**3.1. Genetic control of seed glossiness**

throughout the world [1, 2]. It is naturally distributed from northern Mexico to northern Argentina, with a marked genetic structure. From the classic to the most recent reports, two major gene pools have been recognized for the species, the Mesoamerican and the Andean [3, 4]. Domestication has been independently performed within each gene pool [5, 6], selecting specific genes for growth habit, seed size, color and yield, a phenomenon described as the "domestication syndrome" [7]. Therefore, an enormous panel of diversity can be observed from wild to domesticated genotypes of common bean, making it as a model for understanding crop evolution [8]. The introduction of common bean to other areas than its natural habitat, such as in Brazil [9], led to new combinations of seed and flower colors, shapes and sizes, growth habits, cycle, photoperiod and yield [10]. It has also shaped the interaction between beans and the environ-

In Brazil, common bean is a staple food among most citizens along with rice. Usually, its cultivation is performed in small farming systems along with other crops and animal production systems, providing self-sufficiency for farmers in various regions of the country. Several cycles of selection by local farmers in specific environments have generated new landraces, which are not yet known and available from core collections such as the ones from Centro Internacional de Agricultura Tropical (CIAT, Colombia), United States Department of Agriculture (USDA, USA) and Empresa Nacional de Pesquisa Agropecuária (EMBRAPA, Brazil). Landraces have singular aspects that might assist breeding programs for disease resistance, abiotic stress tolerance, improvement of nutrition facts, among several other desirable aspects of common bean grains. A particular landrace has been discovered with local farmers from the municipality of Cunha, Sao Paulo state, Southeast Brazil. The farmers referred this landrace as "Serro Azul" and have been cultivating it along with other varieties [11]. Serro Azul shows considerable morphological variability, revealing different types of seeds. It shows high genetic variation for seed colors and patterns [12], disease and insect resistance and nodulation ability [11, 13], which

We drive the topic of this chapter highlighting Serro Azul as a case study of how the conservation of landraces might be important for further breeding strategies. First, we describe the importance of landraces of common bean in the discovery of new allele combinations for seed color and pattern genes, using the example of Serro Azul. Then, we briefly discuss the implications of the research of common bean landraces for nutrition and health. Moreover, we outline a significant number of original findings about our landrace in focus, which serve as examples of disease resistance as well as indications of insect resistance. Finally, we guide the reader through other perspectives of the importance of a better knowledge of landraces, their

are among the main aspect studies in breeding programs of common bean.

collection and conservation for future endeavors in breeding programs.

**populations**

**2. The Serro Azul landrace: parental genotypes and segregating** 

Serro Azul is a landrace that was so named by local farmers living in the countryside of the small municipality of Cunha, in São Paulo state, Brazil. From the search along the distinct

ment, leading to new diseases and pests.

180 Rediscovery of Landraces as a Resource for the Future

The genetic control of seed coat color, pattern and glossiness in common bean has been a major issue for scientists in decades. Frequently, the gene nomenclature for loci related to such traits was confusing, leading to a series of meetings for establishing standard nomenclatures (Bean Improvement Cooperative meetings). As regards seed glossiness, Bassett [14] has clarified the differences between glossy and opaque seed coats through a series of genetic crosses, providing genetic stocks (pure lines for specific alleles of seed coat loci) that have been used since then for unraveling the genetic and biochemical aspects of the trait.

Landraces might be the source of additional alleles or allele combinations for studying genes related to color, pattern and glossiness of the seed coat. Moreover, these new sources might be interesting for being added to the breeding programs concerned with such traits. The study of Konzen and Tsai [12] examined the particular aspect of Serro Azul of segregating for seed glossiness and color patterning. The population developed from the cross SAF × SAB was analyzed from the parental genotypes to the F<sup>4</sup> generation for the segregation of these traits. The variation of glossiness was attributed to two alleles at the *Asp* locus, usually referred as the gene controlling glossiness in common bean [14]. SAB carries the dominant allele *Asp*, which confers the intense glossy aspect of the seed coat, while SAF has a dull seed coat [12] (**Figure 1A**). This gene is known to be located in chromosome 7 [15], where genes associated with anthracnose and angular leaf spot resistance [16, 17], common bacterial blight [17] and nodulation [18] are also located.

related to the *J* allele is that it intensifies the glossy aspect of the seed, as seen in both SAB and SAF (**Figure 1A**). Therefore, SAF presents a slight shiny aspect on the seed coat due to the

Genetic Variation of Landraces of Common Bean Varying for Seed Coat Glossiness and Disease…

http://dx.doi.org/10.5772/intechopen.73425

The segregation of the seed coat glossiness along with the color pattern is clearly observed in F2:3 lines (**Figure 1A**) of the cross SAF × SAB and is only due to the *Asp* locus. *J* only contributed to intensify the shiny aspect and is present as the dominant allele in both SAB and SAF. The segregation of glossiness is according to the expected Mendelian proportions (3,1) in F<sup>2</sup>

 generations of the cross between SAF (*asp asp*) and SAB (*Asp Asp*), being attributed to *Asp*. Measurements performed with a colorimeter allowed to validate the categories established (glossy and dull) (**Figure 1B**), based on the L\*a\*b\* color system, in which L\* is the main variable associated with glossiness, while a\* and b\* are measures of distinct colors [12]. In this study, in general, glossy seeds presented higher L\* values than seeds with dull seed coat. However, there was some extent of interaction between the color and the brightness, espe-

Another important aspect of the seed coat glossiness of SAB was shown from microscopy analyses. It followed and confirmed previous findings of classic studies of the glossiness of other common bean genotypes. In general, dull genotypes (*asp asp*) have a rough textured surface of the seed coat, while glossy seed coats have an even surface, as shown from scanning electron micrographs [20] (**Figure 1C**, left image). Moreover, glossy seeds show a thicker palisade epidermis from the seed coat than the dull seed coats (**Figure 1C**, central and right

The seed glossiness has been frequently neglected in breeding programs due to consumer preferences. This is explained by the fact that glossy seeds tend to require higher cooking times than seeds with an opaque seed coat [21]. At first, it seems that since glossiness retards water absorption by the seeds [12, 22] (check **Figure 2**), they take longer to be cooked. However, some line of evidence showed no significant correlation between the cooking time

**Figure 2.** Water uptake on the course of 480 min of the variants Serro Azul Brilhante (SAB, with glossy seed coat) and Serro Azul Fosco (SAF, with dull seed coat). Three replicates of seeds were embedded in distilled water and paper dried

every 30 min for weighing and determining the weight change (adapted from [12]).

and

183

expression of *J* [12].

cially with brown-colored seeds (**Figure 1B**).

**3.2. How important might seed glossiness be?**

F3

figures) [12].

**Figure 1.** Morphological and anatomical aspects of seeds of the common bean landrace Serro Azul. (A) Parental genotypes (Serro Azul Brilhante—SAB and Serro Azul Fosco—SAF) and the four phenotypes of F2:3 families of the cross SAF × SAB. The parent SAB has a glossy brown seed coat, while the SAF seed coat shows a dull gray phenotype. The F2:3 families segregate for both color and glossiness. (B) Principal component analysis with the L\*a\*b\* variables, obtained with a colorimeter for F2:3 families of the cross SAF × SAB. a\*: the amount of green or red; b\*: the quantity of blue or yellow; L\*: the quantity of brightness. (C) Scanning electron profiles of the seed coat surface (left image) and their transversal profile (central image) and histological sections (image on the right) of transversal sections of the seed coat of both SAB and SAF. PE: palisade epidermis; H: hypodermis; PT: parenchymatous tissue. The scale bars indicate 10 μm (left image), 20 μm (central image) and 100 μm (right image) (adapted from [12]).

However, the glossiness of the seed coat in Serro Azul is also related to the expression another gene located in chromosome 10 and referred as the *Joker* (*J*) locus [19]. The dominant allele *J* is responsible for an even distribution of the color shown by each seed, while *jj* genotypes exhibit irregular coloring, especially around the hilum ring [14]. Another peculiar aspect related to the *J* allele is that it intensifies the glossy aspect of the seed, as seen in both SAB and SAF (**Figure 1A**). Therefore, SAF presents a slight shiny aspect on the seed coat due to the expression of *J* [12].

The segregation of the seed coat glossiness along with the color pattern is clearly observed in F2:3 lines (**Figure 1A**) of the cross SAF × SAB and is only due to the *Asp* locus. *J* only contributed to intensify the shiny aspect and is present as the dominant allele in both SAB and SAF. The segregation of glossiness is according to the expected Mendelian proportions (3,1) in F<sup>2</sup> and F3 generations of the cross between SAF (*asp asp*) and SAB (*Asp Asp*), being attributed to *Asp*. Measurements performed with a colorimeter allowed to validate the categories established (glossy and dull) (**Figure 1B**), based on the L\*a\*b\* color system, in which L\* is the main variable associated with glossiness, while a\* and b\* are measures of distinct colors [12]. In this study, in general, glossy seeds presented higher L\* values than seeds with dull seed coat. However, there was some extent of interaction between the color and the brightness, especially with brown-colored seeds (**Figure 1B**).

Another important aspect of the seed coat glossiness of SAB was shown from microscopy analyses. It followed and confirmed previous findings of classic studies of the glossiness of other common bean genotypes. In general, dull genotypes (*asp asp*) have a rough textured surface of the seed coat, while glossy seed coats have an even surface, as shown from scanning electron micrographs [20] (**Figure 1C**, left image). Moreover, glossy seeds show a thicker palisade epidermis from the seed coat than the dull seed coats (**Figure 1C**, central and right figures) [12].

#### **3.2. How important might seed glossiness be?**

However, the glossiness of the seed coat in Serro Azul is also related to the expression another gene located in chromosome 10 and referred as the *Joker* (*J*) locus [19]. The dominant allele *J* is responsible for an even distribution of the color shown by each seed, while *jj* genotypes exhibit irregular coloring, especially around the hilum ring [14]. Another peculiar aspect

(left image), 20 μm (central image) and 100 μm (right image) (adapted from [12]).

182 Rediscovery of Landraces as a Resource for the Future

**Figure 1.** Morphological and anatomical aspects of seeds of the common bean landrace Serro Azul. (A) Parental genotypes (Serro Azul Brilhante—SAB and Serro Azul Fosco—SAF) and the four phenotypes of F2:3 families of the cross SAF × SAB. The parent SAB has a glossy brown seed coat, while the SAF seed coat shows a dull gray phenotype. The F2:3 families segregate for both color and glossiness. (B) Principal component analysis with the L\*a\*b\* variables, obtained with a colorimeter for F2:3 families of the cross SAF × SAB. a\*: the amount of green or red; b\*: the quantity of blue or yellow; L\*: the quantity of brightness. (C) Scanning electron profiles of the seed coat surface (left image) and their transversal profile (central image) and histological sections (image on the right) of transversal sections of the seed coat of both SAB and SAF. PE: palisade epidermis; H: hypodermis; PT: parenchymatous tissue. The scale bars indicate 10 μm The seed glossiness has been frequently neglected in breeding programs due to consumer preferences. This is explained by the fact that glossy seeds tend to require higher cooking times than seeds with an opaque seed coat [21]. At first, it seems that since glossiness retards water absorption by the seeds [12, 22] (check **Figure 2**), they take longer to be cooked. However, some line of evidence showed no significant correlation between the cooking time

**Figure 2.** Water uptake on the course of 480 min of the variants Serro Azul Brilhante (SAB, with glossy seed coat) and Serro Azul Fosco (SAF, with dull seed coat). Three replicates of seeds were embedded in distilled water and paper dried every 30 min for weighing and determining the weight change (adapted from [12]).

and the water absorption rate [23]. Further examination of the genes involved in cooking time is necessary, though.

[23] needs to be further explored by the researchers. Landraces such as Serro Azul are one of

Genetic Variation of Landraces of Common Bean Varying for Seed Coat Glossiness and Disease…

http://dx.doi.org/10.5772/intechopen.73425

185

One of the most important aspects of a breeding program is to find genotypes that are tolerant or even resistant to diseases. The cultivation of common bean is majorly affected by diseases such as common bacterial blight caused by *Xanthomonas axonopodis* pv. *phaseoli*, the angular leaf spot caused by the fungus *Pseudocercospora griseola* and anthracnose by the fungus

**Figure 3.** Screening for anthracnose resistance with races 65 and 73 (*Colletotrichum lindemuthianum*) on SAB and SAF

plants. Controls: Rosinha G2 (susceptible) and G2333 (resistant).

the sources for rescuing the value of seed glossiness.

**4. Serro Azul as a source of disease resistance**

It is well known that the seed coat is the structure that protects the seeds from pathogens and insects, and the glossiness seems to have an important role in such protection. Moreover, seeds with glossiness might have enhanced antioxidant properties due to a higher concentration of specific secondary metabolites in the seed coat, therefore, having an impact in human health [12].

Usually, in the case of landraces, where local selection has been performed, it is more frequent to find common bean accessions that show glossy seed coat [24] (checking the list of genotypes) than in breeding programs. In the case of the landrace Serro Azul, both variants Serro Azul Brilhante (glossy) and Serro Azul Fosco (opaque) have been cultivated [11, 13]. Morphological and biochemical findings are hereafter discussed to show advantages of the seed glossiness for aspects related to human health.

#### **3.3. Biochemical nature of seed coat glossiness and its implications for human health**

The seed coat glossiness has been studied to be mainly conditioned by the *Asp* gene but also influenced by the *J* locus, especially with the dominant allele [14]. A number of studies have also been conducted to better understand the biochemical implications of the expression of such genes on the seed coat.

Classical work has suggested that *J* is essential for the synthesis of proanthocyanidins or condensed tannins [14]. Therefore, the recessive *jj* genotypes have been found to be absent in condensed tannins [25], contrary to the *J\_* genotypes, that are able to synthesize such compounds. Based on the genetic maps that identified the RAPD marker as linked to *J*, a recent study has shown that *J* is linked to a region containing *MYB123* [26], similar to TT2 in *Arabidopsis thaliana* (AT5G35550.1) [27] and *Glycine max* [26], which acts as a key determinant in the proanthocyanidin accumulation of a developing seed [27].

Proanthocyanidins are oligomers or polymers formed by the condensations of flavan-3-ols units such as catechins and epicatechins [28, 29]. In common bean, condensed tannins are mainly composed of catechin monomers [30]. As secondary metabolites, they play important roles as antioxidants, anticarcinogenic and anti-inflammatory [28, 31, 32].

On the other hand, the *Asp* locus is said to be the main gene involved in seed glossiness. Some line of evidence has shown that *Asp* affects the accumulation of anthocyanins due to a structural change that it promotes on the seed coat. Therefore, genotypes with glossy seed coat (*Asp\_*) accumulate more anthocyanins than dull seed coats (*asp asp*) [20]. Anthocyanins have been investigated for their roles in humans such as in anti-inflammatory, lipid peroxidation and membrane strengthening processes [33, 34], as well as in preventing cancer [35].

Therefore, although glossiness has been generally neglected by consumers and, as a result, by selection programs, it might have positive implication for human health. Moreover, the indication that glossiness is not necessarily associated with higher cooking time (since there is a lack of correlation between water absorption and cooking time) as shown by Garcia et al. [23] needs to be further explored by the researchers. Landraces such as Serro Azul are one of the sources for rescuing the value of seed glossiness.
