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Cytogenetic and morphological characterization of lima bean germplasm from the Brazilian Northeast region with a focus on genetic resource conservation

Abstract

In Brazil, lima bean is mainly grown in the Northeast region, where it is widely consumed and is of major economic importance. We evaluated different Phaseolus lunatus accessions from the Northeast of Brazil using agromorphological markers and CMA/DAPI fluorochrome banding techniques. All the accessions showed CMA+ blocks of pericentromeric constitutive heterochromatin (CH). At least one pair of CMA++ terminal marks corresponds to the nucleolus organizer region (NORs). Characterization of the seeds suggests that the two Andean and Mesoamerican Phaseolus cultigroups are represented in the Brazilian germplasm analyzed. Most of them belong to the "Big Lima" group. Characterization studies of lima bean germplasm are important for increasing knowledge of the diversity and variability of the species and for generating useful information for breeding and conservation of this economically important legume.

Keywords:
Phaseolus lunatus; karyotype analysis; CMA and DAPI fluorochromes; heterochromatin; seed characterization

INTRODUCTION

The Phaseolus L. genus, composed of ~75 species, is especially important among the numerous leguminous plants because five of its species are grown for food (Bitocchi et al. 2017Bitocchi E, Rau D, Bellucci E, Rodriguez M, Murgia ML, Gioia T, Santo D, Nanni L, Attene G, Papa R2017 Beans (Phaseolus ssp.) as a model for understanding crop evolution. Frontiers in Plant Science 8:1-17). At least seven independent domestication events have occurred within the genus, which have generated the reproductive isolation of five domesticated species: P. acutifolius A. Gray, P. coccineus L., P. polyanthus Greeman, P. lunatus L., and P. vulgaris L. (Delgado-Salinas et al. 2006Delgado-Salinas A, Bibler R, Lavin M2006 Phylogeny of the genus Phaseolus (Leguminosae): a recent diversification in an ancient landscape. Systematic Botany 31:779-791). Phaseolus lunatus L. (lima bean) is the second most economically important protein source of the genus (Ormeño-Orrilloet al. 2015Ormeño-Orrillo E, Servín-Garcidueñas LE, Rogel MA, González V, Peralta H, Mora J, Martínez-Romero J, Martínez-Romero E2015 Taxonomy of rhizobia and agrobacteria from the Rhizobiaceae family in light of genomics. Systematic and Applied Microbiology 38:287-291).

Mexico is the main diversity center of lima bean (with 90% of the species), while Europe and Brazil are considered secondary diversification centers (revised by Martínez-Nieto et al. 2020Martínez-Nieto MI, Estrelles E, Prieto-Mossi J, Roselló J, Soriano P2020 Resilience capacity assessment of the traditional Lima Bean (Phaseolus lunatus L.) landraces facing climate change. Agronomy 10:1-15). In Brazil, the Northeast region is the main producer of lima bean, where it has a positive economic impact on family farmers, since they are able to sell surplus production. It also has social importance, due to its nutritional benefits, improving quality of life (Assunção-Filho et al. 2022Assunção-Filho JRD, Costa MF, Pinheiro JB, Carvalho LCB, Ferreira-Gomes RL, Lopes ACDA2022 Selection of superior genotypes of lima bean landraces by multivariate approach. Revista Caatinga 35:87-95). The seeds of the P. lunatus L. species have high protein content, as well as carbohydrates, vitamins, and minerals, which make them a nutrient source for humans, especially in developing countries such as Africa and Latin America (Adebo 2023Adebo JA2023 A review on the potential food application of lima beans (Phaseolus lunatus L.), an underutilized crop. Applied Sciences 13:1-19).

Just as in other centers of P. lunatus, the Brazilian Northeast region could be a center of genetic diversity in which breeding programs could be generated for this crop, and this represents a great responsibility for local governments and research institutions. Diversity centers are subject to various risks, including loss of genetic diversity (Lustosa-Silva et al. 2022Lustosa-Silva JD, Ferreira‑Gomes RL, Jaime Martínez‑Castillo J, Carvalho LCB, Oliveira LF, Ortiz‑García MM, Sánchez‑Sosa AG, Silva GR, Costa MF, Silva VB, Lopes ACA2022 Genetic diversity and erosion in lima bean (Phaseolus lunatus L.) in Northeast Brazil. Genetic Resources and Crop Evolution 69:2819-2832). This affects the evolutionary potential of the species, leading to genetic vulnerability or reduction in the ability to adapt to environmental changes, thus increasing the possibility of extinction (Adebo 2023Adebo JA2023 A review on the potential food application of lima beans (Phaseolus lunatus L.), an underutilized crop. Applied Sciences 13:1-19, Lustosa-Silva et al. 2023Lustosa-Silva JD, Oliveira EG, Soares LAC, Ferreira‑Gomes RL, Costa AF, Barros RFM, Almeida RC, Silva VB, Costa MF, Lopes ACA2023 Traditional varieties of lima beans (Phaseolus lunatus L.) in northeastern Brazilian farms: conservation and sustainability. Genetic Resources and Crop Evolution 1:1-12). Therefore, characterization of accessions conserved in germplasm banks is crucial for maintaining genetic resources in diversity centers (Maxted et al. 2012Maxted N, Kell S, Ford-Lloyd B, Dulloo E, Toledo A2012 Toward the systematic conservation of global crop wild relative diversity. Crop Science 52:774-785).

Morphological characterization of seeds is one of the first steps to be carried out in germplasm banks or research centers; it helps in classification and making inferences regarding the origin and diversity of the accessions. Based on seed size, morphology, and variance in 100-seed weight, three commercial lima bean types have been described: Sieva and Potato seeds (with small, rounded seeds that are globular, flat, and kidney-shaped), both of Mesoamerican origin, and Big Lima seeds (which are larger and flat), of Andean origin (Mackie 1943Mackie WW1943 Origin, dispersal, and variability of the lima bean, Phaseolus lunatus. Hilgardia 15:1-29, Baudet 1977Baudet JC1977 The taxonomic status of the cultivated types of lima bean (Phaseolus lunatus L.). Tropical Grain Legume Bulletin 7:29-30, Castiñeiras et al. 1991Castiñeiras L, Esquivel MA, Rivero N, Mariño A1991 Variabilidad de la semilla de Phaseolus lunatus L. en Cuba. Revista del Jardín Botánico Nacional 12:109-114). For example, seed morphological characteristics (size, shape, and color) allowed differentiation of the two Potato and Sieva cultigroups, confirming the presence of both cultigroups in the landraces of lima bean grown in the Yucatan Peninsula (Esquivel-Martínez et al. 2023Esquivel-Martínez GT, Andueza-Noh RH, Garruña R, Villanueva-Couoh E, Martínez-Castillo J, Díaz-Mayo J, Ruiz-Santiago RR, Camacho-Pérez E2023 Morphological differentiation and seed quality of Lima bean (Phaseolus lunatus L.) Genetic Resources and Crop Evolution 71:69-81). In Brazil, Silva et al. (2017Silva RNO, Burle ML, Pádua JG, Lopes ACA, Gomes RLF, Martinez-Castillo J2017 Phenotypic diversity in lima bean landraces cultivated in Brazil, using the Ward-MLM strategy. Chilean Journal of Agricultural Research 77:35-40) used qualitative and quantitative descriptors through the Ward-MLM (Modified Location Model) and characterized several accessions of cultivated lima bean. They showed the presence of accessions with characteristics typical of the Mesoamerican and Andean cultigroups. Jesús-Pires et al. (2022Jesús-Pires C, Costa MF, Zucchi MI, Ferreira-Gomes RL, Pinheiro JB, Viana JPG, Bajay MM, Assunção-Filho JR, Almeida Lopes AC2022 Genetic diversity in accessions of lima bean (Phaseolus lunatus L.) determined from agro-morphological descriptors and SSR markers for use in breeding programs in Brazil. Genetic Resources and Crop Evolution 69:973-986) also investigated the genetic diversity of lima bean landraces using agromorphological and microsatellite markers (SSRs) from the Northeast, West Central, and Southeast regions, and data indicated a considerable diversity in traits related to agronomic performance, such as number of seeds per pod, and 100-seed weight. However, although some studies have evaluated the genetic diversity of lima bean in Brazil using agromorphological and molecular markers (Silva et al. 2017Silva RNO, Burle ML, Pádua JG, Lopes ACA, Gomes RLF, Martinez-Castillo J2017 Phenotypic diversity in lima bean landraces cultivated in Brazil, using the Ward-MLM strategy. Chilean Journal of Agricultural Research 77:35-40, Jesús-Pires et al. 2022Jesús-Pires C, Costa MF, Zucchi MI, Ferreira-Gomes RL, Pinheiro JB, Viana JPG, Bajay MM, Assunção-Filho JR, Almeida Lopes AC2022 Genetic diversity in accessions of lima bean (Phaseolus lunatus L.) determined from agro-morphological descriptors and SSR markers for use in breeding programs in Brazil. Genetic Resources and Crop Evolution 69:973-986) and cytogenetics (Bonifácio et al. 2012Bonifácio EM, Fonsêca A, Almeida C, Santos KGB, Pedrosa-Harand A2012 Comparative cytogenetic mapping between the lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.). Theoretical and Applied Genetics 124:1513-1520, Almeida and Pedrosa-Harand 2013Almeida C, Pedrosa-Harand A2013 High macro-collinearity between lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.) as revealed by comparative cytogenetic mapping. Theoretical and Applied Genetics 126:909-1916, Feitoza et al. 2017Feitoza L, Costa L, Guerra M2017 Condensation patterns of prophase/prometaphase chromosome are correlated with H4K5 histone acetylation and genomic DNA contents in plants. PLoS One 12:1-14), they included only a few or no genotypes collected from the Northeast of Brazil.

Cytogenetics provides the cytological features of a target species, such as number of chromosomes and morphology, the constitutive heterochromatin (CH) distribution pattern, ribosomal DNA sites, among others. Like most Phaseolus species, P. lunatus is a diploid, with 2n= 2x = 22 and mostly metacentric and submetacentric chromosomes. The mapping of 5S and 35S rDNA sites in four cultivated Phaseolus species revealed the presence of one or two 5S rDNA sites and from one to seven 35S rDNA sites located in chromosomes 6, 9, and 10 (Moscone et al. 1999Moscone EA, Klein F, Lambrou M, Fuchs J, Schweizer D1999 Quantitative karyotyping and dual-color FISH mapping of 5S and 18S-25S rDNA probes in the cultivated Phaseolus species (Leguminosae). Genome 42:1224-1233, Pedrosa-Harand et al. 2006Pedrosa-Harand A, Almeida CCS, Mosiolek M, Blair MW, Schweizer D, Guerra M2006 Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.). Theoretical and Applied Genetics 112:924-933). Bonifácio et al. (2012Bonifácio EM, Fonsêca A, Almeida C, Santos KGB, Pedrosa-Harand A2012 Comparative cytogenetic mapping between the lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.). Theoretical and Applied Genetics 124:1513-1520) constructed the first comparative cytogenetic map of P. lunatus, using previously mapped markers from P. vulgaris, and results showed a significant conservation of synteny among species. In addition, chromomycin A3 (CMA) fluorochrome reveled that all the pericentromeric regions of lima bean chromosomes are rich in GC (guanine and cytosine). Feitoza et al. (2017Feitoza L, Costa L, Guerra M2017 Condensation patterns of prophase/prometaphase chromosome are correlated with H4K5 histone acetylation and genomic DNA contents in plants. PLoS One 12:1-14) showed condensation patterns of the prophase/prometaphase chromosome correlated with H4K5 histone acetylation and CMA in some species, among them P. lunatus and P. vulgaris. The CMA+ bands in both species were found to be located in the proximal regions of all the chromosomes, and immunodetection of H4K5ac, an epigenetic mark universally associated with gene expression, revealed signals in the low-condensing region terminal, but not in the pericentromeric CMA+ bands.

Thus, considering that knowledge about the genetic diversity of a germplasm collection is essential for plant breeding programs and for conservation of genetic resources by allowing planning and execution of appropriate strategies, the aim of the present study was to cytogenetically characterize and morphologically classify the seeds of Phaseolus lunatus (lima bean) accessions, a species of great economic importance for the Brazilian Northeast region. The results generated have increased our knowledge about the diversity and variability of the P. lunatus accessions analyzed here and have provided additional assistance regarding the accessions in the germplasm banks of Brazil.

MATERIAL AND METHODS

Plant materials

Twenty cultivated lima bean accessions collected in several states of the Brazilian Northeast and part of thePhaseolus Germplasm Bank of the Universidade Federal de Piauí (PGB-UFPI, Brazil) were used in this study (Table 1). The seeds from the accessions were samples from the bank and were replicated in a greenhouse without cross-pollination for this and further studies.

Table 1
Accession, Provenance, Range of Chromosome Size (RCS), Karyotype Formula (KF), Total Chromosome Length (TCL), Mean Chromosome Length (MCL), Haploid Karyotype Length (HKL), and num ber of CMA/DAPI bands (CMA3/DAPI)

CMA/DAPI fluorochrome staining

Root tips of different lima bean accessions were pretreated in 0.002M 8-hydroxyquinoline for 24 hours at 10 ºC, fixed in a 3:1 ethanol-acetic acid solution, and stored at -20 ºC until use. For the CMA/DAPI fluorochrome staining, we followed Schweizer and Ambros (1994Schweizer D, Ambros PF1994 Chromosome banding: stain combinations for specific regions. In Gosden JR (ed) Chromosome analysis protocols. Human Press, Totowa, p. 97-112), with minor modifications. Root tips were digested with an enzymatic solution of 2% cellulase (Onozuka R-10) and 20% pectinase (Sigma). The slides were stained with 10 µL of CMA (0.5 mg mL-1) for 1h, counterstained with 10 μl of DAPI (2 mg mL-1) for 30 min, mounted in glycerol/McIlvaine (1:1), and stored for three days before analysis.

Image analyses and morphometry

The five metaphases of each accession were photographed using a DF7000GT digital camera coupled to a Leica DM4B microscope. The images were optimized for brightness and contrast using Adobe Photoshop CS3. Chromosome sizes were measured using the Drawid v0.26 (Kirov et al. 2017Kirov I, Khustaleva L, Laere KV, Soloviev A, Meeus S, Romanov D, Fesenko I2017 DRAWID: user-friendly java software for chromosome measurements and idiogram drawing. Comparative Cytogenetics 11:747-757). Idiograms were constructed using Corel DRAW (2017) and chromosome morphologies and parameters (Table 1) were classified according to Guerra (2002Guerra M2002 Como observar cromossomos: um guia de técnicas em citogenética vegetal, animal e humana. FUNPEC, Ribeirão Preto, 131p).

Morphological characterization of the seeds

We measured the length, width, and thickness of 10 randomly selected seeds for each of 16 accessions. The measurements were performed using a digital caliper (in mm), according to IPGRI (2001IPGRI - International Plant Genetic Resources Institute2001 Descritores para Phaseolus lunatus L. International Plant Genetic Resources Institute, Rome, 42p). The 100-seed weight was determined, and the values were established in grams (g) according to Mackie (1943Mackie WW1943 Origin, dispersal, and variability of the lima bean, Phaseolus lunatus. Hilgardia 15:1-29). Seed shape was based on the J and H coefficients, following the established protocols of Puerta Romero (1961Puerta Romero J1961 Variedades de judías cultivadas em España. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria. Ministerio de Agricultura, Subdirección de Capacitación Agraria, Madrid, 798p) and Vilhordo (1996Vilhordo BW, Mikusinski OMF, Burin ME, Gandolfi VH1996 Morfologia. In Araujo RS, Rava CA, Stone LF and Zimmermann MJO (eds) Cultura do feijoeiro comum no Brasil. POTAFOS, Piracicaba, p. 71-99), and seeds were classified as spherical (J = 1.16 - 1.42), elliptical (J = 1.43 to 1.65), oblong/short reniform (J = 1.66 to 1.85), oblong/mean reniform (J = 1.86 to 2.00), and oblong/long reniform (J > 2.00). According to the H coefficient, seeds were classified as flattened (H < 0.69), semi-flattened (H =0.70 to 0.79), and full (H > 0.80). Statistical analysis was performed by the Scott-Knott test, with 5% probability.

RESULTS AND DISCUSSION

All lima bean accessions showed 2n=22 predominantly metacentric chromosomes, and late-condensed terminal chromatin was found in most of the accessions analyzed (Figures 1 and 2). According to the Guerra (1983Guerra M1983 O uso de Giemsa na citogenética vegetal - comparação entre a coloração simples e o bandeamento. Ciência & Cultura 35:190-193) nomenclature proposal, chromosomes were classified as metacentric (m) and submetacentric (sm). Most of the Phaseolus Germplasm Bank accessions (PGB-UFPI 793, 794, 796, 799, 800, 801, 803, 807, 810, and 814) showed karyotype formula 11M, while other accessions had the formula 10M+1SM (PGB-UFPI 790, 795, 804, 809, and 857) and 9M+2SM (PGB-UFPI 797, 806, 815, 816, and 817) (see Table 1).

The chromosome size ranged from 0.85µm (PGB-UFPI 790) to 3.14 µm (PGB-UFPI799), corroborating previous studies (Mercado-Ruaro and Delgado-Salinas 2009Mercado-Ruaro P, Delgado-Salinas A2009 Karyotypic analysis in six species of Phaseolus L. (Fabaceae). Caryologia 62:167-170). Moscone et al. (1999Moscone EA, Klein F, Lambrou M, Fuchs J, Schweizer D1999 Quantitative karyotyping and dual-color FISH mapping of 5S and 18S-25S rDNA probes in the cultivated Phaseolus species (Leguminosae). Genome 42:1224-1233) showed the metaphase mitotic chromosomes ranged from 1.7µm to 3.5µm, and in Sarbhoy (1977Sarbhoy RK1977 Cytogenetical studies in the genus Phaseolus Linn. Cytologia 42:401-413) and others, Phaseolus varieties ranged from 0.70 µm to 3.00 µm. With respect to Total Chromosome Length (TCL), accessions of the Phaseolus Germplasm Bank had sizes ranging from 26.10 µm (PGB-UFPI 790) to 52.91 µm (PGB-UFPI 799). Similarly, Moscone et al. (1999) reported TCL of 27.38 µm for P. lunatus, and in fava bean species, they reported 23.32 µm (P. xanthotrichus), 26.0 µm (P. maculatus), and 29.74 µm (P. coccineus). However, these differences may be related to unequal degrees of chromosome condensation during cell division, to differences in the pretreatment, and/or to different classes of repetitive DNA sequences, as reported for other species (Moscone 1990Moscone EA1990 Chromosome studies on Capsicum (Solanaceae) I. Karyotype analysis in C. chacoense. Brittonia 42:147-154, Pozzobon et al. 2006Pozzobon MT, Schifino-Wittmann MT, Bianchetti LDB2006 Chromosome numbers in wild and semidomesticated Brazilian Capsicum L. Botanical Journal of the Linnean Society 151:259-269, Almeida et al. 2022Almeida BM, Martins LV, Lopes ACA, Gomes RLF, Valente SES, Peron AP, Silva VB, Feitoza LL2022 Karyotype polymorphism of GC-rich constitutive heterochromatin in Capsicum L. pepper accessions. Crop Breeding and Applied Biotechnology 22:e38642113).

We identified the heterochromatin blocks of the P. lunatus accessions using the fluorochromes chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI). All the accessions analyzed showed GC-rich heterochromatin blocks (CMA+/DAPI-) located at the pericentromeric regions, with different sizes and intensities of the signals. We identified at least two terminal CMA++/DAPI- bands in all accessions, probably corresponding to the nucleolar organizer regions (NORs) (Figure 1, Table 1). AT+ blocks were not observed there. Idiograms of each accession are represented in Figure 2.

Figure 1
Double staining with CMA (yellow) and DAPI (blue) fluorochromes in some P. lunatus accessions (merged image). Arrows indicate CMA block in the pericentromeric chromosome regions and arrowheads indicate terminal or subterminal bands. The number of each accession is indicated at the left side of the metaphases. Inserts indicate small CMA+ blocks in a region difficult to detect. Bar = 10μm.

Figure 2
Idiograms representing size, morphology, and distribution of the CMA blocks (yellow bands) located on the chromosome of the P. lunatus accessions analyzed. SM = Submetacentric.

One of the most interesting characteristics associated with plant chromosomes is the amount of constitutive heterochromatin in the pericentromeric regions of the chromosomes. Cytogenetic studies have contributed to the characterization of the heterochromatin and repetitive sequence distribution pattern in Phaseolus and related genera, such as Vigna (Fonsêca and Pedrosa-Harand 2017Fonsêca A, Pedrosa-Harand A2017 Cytogenetics and comparative analysis of Phaseolus species. In Pérez de la Vega M, Santalla M and Marsolais F (eds) The common bean genome. Part of the Compendium of Plant Genomes book series (CPG), Springer, Berlin, p. 57-68, Oliveira et al. 2020Oliveira ARS, Martins LV, Bustamante FO, Muñoz-Amatriaín M, Close T, Costa AF, Benko-Iseppon AM, Pedrosa-Harand A, Brasileiro-Vidal AC2020 Breaks of macrosynteny and collinearity among moth bean (Vigna aconitifolia), cowpea (V. unguiculata), and common bean (Phaseolus vulgaris). Chromosome Research 28:293-306, Ribeiro et al. 2020Ribeiro T, Vasconcelos E, Santos KGB, Vaio M, Brasileiro-Vidal AC, Pedrosa-Harand A2020 Diversity of repetitive sequences within compact genomes of Phaseolus L. beans and allied genera Cajanus L. and Vigna Savi. Chromosome Research 28:139-153). Almeida and Pedrosa-Harand (2013Almeida C, Pedrosa-Harand A2013 High macro-collinearity between lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.) as revealed by comparative cytogenetic mapping. Theoretical and Applied Genetics 126:909-1916) demonstrated that lima bean and common bean (P. vulgaris L.), both with 2n = 22, share common CMA sequences in the pericentromeric and in the NOR regions. Similarly, Bonifácio et al. (2012Bonifácio EM, Fonsêca A, Almeida C, Santos KGB, Pedrosa-Harand A2012 Comparative cytogenetic mapping between the lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.). Theoretical and Applied Genetics 124:1513-1520) observed a karyotype in lima bean composed of 22 predominantly metacentric chromosomes that have pericentromeric regions rich in CMA+/DAPI- heterochromatin, forming blocks of different sizes and intensities, which confirms the strong similarity and karyotypic stability of the genus. In both species, the pericentromeric region had DNA hypermethylation (5mC) and H4K5ac histone hypoacetylation, epigenetic marks associated with the formation and maintenance of constitutive heterochromatin (Fonsêca et al. 2014Fonsêca A, Richard MS, Geffroy V, Pedrosa-Harand A2014 Epigenetic analyses and the distribution of repetitive DNA and resistance genes reveal the complexity of common bean (Phaseolus vulgaris L., Fabaceae) heterochromatin. Cytogenetic and Genome Research 143:168-178, Feitoza et al. 2017Feitoza L, Costa L, Guerra M2017 Condensation patterns of prophase/prometaphase chromosome are correlated with H4K5 histone acetylation and genomic DNA contents in plants. PLoS One 12:1-14).

In addition to cytogenetic characterization, we also traced a morphological profile of the seeds. We observed variation regarding the length, width, and thickness of the seeds. Considering overall length, the accession PGB-UFPI 804 showed statistically significant difference from the accession PGB-UFPI 790, and both had the longest seeds, at 18.38 mm and 19.58 mm, respectively. The accessions with the shortest seeds were PGB-UFPI 817 with 11.97 mm and PGB-UFPI 816 with 11.03 mm, with no statistically significant difference between them (Table 2).

Table 2
Measurement of the length, width, and thickness and classification of the shape, profile, and 100-seed weight of the lima bean seeds from the PGB-UFPI (Phaseolus Germplasm Bank of the Universidade Federal de Piauí)

For seed width, with an overall average of 10.94 mm, the highest values were observed for PGB-UFPI 814 (12.27 mm) and PGB-UFPI 790 (12.77 mm). The smallest values were observed for PGB-UFPI 817 (9.02 mm) and PGB-UFPI 816 (8.20 mm), with flattened seeds, with statistical difference between them (Table 2).

Jesús Pires et al. (2022Jesús-Pires C, Costa MF, Zucchi MI, Ferreira-Gomes RL, Pinheiro JB, Viana JPG, Bajay MM, Assunção-Filho JR, Almeida Lopes AC2022 Genetic diversity in accessions of lima bean (Phaseolus lunatus L.) determined from agro-morphological descriptors and SSR markers for use in breeding programs in Brazil. Genetic Resources and Crop Evolution 69:973-986) investigated the genetic variability of creole varieties of fava beans from agromorphological descriptors and SSR markers and found that the average seed length ranged from 10.10 mm to 16.61 mm. In a similar study using both agromorphological and SSR analyses from the germplasm collection at the Universidade Federal do Piauí (UFPI), different values were found for length, width, and thickness, from 9.65 to 18.52 mm, 7.41 to 11.83 mm, and 5.32 to 6.90 mm, respectively (Melo et al. 2023Melo LF, Silva SCCC, Costa GN, Silva VB, Pinheiro JB, Zucchi MI, Costa MF, Ferreira-Gomes RL, Lopes ÂCA2023 Assessment of genetic diversity in Phaseolus lunatus Landrace germplasm for use in breeding programs. Plant Molecular Biology Reporter 41:292-303). According to Silva et al. (2017Silva RNO, Burle ML, Pádua JG, Lopes ACA, Gomes RLF, Martinez-Castillo J2017 Phenotypic diversity in lima bean landraces cultivated in Brazil, using the Ward-MLM strategy. Chilean Journal of Agricultural Research 77:35-40), there is wide variation regarding the length and width of P. lunatus seeds, which contributes to the genetic variability of this species. The values for these traits found in this study were 8.28 to 22.53 mm and 6.54 to 14.17 mm, respectively. Thus, the results of seed morphology found here indicate there is diversity among the lima bean accessions belonging to the PGB-UFPI, showing the variability of the seeds according to the parameters analyzed (Table 2).

After measurement, the seeds were classified according to the J and H coefficients (Table 2). For the first coefficient, the accessions that had the highest values were PGB-UFPI 804 and PGB-UFPI 806, while the smallest were for PGB-UFPI 803 and PGB-UFPI 799. For the H coefficient, the highest value was obtained for the accessions PGB-UFPI 793, PGB-UFPI 816, and PGB-UFPI 817; and the shortest values were obtained for PGB-UFPI 790 and PGB-UFPI 803.

For the J coefficient, the results showed predominance of seeds with spherical form (PGB-UFPI 793, 795, 797, 799,800, 803, 810, 814, 816, 817, and 857), elliptical form (PGB-UFPI 790, 794, 796, 801, 804, 806, 807, 809, and 815), and flattened profile for all accessions (Table 2). Our results differ from Santos et al. (2002Santos D, Corlett FMF, Mendes JEMF, Wanderley Júnior JSA2002 Produtividade e morfologia de vagens e sementes de variedades de fava no Estado da Paraíba. Pesquisa Agropecuária Brasileira 37:1407-1412), who found different types of forms for the seeds from the analyzed accessions.

Based on molecular information, P. lunatus consists of two major gene pools: the Andean gene pool, which includes the landraces classified within the Big Lima cultigroup (with larger, flat shaped seeds), and the Mesoamerican gene pool (MI and MII), which includes the landraces classified within the Sieva and Potato cultigroups (with smaller, globular, flat, and kidney-shaped seeds) (Andueza-Noh et al. 2013Andueza-Noh RH, Serrano-Serrano ML, Sánchez MIC, Del Pino IS, Camacho-Pérez L, Coello-Coello J, Cortes JM, Debouck DG, Martínez-Castilho J2013 Multiple domestications of the Mesoamerican gene pool of lima bean (Phaseolus lunatus L.): evidence from chloroplast DNA sequences. Genetic Resources and Crop Evolution 60:1069-1086, Martínez-Castillo et al. 2014Martínez-Castillo J, Camacho-Pérez L, Villanueva-Viramontes S, Andueza-Noh RH, Chacón-Sánchez MI2014 Genetic structure within the Mesoamerican gene pool of wild Phaseolus lunatus (Fabaceae) from Mexico as revealed by microsatellite markers: Implications for conservation and the domestication of the species. American Journal of Botany 101:851-864, García et al. 2021García T, Duitama J, Smolenski ZS, Gil J, Ariani A, Dohle S, Palkovic A, Skeen P, Bermúdez-Santana CI, Debouck DG, Martínez-Castillo J, Gepts P, Chacón-Sanchez MI2021 Comprehensive genomic resources related to domestication and crop improvement traits in Lima bean. Nature Communications 12:2-14). Regarding size and weight, seeds can be grouped as Potato (small seeds, from 35 to 50 g for 100-seed weight), Sieva (medium seeds, from 50 to 70 g), and Big Lima (big seeds, from 70 to 110 g), according to Baudet (1977Baudet JC1977 The taxonomic status of the cultivated types of lima bean (Phaseolus lunatus L.). Tropical Grain Legume Bulletin 7:29-30) and Castiñeiras et al. (1991Castiñeiras L, Esquivel MA, Rivero N, Mariño A1991 Variabilidad de la semilla de Phaseolus lunatus L. en Cuba. Revista del Jardín Botánico Nacional 12:109-114). The smallest value found was for PGB-UFPI 816 (36.10 g), and the largest was for PGB-UFPI 806 (91.45 g) (Table 2).

According to the evaluated traits (form, profile, and seed weight), seeds were predominantly in the Big Lima cultigroup (PGB-UFPI 790, 794, 796, 797, 801, 804, 806, 807, 809, 810, 814, and 857), specifically with flattened and elliptical seeds. However, the accession PGB-UFPI 803, also belonging to this group, has a flattened profile and spherical form. The other accessions belong to the Sieva (PGB-UFPI 793, 795, 800, and 815) and Potato (PGB-UFPI 799, 816, and 817) cultigroups (Table 2).

Previous studies morphologically characterized the lima bean seeds belonging to the PGB-UFPI. Santos et al. (2002Santos D, Corlett FMF, Mendes JEMF, Wanderley Júnior JSA2002 Produtividade e morfologia de vagens e sementes de variedades de fava no Estado da Paraíba. Pesquisa Agropecuária Brasileira 37:1407-1412) identified 100-seed weight ranging from 32.6 g (Branquinha and Olho-de-peixe) to 79.5 g (Orelha-de-vó). Nobre et al. (2012Nobre DAC, Brandão Junior DDS, Nobre EC, Santos JMC, Miranda DGS, Alves LP2012 Qualidade física, fisiológica e morfologia externa de sementes de dez variedades de feijão-fava (Phaseolus lunatus L.). Revista Brasileira de Biociências 10:425-429) found an average value of 57.37 g for 100 seeds. Sousa et al. (2015Sousa AMCB, Soares JWM, Silva DMA, Monteiro HNB, Costa MF, Gomes RLF, Lopes ACA2015 Determination of ideal conditions to do artificial crosses in Phaseolus lunatus L. Annual Report of the Bean Improvement Cooperative 58:95-96) analyzed 24 accessions from the germplasm bank and found values of 103g (PGB-UFPI 666) and 101.53 g (PGB-UFPI 622). The smallest average value was found for accession PGB-UFPI 777, with 21.1 g, corroborating considerable polymorphism regarding seed morphology in the PGB-UFPI accessions. Silva et al. (2017Silva RNO, Burle ML, Pádua JG, Lopes ACA, Gomes RLF, Martinez-Castillo J2017 Phenotypic diversity in lima bean landraces cultivated in Brazil, using the Ward-MLM strategy. Chilean Journal of Agricultural Research 77:35-40) observed the formation of three groups in the PGB-UFPI: Group I (composed of 74 accessions), with small, semi-flattened, and spherical seeds, and a medium overall average for length and width. Similar characteristics were found for the Mesoamerican gene pool. Group II (90% of the accessions) exhibited intercalary characteristics of both Andean and Mesoamerican pools. Group III consisted of accessions with the highest values of seed length and width, with predominantly large seeds, notable characteristics of the Andean gene pool.

In this respect, we believe that the accessions analyzed in the present study belong to both Andean (Big Lima) and Mesoamerican (Potato and Sieva) gene pools, with a predominance of the former cultigroup in the selected samples. The big, flattened seeds evaluated here suggest that these accessions belong to the Andean gene pool. These accessions are geographically restricted to Ecuador and the North of Peru and are well-adapted to dry areas (Baudoin et al. 2004Baudoin JP, Rocha O, Degreef J, Maquet A, Guarino L2004 Ecogeography, demography, diversity and conservation of Phaseolus lunatus L. in the Central Valley of Costa Rica. IPGRI - International Plant Genetic Resources Institute, Rome, 84p). As discussed by Silva et al. (2017Silva RNO, Burle ML, Pádua JG, Lopes ACA, Gomes RLF, Martinez-Castillo J2017 Phenotypic diversity in lima bean landraces cultivated in Brazil, using the Ward-MLM strategy. Chilean Journal of Agricultural Research 77:35-40), in Brazil, there is a predominance of species belonging to the Mesoamerican gene pool, corroborating the assumption that this group seems to have a wider distribution than the Andean group (Andueza-Noh et al. 2013Andueza-Noh RH, Serrano-Serrano ML, Sánchez MIC, Del Pino IS, Camacho-Pérez L, Coello-Coello J, Cortes JM, Debouck DG, Martínez-Castilho J2013 Multiple domestications of the Mesoamerican gene pool of lima bean (Phaseolus lunatus L.): evidence from chloroplast DNA sequences. Genetic Resources and Crop Evolution 60:1069-1086). However, Silva et al. (2019Silva RNO, Lopes ACA, Gomes RLF, Pádua JG, Burle ML2019 High diversity of cultivated lima beans (Phaseolus lunatus) in Brazil consisting of one Andean and two Mesoamerican groups with strong introgression between the gene pools. Genetics and Molecular Research 18:1-15) confirmed that both the Andean and the Mesoamerican gene pools of lima bean are widely grown in Brazil, confirming the genetic diversity of the species regarding seed size and shape, which contribute to lima bean conservation in the Northeast of Brazil.

Finally, the present study validates the cytological stability for number of chromosomes, the karyotypic formula, and the CMA-rich pericentromeric heterochromatin pattern, as previously reported by other authors in the P. lunatus species and observed in the accessions preserved in the Phaseolus Active Germplasm Bank of UFPI. The seed morphological profile showed there are representatives of the Andean gene pool, represented by the Big Lima cultigroup (with larger seeds and flat shape), and the Mesoamerican gene pool, represented by the Sieva and Potato cultigroups (with small seeds, that are globular, flat, and kidney-shaped); and it corroborates that the Brazilian Northeast region could be a diversity and domestication center.

ACKNOWLEDGMENTS

We would like to thank the Brazilian agency CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico / process number 457201/2014-2) for financial support and for a scholarship granted to the first author.

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Publication Dates

  • Publication in this collection
    08 Mar 2024
  • Date of issue
    2024

History

  • Received
    09 Sept 2023
  • Accepted
    02 Jan 2024
  • Accepted
    05 Jan 2024
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