Legume Breeding: From Conventional Method to Modern Technique

*Parastoo Majidian*

## **Abstract**

Legume species have various applications in organism's nutrition, medical, and conversion industries because of their high oil, high protein, and high value materials. These crops can prevent soil erosion and increase soil nitrogen for further crop cultivation by bacteria symbiosis as well. Concerning the benefits of these crops, there is a need for more breeding attempts to gain genetic achievements. Accelerated higher genetic gains are required to meet the demand of ever-increasing global population. In recent years, speedy developments have been witnessed in legume genomics due to advancements in next-generation sequencing (NGS) and high-throughput genotyping technologies. A fundamental change in current conventional breeding programs, combined with modern techniques, is of great importance. Thus, a combination of modern and conventional breeding techniques may conduct our goals to reach great achievement on legume breeding regarding industrial and medical uses, human and livestock nutrition faster.

**Keywords:** legume, classical and molecular breeding

## **1. Introduction**

Legumes are of great importance as nutritional and economic values that form part of the diet of millions of people worldwide. Legume seeds include an important source of proteins and peptides (double or triple of most cereals), carbohydrates and dietary fibers, and a good source of some micronutrients such as vitamins, fatty acids, folic acid, and minerals that have significant health benefits [1]. The leguminosae or fabaceae family consists of about 12,000 species distributed throughout the world and adapted to a great variety of habitats [2].

In addition, numerous significant plant species belong to leguminosae family such as beans, faba beans, chickpea, cowpea, clover, pea, peanut, pigeon pea, alfalfa, sweet lupin, and lentil which have various applications for human and livestock nutrition, medical industry, and other conversion industries. In addition, some species are used as ornamental crops and as sources of timber and fuel, especially in tropical regions.

One of the significant criteria of legumes is the capacity to produce symbiotic interactions with bacteria called rhizobia that fix atmospheric nitrogen (N) benefiting the plant, which in turn delivers carbon to the bacteria [3]. This symbiosis

#### *Legumes Research - Volume 1*

reduces the production costs and the risk of environmental pollution due to the use of synthetic N fertilizer. It is estimated that a total of 50–70 MT of N are fixed biologically in agricultural systems annually, 16.4 MT in soybean, and 12–25 MT in pasture and fodder legumes [4].

Legumes crops can be used as an alternative for feeding the global population and contribute to developing sustainable agriculture, taking into account their nutritional, economic, and environmental benefits. However, there is not enough data for these crops than cereals [5]. During the last 50 years, legume production is exposed to the negative effect of biotic and abiotic stresses, which cause a reduction in its yield [6, 7].

The other difficulty in legume production except soybean is the limited availability of genetic resources of legume crops in developing countries [8]. In addition, legume breeding has hindered by the lack of robust doubled haploid protocols for legumes species compared to cereal and oilseed crops [9].

Several studies have been investigated by researchers regarding leguminosae genetic data resources such as DNA chips, databases of Targeting Induced Local Lesions In Genomes (TILLING), Bacterial Artificial Chromosome (BAC) libraries, and several bioinformatics tools as "The Legume Information System" (http://legumeinfo.org/) [10].

Thus, the objective of this chapter is to express and compare classical breeding methods in legume crops as well as modern technologies including marker-assisted selection (MAS), quantitative trait loci (QTLs) mapping, and biotechnology.

## **2. Classical breeding methods**

#### **2.1 Accessions and genetic variation**

Evaluation of crop genotypes and cultivars by phenotypic and genetic traits is basic research in breeding programs in order to group accessions based on their genetics, to make knowledge of their genetic background, and select the parental lines for further crossing breeding projects [11]. In this regard, the characterization of germplasm banks of legume crops worldwide has been crucial for the development of agriculture because they are the reservoirs of genetic diversity [12].

To recognize the core collection of legume species and to distinguish various groups of parental lines for crossing programs, the genetic diversity of this family crop has been expressed in this chapter [13]. Utilization of molecular markers is one of the simple techniques to identify genetic diversity of legume species such as SSR (single sequence repeat), AFLP (amplified fragment length polymorphism), RAPD (random amplification of polymorphic DNA). Due to being highly self-pollination as well as low and very low outcrossing rate value in legume germplasm, most of them has genetically similar values and show low to moderate genetic diversity criteria such as allele by locus, heterozygosity, and polymorphism information content (PIC) at intra-population and intragroup levels. While what is important that the genetic variability among population and group of accessions for further breeding programs. In previous studies, researchers reported on the data obtained from genetic variability parameters including (observed heterozygosity of 2–32%), (alleles by locus of 1.5–19), (PIC of 1–66%) in landraces of common bean, soybean, chickpea, lentil and pea, and varieties of these crops from America, Europe, and Africa [14–16]. In contrast, faba bean collections have shown considerably higher observed heterozygosity (20–36.3%), expected heterozygosity (27%), and PIC values (28.7%) than other legume species [17].

## **2.2 Phenotypic inherited traits**

Some morphological and phenological properties such as growth habit, plant height, pod cross-section, number of pods in plant, pod curvature, hypocotyl color, flower color, days to flowering, node numbers, seed number, seed number per pod, number of flower buds, and 100-seed weight, biological yield display significant differences in most of legume germplasm which is relevant to crop yield and appropriate index for breeding purposes [18]. Morpho-physiological and reproductive traits are consistent in different species of legumes [19–21].

Monogenic traits such as color, shape, texture, presence/absence of certain characters are successfully controlled by conventional breeding approaches. While, multigenic traits (quantitative traits) such as plant yield, resistance to abiotic stresses, and so on are highly affected by environment and by genetic × environment interactions which are time-consuming and less precise in breeding techniques [22].

To quantify the proportion of phenotypic variance among individuals in a population, plant breeders utilize heritability as additive genetic effects in the narrow sense (NSH) [23]. The sum of additive, dominance, and epistasis effects is defined as heritability in a broad sense (BSH). Quantitative genetics as heritability determine the responses of selection and depends on selection method (i.e., mass, pure line, pedigree, bulk, backcrossing, etc.) and the type of selection [23]. In soybean, high heritability values have been estimated for plant height, number of clusters per plant, number of primary branches per plant, seed yield per plant, and number of pods per plant [24].

In common bean, it was determined that high values of BSH, ranging from 0.55 to 0.91 for seven phenological and morphological traits [25]. In other previous studies, it was pointed out the results showed that the BSH values for yield and the yield components ranged from 0.115 to 0.642 higher than BSH for a number of days until flowering [26]. Because of the narrow genetic base of chickpea, it takes time to produce high-yielding cultivars, for example, resistance to *Ascochyta* blight in this crop resulted from eight parental di-allele crosses and their F2 [27].

In lentils, heritability values of various traits have been estimated using traditional genetic improvement. In the last study, some morphological properties including total dry matter per plant, seed yield per plant, number of pods per plant, and number of seeds per plant showed low heritability, while days to 50% flowering, days to maturity, and seed weight indicated higher heritability value of 80% [28]. In another study, other seed quality traits have also been studied. For example, raffinose-family oligosaccharides and sucrose levels were highly heritable (BSH values ≥0.85) [29]. Regarding abiotic stresses, cold tolerance heritability was assessed based on NSH values varied from 0.31 to 0.71 under field conditions and peaked at 1.0 under controlled conditions. Based on the results, additive genes controlled cold tolerance under controlled conditions, while field conditions had a negative effect on cold tolerance and made it sensitive [30].

Regarding pea, BSH as well as NSH values for resistance to two fungal diseases (*Erysiphe pisi* and *Mycosphaerella pinodes*) was estimated as high BSH (0.62–0.81) and moderate NSH (0.43–0.57) values, respectively [31]. Also, high BSH values as about 0.62 were gained for the heritability for days to maturity, plant height, pod length, and 100-seed weight, whereas, moderate heritability values were indicated for plant height, pod length, and 100-seed weight [32].

In faba bean, the least affected agronomic and yield-related traits across the environment were the seed weight and the days to flowering, and the number of pods per plant, while, the strong environmental effects were detected on seed yields and the number of stems per plant [33]. In another study, an important trait for conventional breeding as frost tolerance in faba bean was indicated high heritability

after hardening [34]. Generally, the main objective of breeding programs is to genetically evaluation of legume germplasm in order to select superior lines aiming at improving genetic diversity in their progenies and detect heritability of different traits which are seeking by breeders.

## **3. Bioengineering**

The first plant species that its entire genome sequenced was Arabidopsis thaliana regarding to *Arabiodopsis* Genome Initiative Project 2000. This achievement led to further advances in the field of sequencing technologies by the release of the genome sequence of more than 50 species consisting of rice (*Oryza sativa*), maize (*Zea mays*), and wheat (*Triticum aestivum*), and so on [35]. The Arabidopsis plant model has allowed the study of physiological and metabolic processes during plant growth and in responses to abiotic and biotic stress through genome-wide gene expression analysis [36]. This type of analysis has also enabled the identification of the genes responsible for certain traits such as drought and salinity tolerance [37].

Genomics has made available the use of DNA-based molecular markers for the development of MAS in plant breeding programs [38], which uses genotypic selection instead of phenotypic selection employed in conventional breeding. MAS integrates two main systems such as QTL mapping and candidate gene or major gene localization [39]. These methods are based on analyses of association, in which the traits are studied in a large and diverse population and through linkage disequilibrium (LD), where a segregating progeny of parental lines that contrast in certain traits are studied [40].

In recent years, six legume species from the leguminosae family were thoroughly sequenced such as *Cajanus cajan*, *Cicer arietinum*, *Glycine max*, *Lotus japonicas*, *Medicago truncatula*, and *Phaseolus vulgaris* with the genome length of 833, 738, 1112, 472, 373, and 588 Mb, respectively, which their number of genes and transcripts varied from 28,269–48,680 and 25,640–243,067, respectively.

In addition, other legume species including *Pisum sativum* (4450 Mb), *Lupinus angustifolius* (924 Mb), *Trifolium pratense* (440 Mb), and *Arachis hypogaea* (2800 Mb) were entirely sequenced which were significant for omics studies explaining their genes, proteins, transcription factors, metabolites as well as physiological processes. For example, omics studies on *L. japonicas* resulted in Rhizobium infection and nodulation and salt acclimatization processes based on different techniques including Serial Analysis of Gene Expression, cDNAarray of 18,144 non-redundant ESTs isolated from *L. japonicus*, an Affymetrix GeneChip® with 50,000 probe-sets and real-time RT-PCR, a Microarray profiling using the Lotus Genechip® [41]. In parallel, the first version of the completely common bean genome sequence was recently released [42], and also the genome sequence of chickpea is also available in "The Cool Season Food Legume Genome Database" [43]. Legume genome references have also enabled the application of the RNA sequencing (RNA-seq) approach to conduct global transcriptomic profile studies and to discover new genes and ESTs [44, 45]. Overall, thousands of EST, uni-gene, SSRs, and SNPs have been published for lentils [46], groundnut [47], pigeon pea [48], and pea [49].

Great efforts have been made to compare the genomes between models plant species and crop legumes for an accurate translation of the information gained [50]. It was documented that the genome of lentil species such as *L. ervoides* and *Lens culinaris* has high similarity with *M. truncatula* using comparative genomics which identifies a few major translocations and transfer EST-SSR/SSR sequences from the model *M. truncatula* to enrich an intraspecific lentil genetic map [51]. In pea, it

was reported the construction of a high-density pea SNP map, and the validation of syntenic relationships between pea and other legumes species [52]. In faba bean, there is synteny between its region related to days to flowering with other legumes such as medicago, lotus, pea, lupine, and chickpea. Moreover, QTL mapping studies exhibited the similarity between pod length and a number of seeds per pod of faba bean and L*. japonicas* [53].

## **4. State of the art fabaceae species breeding methods**

Achievement in genomics field such as Quantitative trait loci mapping (QTL), marker-assisted selection (MAS) led to improve our data in legume breeding as 1) cultivar identity/assessment of "purity", 2) evaluation of genetic diversity and parental selection, 3) study of heterosis, 4) identification of genomic regions under selection, 5) marker-assisted backcrossing (MABC), 6) marker-assisted pyramiding, 7) early generation MAS, 8) combined MAS, and 9) multi-parents advanced generation intercrossing [54]. Several techniques, as well as strategies for mapping quantitative traits for the identification of quantitative character genes, have been developed in this century which have accelerated and optimized the cultivar development process [55, 56]. Moreover, relevant technical advances have been accomplished to accelerate the breeding of legumes, such as the increased speed of single seed descent by shorter generation cycles through flowering and fruit set in vitro [57].

Important advances in genomic resources have been made in legumes, encompassing a large number of QTLs and genes mapped for different characters, including agronomic, yield-related, or resistance to biotic or abiotic factors traits. Chickpea, common bean, and soybean are three fabaceae species that have been improved through MAS, showing clear and significant progress in the last years. In lentil and faba bean.

Regarding disease resistance-related genes/QTL, achievements obtained were obtained MAS in breeding lines and cultivars.

Although, the classical breeding techniques can transfer these traits and their useful alleles to the breeding line, the introgression by MAS save time selecting for resistant lines [58]. Also, advanced lines or cultivars of common and snap beans with quantitative traits for certain diseases have been produced using MAS [59]. MAS also allows the use of pyramiding approaches, which has become an important method permitting the introgression of several genes and QTLs on a single line [60]. Fewer achievements on other quantitative traits (i.e., yield) have been reported in the literature. Efforts have been made to successfully introgress QTLs for yield-enhancing traits in soybean [61], and drought tolerance-related traits in chickpea [62]. The advantage of MAS in legumes is to successful translation of quantitative traits of interest (major genes/QTLs that control those characters) in commercial lines regardless of being slow incorporation of QTL using MAS selection. High-quality genome sequence of white lupine (*Lupinus albus* L.) was obtained based on long-read sequencing technologies in order to increase and stabilize lupine yield [63].

## **5. Genetically modified legumes**

Great progress in the regeneration and genetic transformation of certain legumes has been made. Global water scarcity and soil salinization have boosted the research for genetic engineering water stress and/or salt tolerance-related

genes in legume crops such as alfalfa [64], chickpea [65], *M. truncatula* [66], and pigeonpea [67], among others. In soybean, several genes controlling traits, such as soybean cyst nematode resistance [68], seed oil [69] and methionine [70] content, drought resistance [71], among others, have been genetically modified (GM). The most successful case of public knowledge is glyphosate-resistant transgenic soybean, which has been commercialized for over 20 years, and it is undoubtedly the most important genetic modification in soybeans [72]. Other legume species, such as narrow-leaf lupine (*L. angustifolius* L.), have also been successfully genetically transformed to develop glyphosate-resistant lines [73]. Glyphosate is a low-cost, foliar-applied, broad-spectrum herbicide that has molecular targets in essential amino acid biosynthetic pathways, which kill the plant [74]. The activity of this herbicide is to block the shikimate pathway by specific inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) [75]. By inhibition of EPSPS, biosynthesis of aromatic amino acids impairs misregulated the shikimate pathway, affecting plant growth. The development of glyphosate-resistant crops (GRCs) utilized the CP4 gene from Agrobacterium spp., which encodes a glyphosate-resistant form of EPSPS, initially introduced in soybean [76]. The vast majority of the commercial GRCs on the market contain the CP4 EPSPS gene that confers glyphosate resistance [77]. GRCs have simplified weed management practices, reduced crop production costs, and have had positive effects on the environment [78]. While, the potential improvement of weeds resistant to glyphosate cause big concerns due to its high utilization and its genes potential introgression from GM crops into wild relatives (i.e., gene flow) and its high risks of environmental impacts [79]. Although gene flow is a legitimate concern of GM soybean, transgenes frequently represent a gain of function, which might release wild relatives from constraints that limit their fitness [80]. In parallel, several glyphosate resistance management strategies have been proposed by weed specialists to slow down the appearance of weed resistance biotypes to this herbicide [81]. One technology that has been well documented in the development of transgenically stacked-herbicide resistance traits (glyphosate + glufosinate + dicamba) in which the appearance of weeds resistant to any of these herbicides would be greatly diminished [82]. In Latin America, the bean golden mosaic virus (BGMV) from infection of whitefly is a major constraint to bean cultivation. This results in the creation of GM common bean resistance to bean golden mosaic virus (BGMV) by silencing the replicationassociated protein gene (rep) [83].

## **6. Conclusions**

Legume breeding includes different aspects starting from genetic diversity identification and evaluation and improving genetically traits by classical and modern breeding methods. Achievement in legume breeding was gained in fields of phenotypic inherited traits identification, bioengineering, and genetically modified legumes which result in improvement of various traits in legumes such as tolerance to different biotic, abiotic stress, and increase yield. Furthermore, great efforts have been performed to identify and conserve genetic resources of legumes such as wild species, landraces, old cultivars, research materials, breeding lines, and advanced cultivars through classical and state of the art breeding approaches.

## **Conflict of interest**

The authors declare no conflict of interest.

*Legume Breeding: From Conventional Method to Modern Technique DOI: http://dx.doi.org/10.5772/intechopen.101519*

## **Author details**

Parastoo Majidian Mazandaran Agricultural and Natural Resources Research and Education Center, Agricultural Research, Education and Extension Organization (AREEO), Sari, Iran

\*Address all correspondence to: p.majidian@areeo.ac.ir; parastoomajidian63@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 5**
