**6. Genetic diversity based on molecular markers**

The progress achieved in plant breeding programmes culminated in reduced genetic variability in the improved populations [36, 67–69]. This problem may be worse in species with a narrow genetic base, such as Arabica coffee (*C. arabica*). The narrow genetic base of this species is associated with its autogamy, the low number of plants that were initially distributed worldwide, and the recent evolution of the species [30, 36, 70]. Thus, genotype discrimination based on differences in phenotypic characteristics may be difficult because individuals who are genetically distinct may be phenotypically similar, which reduces the selective efficiency. To overcome this difficulty, molecular markers have been used as an important tool in the accurate discrimination of genotypes [71, 72].

DNA markers allow the detection of variations in DNA sequences between individuals of the same species. Because they identify variations in DNA, they are stable and are unaffected by the environment or by pleiotropic or epistatic effects [73]. Thus, molecular markers have been used in breeding programmes as an efficient tool for the discrimination of genotypes and the analysis of genetic

variability, as their analysis is a precise association strategy between phenotypic and genotypic variability.

Genetic diversity assisted by molecular markers has been used in several stages of Arabica coffee breeding programmes. The molecular characterization of coffee accessions is an accurate tool for the conservation and more efficient use of genetic resources by breeders. This molecular information is useful in evaluating the redundancies and deficiencies of the germplasm and generates information on the efficiency of the collection, maintenance, and expansion of a germplasm bank. In addition, the study of molecular diversity provides fundamental information to help breeders choose parents to integrate into cross-breeding schemes, as well as in directing the improvement of the genetic base during the course of a breeding programme.

Different molecular markers, such as simple sequence repeats (SSRs), sequencecharacterized amplified regions (SCARs), and single-nucleotide polymorphisms (SNPs), have been identified and made available for coffee [71, 72, 74–82]. These species-specific markers combined with random markers, such as inter-simple sequence repeats (ISSRs), random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLPs); support the genetic breeding of this crop.

Genetic studies and analyses of diversity and molecular characterizations of different germplasm banks and cultivars of *C. arabica* have benefited from molecular marker technology. Coffee plants belonging to the group of the Híbrido de Timor (HdT) from the Brazilian germplasm bank of the Universidade Federal de Viçosa (UFV) in partnership with Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG) and Empresa Brasileira de Pesquisa Agropecuária (Embrapa Café) have been studied in detail using AFLP and SSR markers [83]. HdT coffee plants are the result of natural hybridization between *C. arabica* and *C. canephora* and are one of the main sources of resistance genes to coffee diseases and pests [84–86]. Through molecular markers, redundancy was observed in the core collection of the HdT, so that two plants with different identifications corresponded to the same genotype. One of them was eliminated, resulting in a core collection containing 151 unique and properly discriminated HdTs. The data obtained allowed fingerprinting of the accessions [83]. The fingerprinting of each genotype allows the identification of individuals through a unique code. This information will provide reliability to breeders for germplasm maintenance, preservation, and exchange.

With 52 alleles from 22 SSRs, it was possible to access the diversity of the Core Collection of HdT [83]. Considerable variability was observed between the accessions, which were separated into 21 groups. This grouping result was analyzed together with the resistance data obtained for the main coffee diseases, rust and coffee berry disease. The concentration of individuals resistant to both diseases was verified in eight groups. Through this analysis, it was possible to identify HdT coffee plants belonging to distinct genetic diversity groups that have not yet been used in genetic breeding. This made it possible to select genotypes in the obtained dendrogram that were as distinct as possible from the sources already explored to date and that have different disease resistance genes. The selected HdT accessions consist of potential parents for breeding aiming resistance to multiple diseases [83].

Molecular markers were also analyzed in the HdT to understand the introgression of the genomes from the coffee species of their origin (*C. arabica* and *C. canephora*), as well as their potential impact on the cup quality on the *C. arabica* cultivars. HdT has the largest portion of the genome corresponding to *C. arabica* [87]; however, the small portion of *C. canephora* provides disease resistance genes. This portion, even though small, raises concern about the possibility of *C. canephora* affect the cup quality, since the beverage quality of *C. canephora* is known to be lower. Thus, the

**71**

*Genetic Diversity of* Coffea arabica

cultivar fingerprinting [90, 91].

cific crosses.

was demonstrated.

*DOI: http://dx.doi.org/10.5772/intechopen.94744*

*arabica* cultivars without affecting beverage quality [89].

effect of introgression of *C. canephora* on HdT derivatives were evaluated [88, 89]. The study also demonstrated the presence of disease-resistant genotypes combined with good cup quality typical of *C. arabica* cultivars. The genetic diversity analysis showed high genetic similarity between HdT with *C. arabica* and clear differentiation among coffee species. The introgression of *C. canephora* in the HdT accessions did not reach 30%. The sensory analysis of the coffee genotypes showed no significant difference in the beverage quality parameters between *C. arabica* cv. Bourbon and HdT-derived cultivars, which demonstrated the possibility of developing *C.* 

Accessions of different species and interspecific hybrids from the germplasm

Using the currently available large-scale genotyping technology, genetic diversity between and within Brazilian coffee breeding progenies was assessed by 49,567 SNPs. The significant number of SNP molecular markers distributed throughout *C. arabica* genome was efficient in discriminating all evaluated accessions by grouping them according to their genealogies. Mixtures within the families were identified. New parents to be introduced in the ongoing breeding were identified, and the parents currently used were analyzed in detail. The population structure and its effect on obtaining the improved varieties of *C. arabica* were discussed [72].

Accessions from the germplasm bank and cultivars launched by the breeding programme of the Instituto Agronômico de Campinas were analyzed with RAPD, AFLP, and SSR markers [92]. The variability observed between accessions was small, and only two groups were formed, one containing genotypes that included most cultivars and the other containing accessions/cultivars derived from interspe-

A more comprehensive analysis of Brazilian coffee plants was performed in 34 cultivars belonging to the Brazilian Cultivar Trial, using SSR markers [93]. The molecular pattern obtained allowed the discrimination of all cultivars and the creation of a fingerprinting data of the main cultivars of the country. The ability of markers to detect varietal mixtures and the diversity between and within cultivars

The genetic variability of *C. arabica* accessions from other countries, such as Costa Rica [94], Mexico [95], Nicaragua [96], India [97–99], Indonesia [100], China [101], Kenya [102] and Ethiopia [34, 103–105], has also been analyzed using markers such as ISSRs, SSRs, sequence-related amplified polymorphisms (SRAPs), AFLPs, and SNPs. In Ethiopia, different studies have shown the presence of great genetic variability in coffee plants. This variability has been attributed to the particular ecological characteristics of the country, such as its rainfall amplitude and its different altitudes, temperatures, and soil fertility, which are suitable for the crop. The presence of indigenous coffee production methods in the country has also contributed to this diversity [5, 106]. Greater genetic diversity has been reported

A broader study of the diversity and fingerprinting of Arabica coffee accessions from various producing regions of the world was done in 2533 genotypes [107]. These genotypes corresponding to the Core Collection of the germplasm of the Tropical Agricultural Research and Higher Education Center, accessions from Southern Sudan, and cultivars/germplasm from North, Central, and South America as well

among wild coffee populations than cultivated genotypes [103].

bank of UFV/EPAMIG/Embrapa were also analyzed with genomic SSRs and expressed sequence tag–SSR markers. The combination of these two types of markers allowed discriminating all accessions, including genotypes traditionally of *C. arabica*, genotypes containing introgression of HdT, *C. canephora*, HdT, *C. racemosa*, and triploids of *C. arabica* and *C. racemosa*. This study also identified unique alleles that are useful for accession discriminating in breeding programmes and for

#### *Genetic Diversity of* Coffea arabica *DOI: http://dx.doi.org/10.5772/intechopen.94744*

*Genetic Variation*

programme.

of this crop.

and genotypic variability.

variability, as their analysis is a precise association strategy between phenotypic

Genetic diversity assisted by molecular markers has been used in several stages of Arabica coffee breeding programmes. The molecular characterization of coffee accessions is an accurate tool for the conservation and more efficient use of genetic resources by breeders. This molecular information is useful in evaluating the redundancies and deficiencies of the germplasm and generates information on the efficiency of the collection, maintenance, and expansion of a germplasm bank. In addition, the study of molecular diversity provides fundamental information to help breeders choose parents to integrate into cross-breeding schemes, as well as in directing the improvement of the genetic base during the course of a breeding

Different molecular markers, such as simple sequence repeats (SSRs), sequencecharacterized amplified regions (SCARs), and single-nucleotide polymorphisms (SNPs), have been identified and made available for coffee [71, 72, 74–82]. These species-specific markers combined with random markers, such as inter-simple sequence repeats (ISSRs), random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLPs); support the genetic breeding

Genetic studies and analyses of diversity and molecular characterizations of

different germplasm banks and cultivars of *C. arabica* have benefited from molecular marker technology. Coffee plants belonging to the group of the Híbrido de Timor (HdT) from the Brazilian germplasm bank of the Universidade Federal de Viçosa (UFV) in partnership with Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG) and Empresa Brasileira de Pesquisa Agropecuária (Embrapa Café) have been studied in detail using AFLP and SSR markers [83]. HdT coffee plants are the result of natural hybridization between *C. arabica* and *C. canephora* and are one of the main sources of resistance genes to coffee diseases and pests [84–86]. Through molecular markers, redundancy was observed in the core collection of the HdT, so that two plants with different identifications corresponded to the same genotype. One of them was eliminated, resulting in a core collection containing 151 unique and properly discriminated HdTs. The data obtained allowed fingerprinting of the accessions [83]. The fingerprinting of each genotype allows the identification of individuals through a unique code. This information will provide reliability to breeders for germplasm maintenance, preservation, and exchange. With 52 alleles from 22 SSRs, it was possible to access the diversity of the Core Collection of HdT [83]. Considerable variability was observed between the accessions, which were separated into 21 groups. This grouping result was analyzed together with the resistance data obtained for the main coffee diseases, rust and coffee berry disease. The concentration of individuals resistant to both diseases was verified in eight groups. Through this analysis, it was possible to identify HdT coffee plants belonging to distinct genetic diversity groups that have not yet been used in genetic breeding. This made it possible to select genotypes in the obtained dendrogram that were as distinct as possible from the sources already explored to date and that have different disease resistance genes. The selected HdT accessions consist of

potential parents for breeding aiming resistance to multiple diseases [83].

Molecular markers were also analyzed in the HdT to understand the introgression of the genomes from the coffee species of their origin (*C. arabica* and *C. canephora*), as well as their potential impact on the cup quality on the *C. arabica* cultivars. HdT has the largest portion of the genome corresponding to *C. arabica* [87]; however, the small portion of *C. canephora* provides disease resistance genes. This portion, even though small, raises concern about the possibility of *C. canephora* affect the cup quality, since the beverage quality of *C. canephora* is known to be lower. Thus, the

**70**

effect of introgression of *C. canephora* on HdT derivatives were evaluated [88, 89]. The study also demonstrated the presence of disease-resistant genotypes combined with good cup quality typical of *C. arabica* cultivars. The genetic diversity analysis showed high genetic similarity between HdT with *C. arabica* and clear differentiation among coffee species. The introgression of *C. canephora* in the HdT accessions did not reach 30%. The sensory analysis of the coffee genotypes showed no significant difference in the beverage quality parameters between *C. arabica* cv. Bourbon and HdT-derived cultivars, which demonstrated the possibility of developing *C. arabica* cultivars without affecting beverage quality [89].

Accessions of different species and interspecific hybrids from the germplasm bank of UFV/EPAMIG/Embrapa were also analyzed with genomic SSRs and expressed sequence tag–SSR markers. The combination of these two types of markers allowed discriminating all accessions, including genotypes traditionally of *C. arabica*, genotypes containing introgression of HdT, *C. canephora*, HdT, *C. racemosa*, and triploids of *C. arabica* and *C. racemosa*. This study also identified unique alleles that are useful for accession discriminating in breeding programmes and for cultivar fingerprinting [90, 91].

Using the currently available large-scale genotyping technology, genetic diversity between and within Brazilian coffee breeding progenies was assessed by 49,567 SNPs. The significant number of SNP molecular markers distributed throughout *C. arabica* genome was efficient in discriminating all evaluated accessions by grouping them according to their genealogies. Mixtures within the families were identified. New parents to be introduced in the ongoing breeding were identified, and the parents currently used were analyzed in detail. The population structure and its effect on obtaining the improved varieties of *C. arabica* were discussed [72].

Accessions from the germplasm bank and cultivars launched by the breeding programme of the Instituto Agronômico de Campinas were analyzed with RAPD, AFLP, and SSR markers [92]. The variability observed between accessions was small, and only two groups were formed, one containing genotypes that included most cultivars and the other containing accessions/cultivars derived from interspecific crosses.

A more comprehensive analysis of Brazilian coffee plants was performed in 34 cultivars belonging to the Brazilian Cultivar Trial, using SSR markers [93]. The molecular pattern obtained allowed the discrimination of all cultivars and the creation of a fingerprinting data of the main cultivars of the country. The ability of markers to detect varietal mixtures and the diversity between and within cultivars was demonstrated.

The genetic variability of *C. arabica* accessions from other countries, such as Costa Rica [94], Mexico [95], Nicaragua [96], India [97–99], Indonesia [100], China [101], Kenya [102] and Ethiopia [34, 103–105], has also been analyzed using markers such as ISSRs, SSRs, sequence-related amplified polymorphisms (SRAPs), AFLPs, and SNPs. In Ethiopia, different studies have shown the presence of great genetic variability in coffee plants. This variability has been attributed to the particular ecological characteristics of the country, such as its rainfall amplitude and its different altitudes, temperatures, and soil fertility, which are suitable for the crop. The presence of indigenous coffee production methods in the country has also contributed to this diversity [5, 106]. Greater genetic diversity has been reported among wild coffee populations than cultivated genotypes [103].

A broader study of the diversity and fingerprinting of Arabica coffee accessions from various producing regions of the world was done in 2533 genotypes [107]. These genotypes corresponding to the Core Collection of the germplasm of the Tropical Agricultural Research and Higher Education Center, accessions from Southern Sudan, and cultivars/germplasm from North, Central, and South America as well

as Africa and Asia. The obtained fingerprinting was efficient. Based on this tool, farmers can verify and trust the identity of the cultivars being planted, and coffee roasters can rely on marketing related to the cultivars they are growing and selling. The seed and nursery sector can become more professional and reliable by using this new monitoring tool to establish and verify the genetic purity of the seed and seedling stock.

Currently, SNP markers are using for genome-wide investigation [72, 82, 108]. In an original work of genome-wide association, candidate genes associated with lipids and diterpenes contents in *C. arabica* were identified [108]. This study detects the domestication and breeding process in *C. arabica*, pointing out the switch in allele frequency, revealing high allelic richness in wild accessions. In this regard, the identification of these candidate genes outlining potential targets for improving beverage cup quality in a coffee breeding programme.
