Identification of Cassava Varieties in Ex-Situ Collections and Global Farmer's Fields: An Update from 1990 to 2020

*Luis Augusto Becerra Lopez-Lavalle, Adriana Bohorquez-Chaux and Xiaofei Zhang*

#### **Abstract**

The identification of cassava cultivars is important for understanding the crop's production system, enabling crop improvement practitioners to design and deliver tailored solutions with which farmers can secure high yields and sustainable production. Across the lowland tropics today, a large number improved varieties and landraces of cassava are under cultivation, making it inefficient for breeders and geneticists to set improvement goals for the crop. The identification and characterization of cassava genotypes is currently based on either morphological characters or molecular features. The major aim of cultivar identification is to catalog the crop's genetic diversity, but a consensus approach has still not been established. Of the two approaches to the identification of variety, morphological characters seem to account for most of the genetic variability reported in cassava. However, these characters must be treated with caution, as phenotypic changes can be due to environmental and climatic conditions as well as to the segregation of new highly heterozygous populations, thus, making the accurate identification of varieties difficult. The use of molecular markers has allowed researchers to establish accurate relationships between genotypes, and to measure and track their heterozygous status. Since the early 1990's, molecular geneticists working with cassava have been developing and deploying DNA-based tools for the identification and characterization of landraces or improved varieties. Hence, in the last five years, economists and social scientists have adopted DNA-based variety identification to measure the adoption rates of varieties, and to support the legal protection of breeder's rights. Despite the advances made in the deployment of molecular markers for cassava, multiple platform adoption, as well as their costs and variable throughput, has limited their use by practitioners of crop improvement of cassava. The postgenomic era has produced a large number of genome and transcriptome sequencing tools, and has increased our capacity to develop and deploy genome-based tools to account for the crop's genetic variability by accurately measuring and tracking allele diversity. These technologies allow the creation of haplotype catalogs that can be widely shared across the cassava crop improvement community. Low-density genome-wide SNP markers might be the solution for the wide adoption of molecular tools for the identification of cultivars or varieties of cassava. In this review we survey the efforts made in the past 30 years to establish the tools for cultivar

identification of cassava in farmer's fields and gene banks. We also emphasize the need for a global picture of the genetic diversity of this crop, at its center of origin in South America.

**Keywords:** cassava, genotype, varieties, SNPs, identification

#### **1. Introduction**

Cassava (*Manihot esculenta*) is a key food commodity in the tropics, being the second most important food staple in the least-developed countries, and the fourth highest source of calories in developing countries [1, 2]. Due to cassava's efficient use of soil nutrients and water resources, poor farmers can still expect reasonable harvests in areas where many other crops will fail to be productive. Thus cassava, as an agricultural commodity, has the potential to have significant global impact on nearly all of the United Nations Sustainable Development Goals (SDGs), with emphasis on SDG-1 (no poverty), SDG-2 (zero hunger), SDG-3 (good health and well-being), SDG-12 (responsible production and consumption), SDG-13 (climate action), and SDG-15 (life on land) [3].

Today, a large number of the varieties of cassava which are under cultivation have persisted from pre-Columbian times, having been perpetuated through vegetative propagation, particularly at its center of origin in South America [4, 5]. From South America, this crop spread to sub-Saharan Africa in the 16th century [6], and from South and Southeast Asia (SEA) in the late 18th and early 19th century to Asia [7]. Crop improvement, led by International Institute of Tropical Agriculture (IITA) in Africa and the International Centre for Tropical Agriculture (CIAT) in Latin America and the Caribbean (LAC), as well as in South Asia and SEA, has made improved varieties more common in farmer's fields [2, 8, 9]. For instance, CIAT and Kasetsart University in Thailand developed what is considered to be the most successful variety ever breed, KU50, which has a notably high fresh root yield and dry matter content [10, 11]. Since its official release in Thailand, this variety has spread throughout SEA. In Vietnam, KU50 was released in 1995 as KM94, and was later introduced in Cambodia as Malay [12–14]. It covers nearly one million hectares today. In cassava, it is quite common for the same variety to be renamed when it is introduced to a new area, leading to the existence of synonymous varieties. The opposite situation also occurs, where different varieties are identified under the same name (homonyms) [15–17].

There is currently little understanding of the number of cassava varieties grown throughout the lowland tropics, but, this number is likely to be in the order of thousands, based on the results obtained by Rabbi et al. [2] and Floro et al. [4]. This number can also be estimated from the total number of the crop accessions (genotypes) kept under conservation in different ex-situ gene banks. In 2010, CIAT commissioned a survey of the status of germplasm conservation of cassava across 50 cassava gene banks [18]. Out of the 50 gene banks surveyed, 34 provided information that allowed the estimation that as many as 14,791 distinct landraces were under conservation in gene banks [18]. The real number, however, is likely be significantly lower, once all varieties are characterized using DNA-based molecular markers [2, 4, 12].

In the past 30 years, a body of knowledge about the varietal identification and genetic diversity of cassava has been developed for genetic materials found in ex-situ collections, experimental field trials, and farmers' fields, using morphological descriptors [17, 19–48], morphological descriptors and molecular markers [16, 49–57], and molecular markers alone [2, 4, 12, 15, 25, 58–126]. The morphological

**5**

*Identification of Cassava Varieties in Ex-Situ Collections and Global Farmer's Fields: An Update…*

descriptors were first defined by CIAT in the late 70's and early 80's [47, 48], and were later revised by Fukuda et al. [21, 46]. Approximately 75 morphological descriptors, also known as traits, have been defined, and 199 alleles have been made available for distinguishing cassava varieties under ex-situ conservation or to catalog the local varietal inventory of farmers, In more than half of these studies, a measure of their genetic diversity was included [19, 23, 25, 29, 30, 33–37, 40, 41, 43]. Additional efforts to identify cassava varieties have been undertaken, combining morphological descriptors and molecular markers, under the assumption that combining knowledge of farmers with DNA-based genetic profiles should more accurately account for the large genetic differentiation observed among cassava varieties in gene banks, breeding programs, and in famers' fields [16, 49–57]. Across the scientific community investigating cassava, the most widely used methods for identification of varieties, and the estimation of its genetic diversity, have involved molecular markers [127]. Since the advent of DNA-based molecular marker technologies, cassava scientists have adopted nearly all of the most popular techniques to elucidate and describe the crop's varietal identities, diversity, domestication, and ancestry [2, 4, 64, 72–74, 78, 126]. These molecular approaches have focused on two primary objectives: (1) to access an adequate number of highly informative DNA-based molecular markers across the cultivated species; and (2) to assess the crop's global ex-situ germplasm, and that of populations produced at publicly funded breeding programs. Thus, the use of molecular markers could allow building a global varietal haplotype catalog, containing the molecular descriptions of the most common varieties of cassava grown during the last 50 years across sub-Saharan Africa, South and SEA, and LAC. This information will facilitate the development, registration, and release of varieties that will effectively replace old

Access to a global catalog of the crop's molecular haplotypes will enable the conducting of studies on the adoption of improved varieties [4]. DNA-based marker technology must be cost-effective, easy to use, and reproducible across laboratories. The reproducibility of molecular marker techniques is extremely important in cassava, due to the presence of fixed somatic mutations, which are potentially caused by clonal propagation, although evidence for this phenomenon is limited [36, 68,

A robust set of highly informative DNA-based markers could be used for variety identification, quality control, and the measurement of genetic diversity, with a potential use in variety registration. Thus, cassava breeders will be able to trace infringements of Plant Breeder's Rights, particularly when the cassava variety is

The need to improve cassava varieties, to fight hunger, malnutrition, and poverty in the tropics, has led to the identification of the problem of discriminating between *M. esculenta* cultigens, particular between landraces and improved types. CIAT and IITA's publicly funded breeding programs have introduced new varieties and cultivars in tropical countries, increasing the number of crop varieties available to farmers in Africa (IITA), Asia, LAC, and SEA (CIAT) [130]. In the late 1970s, CIAT established and evaluated its cassava germplasm collection, developing and using 54 basic morphological descriptors [21], aimed at the efficient selection of parental lines for breeding. In the 2000's, Fukuda et al. [21, 46] revisited the morphological descriptor list by defining 75 descriptors, with the objective of standardizing the characterization data and improving the selection of new exotic

*DOI: http://dx.doi.org/10.5772/intechopen.99110*

varieties with the latest modern cultivars.

licensed for exclusive commercial use.

**2. Morphological descriptors**

95, 128, 129].

#### *Identification of Cassava Varieties in Ex-Situ Collections and Global Farmer's Fields: An Update… DOI: http://dx.doi.org/10.5772/intechopen.99110*

descriptors were first defined by CIAT in the late 70's and early 80's [47, 48], and were later revised by Fukuda et al. [21, 46]. Approximately 75 morphological descriptors, also known as traits, have been defined, and 199 alleles have been made available for distinguishing cassava varieties under ex-situ conservation or to catalog the local varietal inventory of farmers, In more than half of these studies, a measure of their genetic diversity was included [19, 23, 25, 29, 30, 33–37, 40, 41, 43]. Additional efforts to identify cassava varieties have been undertaken, combining morphological descriptors and molecular markers, under the assumption that combining knowledge of farmers with DNA-based genetic profiles should more accurately account for the large genetic differentiation observed among cassava varieties in gene banks, breeding programs, and in famers' fields [16, 49–57].

Across the scientific community investigating cassava, the most widely used methods for identification of varieties, and the estimation of its genetic diversity, have involved molecular markers [127]. Since the advent of DNA-based molecular marker technologies, cassava scientists have adopted nearly all of the most popular techniques to elucidate and describe the crop's varietal identities, diversity, domestication, and ancestry [2, 4, 64, 72–74, 78, 126]. These molecular approaches have focused on two primary objectives: (1) to access an adequate number of highly informative DNA-based molecular markers across the cultivated species; and (2) to assess the crop's global ex-situ germplasm, and that of populations produced at publicly funded breeding programs. Thus, the use of molecular markers could allow building a global varietal haplotype catalog, containing the molecular descriptions of the most common varieties of cassava grown during the last 50 years across sub-Saharan Africa, South and SEA, and LAC. This information will facilitate the development, registration, and release of varieties that will effectively replace old varieties with the latest modern cultivars.

Access to a global catalog of the crop's molecular haplotypes will enable the conducting of studies on the adoption of improved varieties [4]. DNA-based marker technology must be cost-effective, easy to use, and reproducible across laboratories. The reproducibility of molecular marker techniques is extremely important in cassava, due to the presence of fixed somatic mutations, which are potentially caused by clonal propagation, although evidence for this phenomenon is limited [36, 68, 95, 128, 129].

A robust set of highly informative DNA-based markers could be used for variety identification, quality control, and the measurement of genetic diversity, with a potential use in variety registration. Thus, cassava breeders will be able to trace infringements of Plant Breeder's Rights, particularly when the cassava variety is licensed for exclusive commercial use.

#### **2. Morphological descriptors**

The need to improve cassava varieties, to fight hunger, malnutrition, and poverty in the tropics, has led to the identification of the problem of discriminating between *M. esculenta* cultigens, particular between landraces and improved types. CIAT and IITA's publicly funded breeding programs have introduced new varieties and cultivars in tropical countries, increasing the number of crop varieties available to farmers in Africa (IITA), Asia, LAC, and SEA (CIAT) [130]. In the late 1970s, CIAT established and evaluated its cassava germplasm collection, developing and using 54 basic morphological descriptors [21], aimed at the efficient selection of parental lines for breeding. In the 2000's, Fukuda et al. [21, 46] revisited the morphological descriptor list by defining 75 descriptors, with the objective of standardizing the characterization data and improving the selection of new exotic

*Cassava - Biology, Production, and Use*

action), and SDG-15 (life on land) [3].

under the same name (homonyms) [15–17].

in South America.

**1. Introduction**

identification of cassava in farmer's fields and gene banks. We also emphasize the need for a global picture of the genetic diversity of this crop, at its center of origin

Cassava (*Manihot esculenta*) is a key food commodity in the tropics, being the second most important food staple in the least-developed countries, and the fourth highest source of calories in developing countries [1, 2]. Due to cassava's efficient use of soil nutrients and water resources, poor farmers can still expect reasonable harvests in areas where many other crops will fail to be productive. Thus cassava, as an agricultural commodity, has the potential to have significant global impact on nearly all of the United Nations Sustainable Development Goals (SDGs), with emphasis on SDG-1 (no poverty), SDG-2 (zero hunger), SDG-3 (good health and well-being), SDG-12 (responsible production and consumption), SDG-13 (climate

Today, a large number of the varieties of cassava which are under cultivation have persisted from pre-Columbian times, having been perpetuated through vegetative propagation, particularly at its center of origin in South America [4, 5]. From South America, this crop spread to sub-Saharan Africa in the 16th century [6], and from South and Southeast Asia (SEA) in the late 18th and early 19th century to Asia [7]. Crop improvement, led by International Institute of Tropical Agriculture (IITA) in Africa and the International Centre for Tropical Agriculture (CIAT) in Latin America and the Caribbean (LAC), as well as in South Asia and SEA, has made improved varieties more common in farmer's fields [2, 8, 9]. For instance, CIAT and Kasetsart University in Thailand developed what is considered to be the most successful variety ever breed, KU50, which has a notably high fresh root yield and dry matter content [10, 11]. Since its official release in Thailand, this variety has spread throughout SEA. In Vietnam, KU50 was released in 1995 as KM94, and was later introduced in Cambodia as Malay [12–14]. It covers nearly one million hectares today. In cassava, it is quite common for the same variety to be renamed when it is introduced to a new area, leading to the existence of synonymous varieties. The opposite situation also occurs, where different varieties are identified

There is currently little understanding of the number of cassava varieties grown

In the past 30 years, a body of knowledge about the varietal identification and genetic diversity of cassava has been developed for genetic materials found in ex-situ collections, experimental field trials, and farmers' fields, using morphological descriptors [17, 19–48], morphological descriptors and molecular markers [16, 49–57], and molecular markers alone [2, 4, 12, 15, 25, 58–126]. The morphological

throughout the lowland tropics, but, this number is likely to be in the order of thousands, based on the results obtained by Rabbi et al. [2] and Floro et al. [4]. This number can also be estimated from the total number of the crop accessions (genotypes) kept under conservation in different ex-situ gene banks. In 2010, CIAT commissioned a survey of the status of germplasm conservation of cassava across 50 cassava gene banks [18]. Out of the 50 gene banks surveyed, 34 provided information that allowed the estimation that as many as 14,791 distinct landraces were under conservation in gene banks [18]. The real number, however, is likely be significantly lower, once all varieties are characterized using DNA-based molecular

**Keywords:** cassava, genotype, varieties, SNPs, identification

**4**

markers [2, 4, 12].

parental lines for breeding in Africa, The aim of this work was to reduce unavoidably subjective interpretations generated using the morphological method in light of the genetic variability of cassava.

Cassava has large phenotypic variance in the field, with a wide eco-geographic adaptation range, suggesting that there is a significant amount of genetic diversity available for breeding. Thus, the identification and differentiation of commercial and landrace cultivars is very important. It has been necessary until recently to rely on the morphological characteristics of the vegetative parts of cassava. Consequently, a range of vegetative descriptors has been used to distinguish cassava varieties from each other in Africa, Asia, LAC, SEA, and Oceania (**Table 1**). The resolution achieved by Fukuda's et al. morphological descriptors can account for cassava's genetic differentiation between accessions, facilitating the understanding of the crop's genetic resources. Among the 28 morphology-based cassava varietal identification and genetic differentiation studies used in this review, the ranges of qualitative (6–44) and quantitative (0–28) morphological descriptors are significantly different (**Table 1**). Approximately one-third of these studies jointly evaluated qualitative and quantitative morphological descriptors, producing a noticeable increase in the number of genetic targets sampled, and thus improving the assessment of genetic diversity in both natural and segregating populations, allowing for the selection of contrasting parents for breeding.

Although cassava displays strikingly high levels of heterozygosity, clonal propagation has permitted the spread of a small set of superior clones, increasing their frequency of occurrence across different regions. This set of clones is grown under large number of different names. A single genotype cultivated in a given geographical region might be found under different names, resulting in the unintentional presence of duplicated genotypes in any one collection. The results of variety identification based on morphological descriptors in cassava has not revealed the presence of these duplicated entries in the ex-situ collections or under cultivation (**Table 1**), although 20 to 25% genotypic redundancy is expected. This review covers a total of 4,285 cassava accessions from Africa, Asia, LAC, SEA, and the Pacific Islands, but the number of duplicate cassava accessions reported is extremely low (1.4%) (**Table 1**). This result might be explained by the high morphological variability reported in cassava due to changes in soil, climatic, and biotic factors, making it difficult to precisely describe the morphological characteristics of this crop. The inability to identify genetic duplicates in a germplasm collection has profound implication for cost-effective germplasm conservation, as well as for germplasm use by breeding programs. Thus, the accurate and reliable identification and elimination of duplicates within a germplasm collection will facilitate genetic resource management and use, while reducing maintenance costs.

These studies have revealed an important heterogeneity within cassava cultivars, particularly those held by farmers [31, 33, 37, 39, 43]. The use of morphological descriptors in the early characterization and identification of cassava varieties is useful to identify new genetic variability, but it can be a lengthy process, taking more than a year to obtain and analyze this type of data. The number of potentially uncharacterized varieties still used in traditional farming is estimated to be as high as 15,000 [18]. Thus, it is likely that the available number of morphological descriptors is inadequate to account for the crop's large genetic variability, as well as the number of cassava cultigens which are affected by environmental factors that influence their phenotypes. This situation highlights the need to develop a method to measure the crop's genetic variability, reducing or eliminating the need to use morphological descriptors. Molecular markers, due to their nature, could provide an immense advantage in the identification of varieties and the characterization of genetic variability, by providing more detailed information about its

**7**

**Region**

Africa

Côte d'Ivoire

Nigeria

Benin Côte d'Ivoire

Chad Cameroon Côte d'Ivoire

Angola

> LAC

Brazil Brazil Costa Rica

Brazil Brazil Brazil Brazil Brazil Brazil

**Location**

**Cassava** *(M. esculenta)*

**Source** Collection maintained at CNRA's research station at Bouaké

Collection maintained at IITA, Ibadan, Nigeria

Collection maintained at IITA, Ubiaja

Field collected in 55 villages surveyed in the southern region

Field collected across 26 villages in the Centre-west, South-west and West region Field collected in Mandoul, Moyen Chari, Tandjilé, Logone Occidental and Oriental region

Field collected across de Humid Forest & Guinea Savannah Agroecologies

Field collected in the forest zone of the Ivory Coast

Collection maintained at the Agronomic Investigation Institute

Collection maintained at Embrapa Mandioca e Fruticultura, Cruz das Almas

Collection maintained at Mandioca do Cerrado (BGMC) - Embrapa

Collection maintained at Centro Agronómico Tropical de Investigación y

Enseñanza (CATIE)

Collection maintained at Embrapa Mandioca e Fruticultura, Cruz das Almas

Collection maintained at Embrapa Mandioca e Fruticultura, Cruz das Almas

Collection maintained at Embrapa Mandioca e Fruticultura, Cruz das Almas

Regional Germplasm Bank of Eastern Amazon, situated in Belém, Pará,

Brazil,

Collection maintained at Mato Grosso's State University (UNEMAT -

Cáceres) and Embrapa Agrossilvipastoril)

Field collected in the Brazilian Middle North Regions, Viçosa-MG

10

8 MAP

24

0

0

[31]

95 262 158

6–8 & 12

29

9

0

[23]

MAP

11–12 MAP

21

0

0

[28]

11–12 MAP

35

13

0

[32]

95

11–12 MAP

32

0

0

[38]

200

NP

19

16

0

[26]

16 37

NP

44

28

0

[40]

12 MAP

33

0

0

[41]

14

8 MAP

10

4

0

[43]

40

12 MAP

12

10

0

[25]

44

NP

20

4

0

[30]

89

3, 6, 9 & 12 MAP

35

14

0

[29]

**Morphological Descriptors**

**Scoring Schedule**

> **No.**

340 1766 1890

125 159

59

3, 6, 9 & 12 MAP

32

13

3

[35]

5–12 MAP

14

0

16

[19]

NP

20

0

0

[37]

NP

28

8

0

NP

32

8

0

[39]

5–12 MAP

14

0

35

[42]

**No. of variables**

**QLT**

**QNT**

**No. of Dupl.**

**Rev. Ref.**

*Identification of Cassava Varieties in Ex-Situ Collections and Global Farmer's Fields: An Update…*

*DOI: http://dx.doi.org/10.5772/intechopen.99110*


*Identification of Cassava Varieties in Ex-Situ Collections and Global Farmer's Fields: An Update… DOI: http://dx.doi.org/10.5772/intechopen.99110*

*Cassava - Biology, Production, and Use*

of the genetic variability of cassava.

the selection of contrasting parents for breeding.

parental lines for breeding in Africa, The aim of this work was to reduce unavoidably subjective interpretations generated using the morphological method in light

Although cassava displays strikingly high levels of heterozygosity, clonal propagation has permitted the spread of a small set of superior clones, increasing their frequency of occurrence across different regions. This set of clones is grown under large number of different names. A single genotype cultivated in a given geographical region might be found under different names, resulting in the unintentional presence of duplicated genotypes in any one collection. The results of variety identification based on morphological descriptors in cassava has not revealed the presence of these duplicated entries in the ex-situ collections or under cultivation (**Table 1**), although 20 to 25% genotypic redundancy is expected. This review covers a total of 4,285 cassava accessions from Africa, Asia, LAC, SEA, and the Pacific Islands, but the number of duplicate cassava accessions reported is extremely low (1.4%) (**Table 1**). This result might be explained by the high morphological variability reported in cassava due to changes in soil, climatic, and biotic factors, making it difficult to precisely describe the morphological characteristics of this crop. The inability to identify genetic duplicates in a germplasm collection has profound implication for cost-effective germplasm conservation, as well as for germplasm use by breeding programs. Thus, the accurate and reliable identification and elimination of duplicates within a germplasm collection will facilitate genetic

resource management and use, while reducing maintenance costs.

These studies have revealed an important heterogeneity within cassava cultivars,

particularly those held by farmers [31, 33, 37, 39, 43]. The use of morphological descriptors in the early characterization and identification of cassava varieties is useful to identify new genetic variability, but it can be a lengthy process, taking more than a year to obtain and analyze this type of data. The number of potentially uncharacterized varieties still used in traditional farming is estimated to be as high as 15,000 [18]. Thus, it is likely that the available number of morphological descriptors is inadequate to account for the crop's large genetic variability, as well as the number of cassava cultigens which are affected by environmental factors that influence their phenotypes. This situation highlights the need to develop a method to measure the crop's genetic variability, reducing or eliminating the need to use morphological descriptors. Molecular markers, due to their nature, could provide an immense advantage in the identification of varieties and the characterization of genetic variability, by providing more detailed information about its

Cassava has large phenotypic variance in the field, with a wide eco-geographic adaptation range, suggesting that there is a significant amount of genetic diversity available for breeding. Thus, the identification and differentiation of commercial and landrace cultivars is very important. It has been necessary until recently to rely on the morphological characteristics of the vegetative parts of cassava. Consequently, a range of vegetative descriptors has been used to distinguish cassava varieties from each other in Africa, Asia, LAC, SEA, and Oceania (**Table 1**). The resolution achieved by Fukuda's et al. morphological descriptors can account for cassava's genetic differentiation between accessions, facilitating the understanding of the crop's genetic resources. Among the 28 morphology-based cassava varietal identification and genetic differentiation studies used in this review, the ranges of qualitative (6–44) and quantitative (0–28) morphological descriptors are significantly different (**Table 1**). Approximately one-third of these studies jointly evaluated qualitative and quantitative morphological descriptors, producing a noticeable increase in the number of genetic targets sampled, and thus improving the assessment of genetic diversity in both natural and segregating populations, allowing for

**6**

