ± 6.2	61.9 ± 17.5
	% error	51.5 ± 8.8	38.0 ± 6.5	31.8 ± 5.2
UPL 05	% accuracy	50 ± 13.2	72.2 ± 12.4	63.8 ± 12.1
	% error	43.2 ± 5.4	24.4 ± 7.4	24.5 ± 7.3
UPL 06	% accuracy	61.1 ± 21.9	73.5 ± 12.8	63.2 ± 20.5
	% error	30.7 ± 12.1	19.2 ± 6.1	26.5 ± 7.0

• Standard deviation from the mean*

At flowering, the highest accuracy rate (98.6%) was observed for the cultivar BRS Cícero. In addition to the characteristics of the leaves, the color of the flower was indicated by the evaluators as a determining factor to differentiate this cultivar from the others. For the cultivar BRS Toro, the accuracy rate was 46.4%, the lowest percentage among the cultivars, which can be attributed to the lack of synchronization in flowering, compared to the other cultivars. At this stage, there was an accuracy rate greater than or equal to 50% for seven cultivars. At 40 days, most cultivars were differentiated with a higher accuracy rate at flowering, when compared to the vegetative stage.

At 140 days after sowing, a significant reduction in the accuracy rate (< 50%) was observed for all cultivars of the kabuli group, except for BRS Cícero. For the cultivars of the desi group, an increase in accuracy rate, in relation to flowering, was observed only in 10108 and 10209, a result that can be explained by the difference between the cycles of the cultivars. Thus, for the distinction of chickpea cultivars, it is necessary to use several morphological characteristics of seeds and plants at different phenological stages.

The results of the chi-square test (χ²) showed significance for the cultivars BRS Cristalino in the pre-harvest (χ² = 8.41) and 10209 in the vegetative stage (χ² = 10.56). For the evaluation carried out at flowering, there was no significance of the deviations, which indicates that the errors were due to chance. In the evaluations carried out for the cultivar BRS Cícero in the vegetative and flowering stages, very low chi-square values and high accuracy rates were observed.

When comparing the chi-square results with those of the error and accuracy rates, the significance of the deviations were observed in stages and cultivars with the lowest accuracy rates, which can be explained mainly by the absence of morphological descriptors for the differentiation of the evaluated cultivars.

It is worth pointing out that, despite the possibility of using morphological markers to differentiate cultivars, also aiming at the RNC, it is important to use other descriptors of biochemical or molecular nature, considering that some cultivars are very similar morphologically, which contributed to the low accuracy rates and high chi-square values.

In general, among the descriptors evaluated, anthocyanin coloration in the stem, flower color, seed color, seed color intensity, and seed shape were the safest morphological descriptors for distinguishing the kabuli and desi groups (Table 1).

Regarding the protein analysis, for the enzyme alcohol dehydrogenase (ADH) (Figure 3) it was possible to separate only cultivar 10209 from the others. The genes that code for ADH are expressed in plant tissues at different developmental stages and their activity can be induced by different environmental stresses (Shi et al., 2017).

Figure 3
Zymogram of the isoenzyme alcohol dehydrogenase (ADH) in seeds of chickpea cultivars.

By means of the isoenzyme malate dehydrogenase (MDH) it was possible to separate the cultivars of the kabuli group from those of the desi group (Figure 4). In the first group, composed of the cultivars BRS Kalifa, BRS Cícero, BRS Toro, BRS, Aleppo and BRS Cristalino, an isoform of higher molecular weight was observed. For the second group, composed of cultivars 10108, BRS Hari, 10209, UPL 05 and UPL 06, this isoform was absent. Yudina (2012) reports that this isoenzyme can be considered a safe marker in genetic studies, as it has multiple molecular forms and high activity in several plant organs and tissues.

Figure 4
Zymogram of the isoenzyme malate dehydrogenase (MDH) in seeds of chickpea cultivars.

For catalase (CAT) (Figure 5), it was possible to distinguish only cultivar 10108 from the others, through the absence of an isoform with higher molecular weight. The presence of multiple catalase isoenzymes suggests the structural and functional versatility of this system in a variety of plant species. For chickpea crop, this isoenzyme system is widely applied for the classification of cultivars in relation to tolerance to low temperatures (Kaur et al., 2012), drought (Raheleh et al., 2012) and infection by pathogens (Gayatridevi et al., 2012).

Figure 5
Zymogram of the isoenzyme catalase (CAT) in seeds of chickpea cultivars.

By means of esterase (EST) (Figure 6), it was possible to separate the cultivars into three groups: the first composed of BRS Kalifa, BRS Cícero, BRS Toro, BRS Aleppo and BRS Cristalino, the second of cultivar 10108, and the third of the cultivars BRS Hari, 10209, UPL 05 and UPL 06. It is worth pointing out that the isoenzyme esterase has an affinity for different substrates, which often explains the greater polymorphism in plants (Scandalios, 1969; Gillespie and Langley, 1974; Weeden and Wendel, 1990). Begum and Alam (2019) observed a distinct polymorphic pattern of esterase among nine chickpea cultivars.

Figure 6
Zymogram of the isoenzyme esterase (EST) in seeds of chickpea cultivars.

For storage proteins (Figure 7), about 10 molecular weight bands ranging from 100 kDa to 10 kDa were detected. The protein profiles observed for cultivars 10108 and BRS Hari were the most distinct. For the cultivar 10108, there was absence of the molecular weight bands of 90 kDa and 70 kDa. In the cultivar BRS Hari, there was absence of the 90 kDa peptide and presence of the 70 kDa peptide, making it different from the 10108 cultivar and the others.

Figure 7
Zymogram of storage proteins in seeds of chickpea cultivars.

Analysis of storage proteins using the SDS PAGE electrophoretic system is widely used and recommended for chickpea crop, for the identification of cultivars and in studies of genetic diversity and variability (Hameed et al., 2012; Jha et al., 2012; Wakasa and Takaiwa, 2013; Begum and Alam, 2019).

In chickpea seeds, protein bands of 10, 11 and 12 kDa can be identified as albumins (2S) (Vioque et al., 1998), and bands of 15, 18, 33, 35, 37 and 50 kDa are attributed to the protein vicilin (7S) (Gueguen, 1991). In addition, peptides with weights of 24, 25 and 42 kDa may be associated with legumins (11S), while those with weights of 55 and 58 kDa may be associated with glutelins, and bands with high molecular weight of ~97 kDa can be attributed to lipoxygenases (Agrawal et al., 2013; Chang et al., 2022).

For heat-resistant proteins (Figure 8), low polymorphism was observed. In general, four bands were obtained, ranging from 90 kDa to 15 kDa. It was possible to separate only cultivar 10108 from the others, due to the absence of the 90 kDa molecular weight band.

Figure 8
Zymogram of heat-resistant proteins in seeds of chickpea cultivars.

In the clustering of cultivars (Figure 9), it was possible to observe the formation of two groups, one formed by cultivar 10108, and the other formed by the other cultivars with a similarity coefficient of 0.89. In the latter, two subgroups were observed, consisting of cultivars of the kabuli group, namely BRS Kalifa, BRS Cícero, BRS Toro, BRS Aleppo and BRS Cristalino, with a similarity of 0.98, and cultivars of the desi group, namely BRS Hari, UPL 05, UPL 06 and 10209, with a similarity of about 0.95.

Figure 9
Dendrogram of the 10 chickpea cultivars studied, obtained by UPGMA cluster analysis, based on the Jaccard’s coefficient of similarity, using protein markers.

Maximum similarity coefficients were observed between the cultivars BRS Kalifa, BRS Cícero, BRS Aleppo and BRS Toro, from the kabuli group, and between the genotypes UPL 05 and UPL 06, from the desi group. The lowest genetic similarity was observed between cultivar 10108 (0.89), from the desi group, and the others evaluated.

Regarding the stability of the protein patterns, an important characteristic for the identification of cultivars and certification of the genetic purity of seed lots involving storage and heat-resistant proteins, no differences were found in the patterns for the three chickpea cultivars, BRS Aleppo, BRS Kalifa and BRS Toro, produced under two levels of nitrogen: 50 kg.ha^-1 and 200 kg.ha^-1.

Regarding the DNA analyses, among the 50 SSR primers tested, 24 were polymorphic, and for the others, monomorphic patterns were observed, or they were not amplified.

Only the electrophoretic profiles of those that allowed the distinction of the largest number of cultivars (TA-180, TA-11 and TA-203) were presented (Figure 10). By means of TA-180, the same pattern was observed for BRS Kalifa, BRS Cícero, BRS Cristalino, BRS Hari and 10209, as well as between cultivars 10108 and UPL 05, while the cultivars BRS Aleppo and BRS Toro were differentiated from the others. This primer has been used in genetic diversity studies for chickpea crop (Solanki et al., 2022). Ningwal et al. (2023) also verified the efficiency of the aforementioned primer for the distinction of 57 lines of the desi group. When investigating hybridism in chickpeas, Thakur et al. (2021) found that, of 51 pairs of SSR primers, hybrid plants were confirmed only through TA-180 (1.2% of the total SSRs).

Figure 10
Patterns observed from the amplification of TA-180, TA-11 and TA-203 primers in chickpea cultivars.

By the primer TA-11 it was possible to observe four distinct patterns, one for BRS Kalifa, BRS Aleppo, BRS Cristalino and UPL 05, another for the cultivars BRS Cícero, BRS Toro and UPL 06, and another for 10108 and BRS Hari. The highest number of bands was observed for the cultivar 10209, whose fragments varied between 900 bp and 400 bp. When using this primer, Castro et al. (2011) and Kosgei et al. (2022) observed a high level of polymorphism between cultivars for chickpeas.

By the primer TA-203, it was possible to distinguish all the cultivars used in this study. Therefore, this primer can be considered recommended for use as a marker to differentiate chickpea cultivars, for the national registry of cultivars and/or for the certification of the genetic purity of seed lots.

Valadez-Moctezuma et al. (2020) recommend the use of primer TA-203 for its efficiency in distinguishing chickpea cultivars from Mexican germplasm. In a study conducted by Südüpak (2013), this primer contributed to the study of allelic variation, with a high value of polymorphism information content (PIC) in Turkish varieties of chickpeas.

Regarding the genetic similarity study (Figure 11), four groups were formed as follows: Group 1 - BRS Kalifa, BRS Aleppo, BRS Cristalino and BRS Toro; Group 2 - BRS Cícero; Group 3 - BRS Hari and 10209, and Group 4 - 10108, UPL 05 and UPL 06.

Figure 11
Dendrogram of the 10 chickpea cultivars studied, obtained by UPGMA cluster analysis, based on Jaccard’s coefficient of similarity, based on microsatellite analysis.

The highest value of similarity (0.67) was observed between the cultivars UPL 05 and UPL 06, and the similarity index of these two cultivars in relation to cultivar 10108 was approximately 0.55. Although it was not possible to have access to the genealogy data of all cultivars, it was possible to observe that BRS Cristalino and BRS Kalifa share the same female parent (CNPH 035). The cultivar BRS Cristalino is also related to BRS Toro, as they have in common the parent GB 0528.

As observed in the proteomic analysis, the cultivars of the kabuli group, BRS Kalifa, BRS Aleppo, BRS Cristalino and BRS Toro, were classified in the same group by microsatellite analysis (Figure 11). In this group, the most related cultivars were BRS Aleppo and BRS Cristalino with approximate similarity of 0.66. These two cultivars have an approximate similarity of 0.62 with the cultivar BRS Kalifa. In this group, the greatest similarity was observed for BRS Toro, 0.53, compared to the other cultivars that are part of this group. Differences in similarity clusters were observed when using proteins and DNA by microsatellites. For the cultivar BRS Cícero, for example, there were high values of similarity between it and the other cultivars of the kabuli group, through proteomic analysis. However, when using the SSR technique, the cultivar BRS Cícero was one of the least similar, 0.41, compared to the group of the least similar cultivars in the desi group.

In accordance with Ordinance No. 502, which provides for the use of molecular markers as descriptors, aiming at RNC, SSR markers are interesting tools that can collaborate and complement the deposit of new materials with MAPA. The use of these markers allowed obtaining results of easy interpretation, making it possible to identify with clarity and precision a large number of cultivars, when compared to the morphological and protein descriptors.

Considering the guidelines provided by law, DNA markers become important tools for the registration of cultivars, since they are stable and therefore are not altered as a function of environmental conditions. In addition, there is a greater probability of distinguishing similar materials.

CONCLUSIONS

Chickpea cultivars can be distinguished and identified using a combination of morphological and molecular markers. The most appropriate developmental stage for identification varies among cultivars. The primer TA-203 stands out as an important marker to distinguish Brazilian chickpea cultivars, thus contributing to their registration.

ACKNOWLEDGMENTS

The authors would like to thank the research funding agencies Coordination for the Improvement of Higher Education Personnel (CAPES - Brazil), Minas Gerais State Research Support Foundation (FAPEMIG - Brazil) and National Council for Scientific and Technological Development (CNPq).

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• DATA AVAILABILITY
  Additional data will be made available by the authors upon reasonable request.

Editado por

• Editor:
  Denise Cunha Fernandes dos Santos Dias

Disponibilidade de dados

Additional data will be made available by the authors upon reasonable request.

Datas de Publicação

• Publicação nesta coleção
  13 Out 2025
• Data do Fascículo
  2025

Histórico

• Recebido
  16 Jun 2025
• Aceito
  19 Ago 2025