Faba bean genomics

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Faba bean genomics: Current status and future prospects Article in Euphytica · August 2012 DOI: 10.1007/s10681-012-0658-4

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Euphytica (2012) 186:609–624 DOI 10.1007/s10681-012-0658-4

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Faba bean genomics: current status and future prospects Salem S. Alghamdi • Hussein M. Migdadi Megahed H. Ammar • Jeffrey G. Paull • K. H. M. Siddique



Received: 6 August 2011 / Accepted: 3 March 2012 / Published online: 21 March 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Faba bean represents a crucial source of protein for food, especially for Mediterranean countries, and local demand for faba bean grains is increasing. The crop is also gaining increased attention as an elite candidate for conservation agriculture. However, the complexity of the faba genome has made progress in breeding programs and molecular studies relatively slow compared with other legume crops. Recent advances in plant genomics have made it feasible to understand complex genomes such as faba bean. With the increase of faba bean consumption in the Middle East region, there is an urgent need to develop elite faba genotypes suitable for arid and semi arid environments, with high yield potential and acceptable nutritional quality. This article highlights the recent advances in legume and faba genomics and its potential to contribute to the above mentioned goal.

S. S. Alghamdi (&)  H. M. Migdadi  M. H. Ammar  K. H. M. Siddique Legume Research Group, Plant Production Department, Faculty of Food and Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia e-mail: [email protected] J. G. Paull Schools of Agriculture, Food and Wine, The University of Adelaide, Adelaide, SA 5005, Australia K. H. M. Siddique The UWA Institute of Agriculture, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia

Emphasis is given on prospects on faba improvements strategies from the Middle East point of view. Keywords Faba bean  Genomics  QTL Functional genomics  Genetic diversity  A biotic stress

Introduction Legumes along with cereals have been fundamental to agriculture, providing protein in human diet, edible oils, fodder and forage for animals. Furthermore, legumes supply an important added value to the crop by fixing atmospheric nitrogen in symbiosis with soil bacteria, thus, reducing costs and minimizing impact on the environment. Faba bean (Vicia faba L.) is the third most important cool season food legume crop worldwide. It originated in the Near East and was domesticated during the early stages of the development of agriculture (Cubero 1974). Due to its multiple uses, high nutritional value, and ability to grow over a wide range of climatic and soil conditions, faba bean is suitable for sustainable agriculture in many marginal areas (Nadal et al. 2003), and the crop has gained greater global attention in recent years. Faba bean is a member of the Fabaceae family (grain legume) and native to North Africa and Southwest Asia. It has been extensively cultivated in the Middle East for millennia and is an important protein crop, also known as ‘field

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bean’, in Africa, Asia, Europe, Australia and North America. China alone provides half of the 4.3 million tons produced worldwide (Li-Juan et al. 1993). The crop has a tremendous genetic potential for developing resistance to biotic and abiotic stresses. In addition, breeding of cultivars with tannin-free seeds and, more recently, with low vicine–convicine content, offers new perspectives in animal nutrition (Torres et al. 2010). Faba bean plays a critical role in crop rotation through symbiotic N2-fixation and in improving soil physical conditions after harvest, and is also utilized as green manure, especially in organic farming. It also provides vegetable proteins in animal and poultry feed supplies and contributes to human nutrition because of its high protein content and other essential nutrients (Haciseferogullari et al. 2003). Although faba bean is less consumed in western countries as human food, it is considered as one of the main sources of cheap protein and energy for many people in Africa, parts of Asia and Latin America, where many people cannot afford to buy meat. The crop is becoming increasingly important in Saudi diets and it is consumed fresh as a vegetable and as dry seeds (Alghamdi 2008). The protein content among faba bean genotypes ranges from 24 to 35 % of seed dry matter (El-Sherbeeny and Robertson 1992). Faba beans are rich in lysine and therefore considered a valuable supplement to cereal proteins (El Fiel et al. 2002). The seeds however, are poor in sulfur amino acids and tryptophan when compared with soybean seeds. Genetic and breeding improvements in faba bean have resulted in new plant types adapted to environmental stresses, having high yield potential, high protein content, disease resistance and free from major anti-nutritional factors (tannins, vicine–convicine) in the seeds (Duranti and Gius 1997; Torres et al. 2010). From the center of origin, the crop spread around the world leading to specific adaptation factors and selection for different agronomic traits related to plant architecture along with size, weight and shape of seeds. According to seed characters, four groups have been defined: major, equina, minor and paucijuga (Cubero 1974). These groups can still be recognized in the major areas of production. The largest seeds emerged in South Mediterranean countries and China. Medium seeded types (equina) are grown throughout the Middle East, North Africa and in Australia while small seeds are found in Ethiopia and are the favored type in North European agriculture (Duc 1997).

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Mature faba bean is a staple in the diets of many societies in the Middle East and North Africa, while green faba beans are a favored vegetable in many countries. Though the agronomic and economic importance of faba bean is well demonstrated, its cultivation is still limited due to a range of factors such as the high susceptibility to diseases and pests and the sensitivity of the crop to adverse environmental conditions. Faba bean contributes to sustainable agriculture for management of soil fertility and plays an essential role in crop rotation. Faba is grown in about half a million ha in the North African and Middle East countries. Morocco grows the largest area of close to 200,000 ha, and the highest productivity per unit area in the region is in Egypt with an average yield of 3.4 t ha1 and a total production of 244,109 tons (FAO 2009). The Kingdom of Saudi Arabia imports approximately 100 thousand tons of food legumes annually with an annual cost of about 145 million SAR to fill the gap between local production and consumption (Ministry of Agriculture, KSA 2007). Faba bean is consumed fresh and as dry seeds and local demand is significantly increasing. However, many factors hamper large scale production of faba bean in the region including biotic and abiotic stresses. Due to the arid and semi-arid environmental conditions, the abiotic stresses of drought, salinity, heat and frost are among the main production limiting factors, while a relatively low levels of infestations with biotic stresses is observed in the Kingdom. Furthermore, in some pockets in the region, where temperate and humid weather occur, as northern parts of Egypt, Morocco and Sudan, biotic stresses are also of a great concern. Developing stress tolerant, high yield potential, early maturing varieties with acceptable nutritional quality will significantly enhance the area of cultivation of faba bean in the Mediterranean region. Farmers in areas of unfavorable agricultural environments rely upon their local landraces and there is often poor adoption of new cultivars that were selected for adaptation to less favorable conditions. Reasons for poor adoption include lack of access to these varieties, the varieties might not have the attributes required by farmers, or they might not appear as productive as expected. A farmer’s participatory approach of variety development and extension might encourage the adoption of higher yielding varieties by low-resource farmers. Since application of genomic

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tools does not eliminate the need for breeding programs to cope with genotype by environment interaction (Witcombe 1996), the possible selection for yield based on molecular markers may require preliminary definition of adaptation and yield stability targets, (Hayes et al. 1993). This approach has been applied in many regions including Colombia, India, Namibia, Nepal, Rwanda, Australia, Syria and Jordan (Weltzien 2000; Ceccarelli et al. 2000; Sperling et al. 2001; Witcombe et al. 2003; Bishaw 2004). The genome size of faba bean (2n = 29 = 12 chromosomes) is 13,000 Mb (Ellwood et al. 2008), much larger than other legume crops such as soybean (Glycine max) (*1,200 Mb), pea (Pisum sativum) (*4,000 Mb) or (Medicago sativa) (*450 Mb). Due to the genome complexity compared to other legume species, relatively low progress has been made in faba bean genomics and this has made it difficult to breed elite cultivars for adverse environmental conditions. It is worth to note that the genome complexity of Vicia faba compared to other Vicia species may explain why it is reproductively isolated from other Vicia species. Marker technology and faba genomics The large genome size of faba bean may be largely explained by a high number of retrotransposon copies, microsatellites and even genes which are the basis of the sequence variability that can be explored in genomes (Pearce et al. 2000). Morpho-agronomical genetic diversity based assessment is classic and imperative for their effective exploitation in plant breeding schemes and their efficient conservation and management. Molecular marker systems have provided a new avenue for evaluating germplasm and assessing the genetic diversity in faba bean populations. They are not affected by environmental factors or by development stages and can save time and costs in selection (Pozˇa´rkova´ et al. 2002; Zeid et al. 2003; Roman et al. 2004; Terzopoulos and Bebeli 2008; Torres et al. 2010; Zong et al. 2009), and can contribute to a more holistic picture of genetic diversity within a collection of populations (Curley and Jung 2004). The development of a wide range of molecular tools and their utilization will have a significant impact on the development of elite faba bean lines with superior agronomic attributes, and identification of genes/ genotypes for specific traits of interest. Molecular markers and isozyme polymorphisms have been

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reported for faba beans (Table 1), but to a limited extent compared to other major crop species. Biochemical markers have been used in Vicia for numerous applications such as evolutionary and taxonomic studies, inbreeding, estimating crossing frequencies, varietal fingerprinting, synthetic value and cultivar identification (Suso et al. 1995; Maalouf et al. 1999) and further studies on isozyme inheritance and variation include those of Van de Ven et al. (1991), who constructed the first linkage map of V. faba using Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorhic DNA (RAPD) and isozyme markers. Torres et al. (1993) used 66 segregating allozyme and DNA polymorphisms to construct a preliminary linkage map for faba bean and Suso et al. (1993) added new isozyme markers to this map. Torres et al. (1995) studied the genetics, location, and linkage of isozyme markers in 13 F2 populations trisomic for four of the six chromosomes (nos. 3, 4, 5, and 6) of the species. Five loci were assigned to a specific chromosome: Est-2 to chromosome 3, Fk-2 to chromosome 4, Prx-1 to chromosome 5, and Sod-1 and Pgd-p to chromosome 6, while additional isozyme markers were assigned to linkage groups in a subsequent study (Torres et al. 1998). Genetic diversity of faba bean has been studied with a number of molecular marker systems. Van de Ven et al. (1990) used RFLP markers to study genetic variability in 16 accessions of V. faba and four wild Vicia species. The majority of polymorphisms detected were attributed to the wild species, but as interspecific crosses cannot be produced between faba bean and other species, it is important to identify genetically diverse faba bean parents to create a linkage map based on molecular markers. Link et al. (1995) used RAPD markers to study the genetic diversity within European (minor and major) and Mediterranean faba bean germplasm. Cluster and principal coordinate analyses identified European small-seeded lines and Mediterranean lines as distinct groups with European large-seeded lines located in between, consistent with published archaeo-botanical findings. They concluded that RAPDs are useful for classification of germplasm and identification of divergent heterotic groups in faba bean. Alghamdi (2003) used RAPD markers to distinguish between faba bean genotypes, comprising eight inbred lines and twenty-eight F1 hybrids derived from eight parents. Amplification conditions and reproducibility

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Table 1 Summary of molecular markers and their applications in faba bean Application

Marker type

Main output

Reference

Taxonomy

RFLP, RAPD

V. faba is more closely aligned to species from the sections Hypechusa and Peregrinae than to those in the narbonensis complex

Ven et al. (1993)

Genetic diversity

RFLP

Importance of genetically diverse faba bean parents to create a linkage map based on molecular markers Classification of germplasm and identification of divergent heterotic groups

Van de Ven et al. (1990)

Varietal identification and genetic purity assessment of F1 hybrid seeds

Alghamdi (2003)

RAPD

AFLP

ISSR

Verification of pedigree relationships

Zeid et al. (2003)

Genetic diversity assessment of Chinese winter and spring faba bean

Zong et al. (2009, 2010)

Genetic diversity analysis of Mediterranean faba bean

Terzopoulos and Bebeli (2008), Alghamdi et al. (2011)

Genetic diversity assessment in faba bean

Mapping

Marker development for gene tagging and MAS

QTL mapping

Genetic variation among eight faba bean genotypes differing in drought tolerance

Al-Ali et al. (2010)

SSAP

Distinction between geographic origins of V. faba genotypes,

Sanz et al. (2007)

RFLP, RAPD, SSR, isozyme and allozyme markers

Construction linkage map of V. faba

Torres et al. (1993, 1995, 1998), Patto et al. (1999) Pozˇa´rkova´ et al. (2002)

Development and characterization of microsatellite markers from chromosome 1-specific DNA libraries Development of a composite map in Vicia faba

Roman et al. (2004),

Comparative genetic map

Ellwood et al. (2008)

CAPs and SCARs,

Vicine,convicine and tannins contents in faba bean

SSR

Growth habit

Gutierrez et al. (2006, 2007, 2008)Avila et al. (2006, 2007)

RFLP, RAPD, SSR, isozymes and allozymes markers

New loci from Orobanche resistant

Zeid et al. (2009)

Development and characterization of 21 EST-derived microsatellite

Ma et al. (2011)

First QTL map in faba

Ramsey et al. (1995)

Mapping of quantitative trait loci controlling broomrape resistance

Roman et al. (2002, 2003)

Locating genes associated with Ascochyta fabae resistance

Diaz et al. (2004, 2005)

RAPD markers linked to the Uvf-1 gene conferring hypersensitive resistance against rust

Avila et al. ( 2003)

Ascochyta blight resistance

Avila et al. (2004)

Frost tolerance

Arbaoui et al. (2008)

of RAPD patterns were examined. Comparison of polymerase chain reaction (PCR) products obtained using 25 decamer arbitrary primers allowed identification of all the lines analyzed. With several primers, line specific RAPD markers were identified while with

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Link et al. (1995)

others polymorphism was more extensive, revealing several RAPD markers for several lines. Zeid et al. (2003) applied Amplified Fragment Length Polymorphism (AFLP) markers to assess genetic diversity among elite faba bean inbred lines

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from Europe (minor, major, winter and Southern European types), North Africa and Asia (China and Japan). Based on clustering and Principal Coordinate Analysis they found that only the Asian lines were distinct as a group and several known pedigree relationships were verified. The history of spread and cultivation of faba bean in the studied regions were corroborated with a priori grouping of inbred lines (on the basis of geographic origin and seed size) and AFLP data. High level of genetic diversity was identified among local populations of Greek germplasm by Inter Simple Sequence Repeats (ISSRs) (Terzopoulos and Bebeli 2008). The Mediterranean-type faba beans could be subdivided into at least two different germplasm pools with promise for the production of synthetic varieties. Furthermore, Alghamdi et al. (2011) used twelve ISSR primers to assess the genetic diversity among 34 local Saudi Arabian and exotic faba bean accessions. The ISSR primers produced a total of 71 alleles, all of which were polymorphic using the 34 genotypes. The results of clustering by Nei’s genetic distance using UPGMA algorithm at the 0.6 dissimilarity separated genotypes to four main clusters with many sub clusters. Genotypes collected from Egypt and King Saud University grouped in one cluster, ICARDA’s genotypes in another cluster and one local determinate hybrid line formed a single cluster. The remaining genotypes were clustered in the fourth group. The high number of sub-clusters formed in their study indicated high genetic variability related to collection sites and should be utilized in faba bean improvement. Al-Ali et al. (2010) detected a considerable amount of genetic variation among eight faba bean genotypes differing in drought tolerance. A high level of co-linearity was observed between morphological and molecular data indicating the potential to develop molecular markers to assist selection in faba bean breeding. ISSR proved to be a good tool in varietal identification and fingerprinting due to the presence of varietal specific bands. The genetic diversity of winter and spring faba bean landraces from different provinces in China was compared with germplasm from the rest of the world, using AFLP analyses (Zong et al. 2009, 2010). The most diverse set of spring faba bean accessions was from Canada, followed by accessions from ICARDA. The other landraces were clustered in four gene pools—a separation of Asian/Chinese from European/ African accessions, and further separation between

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China and Asia (non-Chinese), and between Africa and Europe. The winter faba bean accessions from China were found to be distinct from the winter gene pools in the rest of the world and from the spring types in China. African and Asian faba bean landraces showed some clustering, and these were broadly associated with European sources. Genomic microsatellites (SSRs) was used to characterize genetic variability of a global composite collection of 1,000 accessions of faba bean developed in collaboration between ICARDA (Syria), INRA (France) and CSIC (Spain). A preliminary test made with 2 primers designed for genomic SSR motifs and applied to 750 genotypes, revealed 127 alleles which indicated a high level of polymorphism between faba bean accessions (Duc et al. 2010, cited after Sadiki et al. 2006). Due to the limited knowledge of the progenitor species of V. faba, some studies have been undertaken to identify closely related species by means of molecular markers. For instance, the taxonomic relationships between 52 accessions of 12 Vicia species and three accessions of Lathyrus spp. were examined using nuclear RFLP- and PCR-generated data (Ven et al. 1993). An analysis of mitochondrial DNA phenotypes was both consistent with and complemented the results from the nuclear data that suggested that V. faba is more closely aligned to species from the sections Hypechusa and Peregrinae than to those in the narbonensis complex. Shiran and Mashayekh (2004) concluded that no variability could be detected among V. faba genotypes at the cytoplasmic level on chloroplast DNA, which appeared to be highly conserved during domestication, with a narrow variability at the origin. In contrast, Flamand et al. (1992, 1993) demonstrated that mitochondrial DNA displays variability in sequence and plasmid size; in some cases, associated with diversity in nuclear genomes. Long Terminal Repeat sequence retrotransposon based markers were compared for their usefulness in sequence specific amplified polymorphism (SSAP) marker development in two Vicia species (V. narbonensis and V. faba). SSAP could distinguish between geographic origins of V. faba genotypes, but not between paucijuga, minor, equina or major types (Sanz et al. 2007). Faba bean possesses one of the largest and least studied genomes among cultivated crop plants, including other legume species. The development of a detailed linkage map for V. faba will greatly increase

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the efficiency of genetic and breeding studies, and enable the establishment of marker-assisted selection for economically important traits (Torres et al. 2010). These technologies have proven useful in many species other than V. faba. In species with a large genome size and little pre-existing genomic information such as in faba bean, development of genetic markers is complex and costly, and the development of high-density linkage maps, and identification and locating genes of interest is complicated without reliable and informative molecular tools. Hence the development of a wide range of molecular markers is crucial to faba bean breeding and for further use in marker assisted breeding. One of the earliest studies on the faba genome was carried out by Sirks (1931) who found that 19 genetic factors formed four linkage groups. Sirks’ material was lost during the Second World War. Later, several linkage maps based on morphological, isozyme, RFLP, microsatellite and RAPD markers were constructed (Torres et al. 1993, 1998; Satovic et al. 1996; Patto et al. 1999; Pozˇa´rkova´ et al. 2002; Roman et al. 2002, 2003, 2004) and the first attempt at mapping quantitative trait loci (QTL) in faba bean was reported by Ramsey et al. (1995). Moreover, the first attempts to map genes and QTLs for resistance to the parasitic plant broomrape (Orobanche crenata) and fungal diseases (Ascochyta fabae and U. viciae-fabae) have been reported (Roman et al. 2002, 2003; Avila et al. 2003, 2004). The first exclusively gene-based genetic map of faba bean was produced by Ellwood et al. (2008) using gene-based orthologous markers and an F6 recombinant inbred line population. The linkage analysis using intron-targeted amplified polymorphic (ITAP) markers revealed seven major and five small linkage groups (LGs) with evidence of a simple and direct macrosyntenic relationship between faba bean and Medicago truncatula, and found to share a common rearrangement with lentil. They concluded that the composite map, anchored with orthologous markers mapped in M. truncatula provides a central reference map for future use of genomic and genetic information in faba bean genetic analysis and breeding. Roman et al. (2004) used isozymes, RAPDs, seed protein genes and microsatellites to construct a composite map of the V. faba genome. The 14 major linkage groups (five of which were located in specific chromosomes) included 192 loci and covered 1,559 cM

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with an overall average marker interval of 8 cM. This would provide an efficient tool in breeding applications such as disease-resistance mapping, QTL analyses and marker-assisted selection. Linkage analysis of seven F2 families of faba bean descended from trisomic plants, analyzed for isozyme, morphological, RAPD and seed-protein genes revealed 14 linkage groups, 8 of which were unambiguously assigned to specific chromosomes. The most important QTLs for seed weight, located on chromosome 6, explained approximately 30 % of the total phenotypic variation (Patto et al. 1999). Primary trisomics available in V. faba have been used to establish the chromosomal location of many genes and to consolidate the linkage map of this species (Torres et al. 1993, 1998). Resistance to several major pathogens or pests of V. faba has been identified by QTL analysis. Roman et al. (2002) developed the first QTL map controlling crenate broomrape response in V. faba. Three QTLs explained more than 71 % of the phenotypic variance and two of the QTLs showed considerable dominant effects in the direction of resistance suggesting that broomrape resistance in faba bean can be considered a polygenic trait with major effects from a few single genes. The strong dominant effects of the QTLs found in this study do not preclude their use in faba bean breeding programs since the introgression of different resistance genes into breeding lines may generate even more resistant, heterozygous plants. Two out of three QTLs have been further validated in RILs, moreover, new genome regions on chromosome I conferring resistance were also detected (Diaz et al. 2004, 2005a). Further validation of putative QTLs across multiple test environments is being carried out as well as the saturation of the genomic regions associated with broomrape resistance (Garcı´a-Ruı´z 2007; Zeid et al. 2009). Using the QTL approach, Roma´n et al. (2003) identified two QTLs (Af1 and Af2) for partial resistance against one isolate of Ascochyta blight (Ascochyta fabae) ascribed to chromosomes 3 and 2. The QTLs explained 25.2 and 21.0 % of the phenotypic variability, respectively. Avila et al. (2004) used bulk segregant analysis (BSA) to identify the first molecular markers associated with the gene Uvf-1 controlling a race-specific resistance to rust in faba bean. Furthermore, they studied the resistance to A. fabae on leaves and stems and located 6 QTLs, Af3 to Af8. Both isolatespecific and organ-specific QTLs were detected.

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Mapping conserved markers in the region of the QTLs will help to establish the possible homology of these QTLs and will demonstrate the stability of QTLs for A. fabae resistance across different genetic backgrounds. Subsequently, Diaz et al. (2005b) have developed RILs and new molecular maps are being constructed. Preliminary results revealed the conservation of Af1 and Af2 across early (F2) and late (F6) selfing generations. Avila et al. (2003) mapped rust resistance in faba bean using Bulk Segregant Analysis (BSA). Three RAPD markers (OPD13736, OPL181032 and OPI20900) were mapped in coupling phase to the resistance gene Uvf-1. No recombinants between OPI20900 and Uvf-1 were detected. Two additional markers (OPP021172 and OPR07930) were linked to the gene in repulsion phase at a distance of 9.9 and 11.5 cM, respectively. Although Quantitative Trait Loci (QTL) mapping studies have been performed for almost all grain legumes, in most cases no markers are readily available for Marker Assessed Selection (MAS). The limited saturation of the genomic regions bearing putative QTLs makes it difficult to identify the most tightly-linked markers and to determine the accurate position of QTLs (Torres et al. 2010). Linkage disequilibrium (LD) is emerging as alternative tool for identifying QTLs in plants. LD, the nonrandom association of alleles at different loci, plays an integral role in association mapping, and determines the resolution of an association study. Recently, association mapping has been exploited to dissect QTL. LD patterns and distributions are of fundamental importance for genome-wide mapping associations (Agrama et al. 2006; Samah 2009). Faba bean is one of the most drought sensitive legumes and development of drought-tolerant cultivars is essential to improve yield stability of the crop. Recently, expressed sequence tagged sites (ESTs) involved in drought avoidance were identified in chickpea, pea and M. truncatula (Jayashree et al. 2005; Buhariwalla et al. 2005). Determination of the genetic variability of these genes and their integration into current faba bean maps through comparative genomics and synteny studies may be the next steps towards a targeted molecular breeding effort and a more efficient faba bean germplasm management (Torres et al. 2010). Winter-hardiness is a complex trait, which depends not only on the frost tolerance but also on tolerance to other abiotic and biotic

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constraints. The superiority of winter types over spring types for grain yield, and the crucial impact of complete overwintering on grain yield emphasize the importance of improving faba bean winter hardiness (Arbaoui et al. 2008). Accumulation of polyunsaturated fatty acid is considered an important adaptation mechanism to low non-freezing and freezing temperatures. Thus, 101 faba bean recombinant inbred lines derived from the cross between two frost-tolerant lines were tested for frost tolerance and leaf fatty acid content before and after hardening (Arbaoui and Link 2008). For frost tolerance, five putative QTLs were detected; three for unhardened frost tolerance that explained 40.7 % of its genotypic variance and two for hardened frost tolerance that explained 21.8 %. Three QTLs were detected for oleic acid content in unhardened leaves, explaining a total of 40.6 % of the genotypic variation after cross-validation (Arbaoui et al. 2008). The first molecular marker (cleaved amplified polymorphism (CAP)) with 100 % discriminating efficiency for the selection of determinate growth habit in faba bean was developed by Avila et al. (2006, 2007) using the candidate gene approach. They suggested that this co-dominant marker could facilitate the breeding process and be very useful for marker assisted selection, quality control and varietal registration. A gene tagging approach has been carried out to facilitate indirect selection for seed quality traits zero tannin and low vicine and convicine content in faba bean seeds. Bulk segregant analysis (BSA) identified RAPD markers linked to the genes conferring these traits. These markers were cloned and sequenced to develop more robust and reliable SCARs. The SCAR markers developed will facilitate gene pyramiding and accelerate the production of new faba bean cultivars with improved nutritional value for human and animal consumption (Gutierrez et al. 2006, 2007, 2008). Summary of the utilization of various molecular markers in vicia faba are presented in Table 1.

Faba bean functional genomics As a result of the rapid accumulation of DNA sequence information, including the complete genome sequences for several plant species, plant research is focusing increasingly on analysis of gene function.

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Genomics approaches are essential to understand complex traits and various metabolic pathways (Gepts et al. 2005). Breakthroughs in understanding the relationship between genotype and phenotype will come about through proteomics and metabolomics. Due to the complex nature of the faba genome, there has been slow progress in structural faba bean genomics and subsequently functional genomics is far behind many other crops. A very few and scattered efforts have been made in this regards. Thus, an indirect approach of using model plant systems has been proposed. Comparative genomics with model plants to assess synteny can facilitate back-and forth use of genomics resources between different legume species, making the research cost-effective and efficient. In addition, it can speed up gene discovery in species that are less understood because of their large genome or they are less easily transformed. The model plant A. thaliana played a key role in elucidating the functions of many plant genes, especially in monocots. However, A. thaliana cannot be considered as the universal plant model, particularly for legumes since it is not the natural host of many pathogenic or symbiotic bacteria and fungi (Handberg and Stougaard 1992). M. truncatula and Lotus japonicus represent models for the cool-season legumes. Their genomes are relatively small, estimated around 500 and 470 Mb respectively (Sato et al. 2007), and are significantly smaller than most legume species, e.g. 4,000 Mb for pea or 13,000 Mb for faba bean. Furthermore, they are ambient for plant transformation techniques using Agrobacterium tumefaciens or Agrobacterium rhizogenes (Rispail et al. 2010). Considerable synteny at the genome level among legume species has been reported (Lee et al. 2001; Kalo et al. 2004; Choi et al. 2004; Zhu et al. 2005). More than 200,000 and 100,000 ESTs are available from public database for M. truncatula and L. japonicus respectively, and nearly 190 and 315.1 Mb of their respective genomes have been sequenced (Cannon et al. 2006; Ane´ et al. 2008). A high level of co-linearity was found between the faba bean, lentil and M. truncatula genomes based on the macro-synteny established between faba bean and M. truncatula. Despite the large differences in genome sizes between M. truncatula and V. faba, a simple and direct relationship between the two genomes was identified with strong evidence for extensive colinearity between linkage group pairs of the two species (Ellwood et al. 2008). The large synteny

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among the model legume species and V. faba suggests their possible use in orthologous gene identification, marker development and establishing high density physical maps. This will greatly help in dissection of the complex faba genome and will help to understand the various cellular pathways in response to environmental stimuli. The EST sequence data available in the public domain from three cereals, namely rice (Oryza sativa), maize (Zea mays) and sorghum (Sorghum bicolor), and three legumes, namely soybean (G. max), medicago (M. truncatula) and lotus (L. japonicus), were used by Jayashree et al. (2005) to fish out SSR motifs and validate them in V. faba genotypes The datasets obtained from public resources represented 98.9 Mb of sorghum (187282 ESTs), 183.7 Mb of maize (407423 ESTs), 143.9 Mb of rice (272567 ESTs), 45.4 Mb of lotus (109618 ESTs), 135.86 Mb of soybean (330436 ESTs) and 121.1 Mb from Medicago (226923 ESTs). Their results showed that on an average, 19 % of the ESTs from cereals (35–65,000 ESTs) and 10.6 % of the ESTs from legumes (10–35,000 ESTs) were found to contain SSRs in the complete redundant set of ESTs analyzed. The frequency of SSRs observed under the conditions in this study amounted to 1 SSR/1.79 kb in sorghum, 1 SSR/2.21 kb in maize, and 1 SSR/1.72 kb in rice while in the three legumes considered in this study the frequency of occurrence was 1 SSR/3.5 kb. Thus the three cereal crops had a higher relative abundance (number of SSRs/kb of sequence) of SSRs compared to the legumes. Recently, some faba bean sequences were made available on the public database, and more than 5,000 ESTs are now available, in addition to some other scattered genomic sequences. These emerging sequences, together with the tremendous progress in sequencing technologies, i.e. second generation sequencers, opened a new avenue for faba bean genomics and many working groups around the globe have initiated some sequencing efforts in faba bean. This will make it feasible to construct a high density physical map and hence facilitate gene tagging and subsequent map-based cloning. Twenty-one ESTderived microsatellite simple sequence repeat (SSRs) markers were developed in faba bean by screening the NCBI database and Markers were validated and explored for size polymorphism among faba bean accessions (Ma et al. 2011).

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Transcriptome analysis If sequencing complex genomes such as faba bean genome is a challenging task, assigning the functions for putative genes will be an even more difficult one. DNA microarray technology is a key element in today’s functional genomics toolbox. Transcriptomic tools have been developed in model legume species for the study of such complex genomes (Gallardo et al. 2007; Sanche´z et al. 2008a, b). For instance, 384 salt stress-related genes were used to study M. truncatula response to salt stress (Merchan et al. 2007). Affymetrix chips with bioinformatically optimized oligonucleotides are also commercially available for model legumes (http://www.affymetrix.com; Sanchez et al. 2008) and a novel generation of M. truncatula gene chips with probe sets for 1,850 M. sativa transcripts that will facilitate transcriptomic analysis of closely related species are now available (Ane´ et al. 2008). Quantitative Polymerase Chain Reaction (qPCR) has been established for simultaneous monitoring of transcription factors in M. tranculata (Kakar et al. 2008). Furthermore, Avila et al. (2006) demonstrated that an ortholog of CEN/TFL1-like genes is responsible for the growth habit in faba bean. CEN/TFL1like genes are extensively recognized as a group of homologous genes responsible for growth habit in different plant species. Based on this gene information, Avila et al. (2007) developed a CAPs marker for growth habit with potential for marker assisted selection in faba bean. They reported that the marker is expected to facilitate efficient detection of genotypes with determinate growth habit in faba bean breeding populations. Moreover it will play an important role during selection in pyramiding additional suitable genes to develop new cultivars for green pod production. Proteome analysis Several proteomic approaches, based on different protein separation techniques and identification by mass spectrometry, have been developed and applied for legume model species. These original approaches targeted the establishment of reference protein and peptide maps in M. truncatula (Watson et al. 2003) and the analysis of the symbiotic compartment in both L. japonicus and M. truncatula (Wienkoop and Saalbach 2003; Valot et al. 2006; Larrainzar et al.

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2007; van Noorden et al. 2007). More recently, the range of application of proteomic approaches has been broadened to include grain filling (Repetto et al. 2008) and pathogen interactions (Colditz et al. 2004). Second and third generation proteomic tools such as Differential In-Gel Electrophoresis (DIGE) and isobaric Tag for Relative and Absolute Quantitation (iTRAQ) are being developed in M. truncatula along with approaches targeting the post-translational modifications including nitrosylation and phosphorylation on a large scale. So far, there are no large scale proteomic studies on faba bean. Utilization of such approaches will help to understand faba bean genome and elucidate gene function, particularly those genes governing the abiotic stress and nutritional values. This will significantly facilitate crop manipulation and improvement. Knock out and transgenic approach: Development of novel methods to introduce genes into grain legumes through plant transformation promises to give plant breeders the opportunity to overcome hybridization barriers and limitations related to those traits for which little or no natural resistance has been identified. In addition, transformation provides the means to study gene function and genome organization (Rispail et al. 2010). Faba bean appears to be recalcitrant towards in vitro regeneration (Khalafalla and Hattori 2000). Due to difficulties in regeneration from callus, and the releasing of phenolic compounds, studies on in vitro culture of faba bean are therefore difficult to carry out (Bo¨ttinger et al. 2001). However, some regeneration systems of faba bean have been reported (Bo¨ttinger, et al. 2001; Hanafy et al. 2005; Bahgat et al. 2009) thus, making faba bean transformation feasible. The first attempts to transfer foreign genes into V. faba were published by Schiemann and Eisenreich (1989). Seedlings were inoculated with Agrobacterium rhizogenes containing the binary vector pGSGluc1 transferring nptII and GUS under control of the bidirectional TR1/2 promoter. GUSpositive roots developing at the inoculation sites were propagated on hormone-free medium. Callus established from these roots maintained GUS activity. Regeneration of shoots was not reported. A similar study was performed by Ramsay and Kumar (1990), using an A. rhizogenes strain containing pBin19 for inoculation of V. faba cotyledons and stem tissue,

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leading to successful transfer of nptII. The transgenic nature of established root clones was confirmed by hormone autotrophy and NPTII dot blot assays. To date there are few reports on the successful transformation of V. faba with the recovery of fertile transgenic plants. One of legume transformation landmark experiments was the production of transgenic lines of V. narbonensis via Agrobacteriummediated with a gene for the methionine-rich 2S albumin from Brazil nut (Bertholletia excels). The trangene was controlled by a seed-specific promoter and the coding region of the 2S albumin gene that has been completely synthesized was fused to the seedspecific leguminB4 promoter from Vicia faba (Pickardt et al. 1995). Bo¨ttinger et al. (2001) established stable transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lys C gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine, and (3) the coding sequence for a methionine-rich sunflower 2S-albumin, each in combination with nptII as selectable marker (Bo¨ttinger et al. 2001). Hanafy et al. (2005) developed an efficient Agrobacterium-mediated transformation system in faba bean based upon direct shoot organogenesis of meristematic cells derived from embryo axes. They reported the production of transgenic faba bean containing the SFA8 gene from sunflower and bar gene from Streptomyces hygroscopicus as a selectable marker. Transformed Albatross and Giza 2 cultivars were regenerated and confirmation of transgene was assayed. Many reverse genetic approaches have been developed in legume model plants. To this purpose, several collections of chemical or insertional mutants including T-DNA and transposon tagged lines have been created (Penmetsa and Cook 2000; Webb et al. 2000; Kawaguchi et al. 2002; Tadege et al. 2008). These collections have already been used to identify new genes required for symbiosis but may also be screened for other interesting traits. The improvement of PCRbased techniques for screening for mutation in a gene of interest allowed the development of novel approaches for efficient reverse genetic analysis. Several of these novel approaches have been, or are being, developed in M. truncatula and L. japonicus

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including Targeted Induced Local Lesions in Genome (TILLING), saturating Tnt1-insertion mutagenesis and fast-neutron mutagenesis. TILLING relies on chemical mutagenesis, for example with ethyl methylsulfonate (EMS), and provides an allelic series ranging from silent mutations to complete loss-offunction of the gene of interest. This method was first developed for legume in L. japonicus (Perry et al. 2003) already allowing the identification of novel symbiotic genes in this species and could be extended in Vicia faba to uncover the gene function of economically important traits. In addition to help identifying new genes involved in plant biology, these methods can serve to identify the exact function of these genes, which is a prerequisite step before gene transfer into other legume crops such as faba bean. Mutagenesis with fastneutron, that cause deletions, and transposable elements such as Tnt1 and Tos17 and/or T-DNA tagging, when inserted within the gene of interest, all are likely to produce gene knockouts. These approaches are ideal to identify gene function in grain legumes including Vicia faba. Furthermore, point mutants identified by TILLING could be more useful as a source of favorable alleles for subsequent selection. Transformation-based methods such as RNA interference (RNAi) and/or Virus-Induced Gene Silencing (VIGS) were also established in model legumes (Limpens et al. 2004; Maeda et al. 2006) for gene knock out. Understanding and implementation of these approaches will greatly help in faba improvement programs through identification of gene function and subsequent crop manipulation. Perspectives The development of genetic markers is complex and costly in species with little pre-existing genomic information. Faba bean possesses one of the largest and least studied genomes among cultivated crop plants and few gene-based genetic maps have been reported (Ellwood et al. 2008). The development of new markers in faba bean will be an essential step for MAS to be adopted as a routine procedure in faba bean breeding programs. Many regional working groups are now engaged in developing molecular markers in faba bean. This includes the utilization of SCAR, SRAP, ISSR, AFLP, SSR and SNP markers. Developing new SSRs based on SSR enriched libraries from locally

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adopted genotypes, EST-based SSRs or cross legume SSRs may be deployed. The development of SSRs together with increasingly larger sets of transferable markers such as ESTs in faba bean should provide direct bridges among genetic maps, allowing not only to streamline high-resolution mapping and positional cloning of major QTLs or genes of interest, but also the development of many types of DNA markers such as STSs, SCARs or SNPs that will greatly help in establishing MAS systems (Torres et al. 2010). Evaluation of the extent of linkage disequilibrium in exotic and domesticated germplasm is required. Phenotypic evaluation of multiple populations per species should be conducted so that the locations of quantitative trait loci for important agronomic traits can be identified by genetic and association mapping. The accumulation of mapping information will facilitate the exploration of syntenic regions across legumes (Gepts et al. 2005). These genetic tools will also help in construction of physical maps of chromosomes in faba bean. Construction of physical maps will allow better understanding of such a complex genome and facilitate cloning and manipulation of traits with economic interests. This will also help to better understand the secondary metabolism involved in interactions between legumes and pathogens, symbiotic organisms, predators, and pollinators and will lead to faba bean varieties with enhanced yield potential, nutritional benefits, resistance to pests and diseases, and tolerance of adverse environmental conditions. Using molecular marker technology, it is now feasible to analyze quantitative traits such as salt tolerance, and identify the chromosomal regions associated with such characters (QTL’s). Identifying such regions will significantly help to increase the selection efficiency in the breeding programs. Molecular marker assisted selection is considered to be faster, more efficient, and probably more cost effective than conventional screening particularly for abiotic stresses where expression of the trait is subject to significant environmental effects. It will also help narrow down the possible candidate genes and ultimately will lead to map based cloning of the major genes controlling the trait of interest and opening a new avenue for genetic manipulations using the real candidate genes (Ammar 2004). With the recent advances in DNA sequencing and single nucleotide polymorphism (SNP) genotyping, new approaches to QTL mapping and quantitative trait nucleotide (QTN)

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identification are now available and this could be applied to faba been for identification of phenotyperelated SNPs. Once genes responsible for quantitative variation are identified, information can be passed onto faba bean breeding programs to enable implementation of MAS. This will greatly help in accelerating the breeding program. In addition, traditional breeding efforts will be greatly enhanced through integrated approaches using functional, comparative and structural genomics. It should be kept in mind, however, that optimization of marker genotyping methods in terms of cost-effectiveness and a greater level of integration between molecular and conventional breeding represent the main challenges for the future adoption and impact of MAS on faba bean breeding (Torres et al. 2010). The use of molecular markers and the development of suitable mapping populations will allow significant progress in mapping to enhance breeding strategies in faba bean. Local variety Hassawi 2, with drought tolerance and excellent cooking quality was used with an introduced small black seeded Pakistani variety for developing a mapping population in an attempt to map QTLs for drought tolerance in V. faba. Recent studies (Al-Ali et al. 2010) proved that some physiological parameters such as stomatal conductance, leaf rolling, and leaf temperature as will as grain yield under stress are well associated with drought tolerance. These parameters along with water use efficiency and proline content could be utilized in plant phenotyping. Breeding programs for drought tolerant in faba bean should consider the genetic diversity in the tested genotypes for physiological, morphological and agronomical traits and the important correlations among these traits. Significant correlations allow the utilization of relatively simple traits as indirect selection criteria for drought tolerance in faba bean breeding. Other drought tolerant traits investigated in a number of field legumes include: dry matter accumulation under stressed and unstressed environments, relative water content (RWC), stomata frequency, stomata size, transpiration efficiency, carbon isotope discrimination (D13C), leaf temperature and osmotic potential. These traits have been detected to have significant linkage with drought tolerance and could be utilized in drought breeding selection (Ricciardi et al. 2001; Sa´nchez et al. 2001; Stoddard et al. 2006; Khan et al. 2004; O’Neill et al. 2006; Khan et al. 2007; Al-Ali et al. 2010).

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There is an urgent need to identify chromosomal regions associated with economically important traits in faba bean. Identification of expression QTLs (e QTLs) will help in narrowing down candidate genes for traits of interest and lead to an increase number of QTLs for agronomically important traits for faba bean improvement. One of the functional genomic approaches to identify candidate genes responsible for a trait of interest is through differential expression strategies. DNA chips and subtractive hybridization are among the tools of choice to identify abiotic stress responsive genes. Many genes are expected to be drought responsive, among which, a fewer number are the real candidate genes. Combining the QTL approach with differential display strategy will allow narrowing down the possible candidate genes by focusing only on those responsive genes in the major QTL regions (Ammar et al. 2009). In summary, the bioinformatics tools and analysis of gene motifs, real candidate genes could be identified in faba bean. Further PCR-based validation using such candidate genes designed primers will demonstrate the efficiency of the genes identified. This will allow trait manipulation and eventually will lead to the development of stress tolerant faba bean genotypes. The availability of second generation sequencing and highthroughput technology in parallel with other genomic approaches will facilitate the analysis of transcripts, proteins, insertional and chemically induced mutants and will allow understanzding the gene functionphenotype relationship. Furthermore, developing efficient regeneration protocols will allow successful in vitro culture and genetic transformation in faba bean. This will facilitate the development of transgenic faba beans for biotic and abiotic stress tolerance and open a new avenue for faba bean functional genomics and crop manipulation. Ultimately this will help in developing better faba bean genotypes suitable for local and regional ecosystem and enhancing the role of faba bean for conservation agriculture in arid and semi-arid regions of the Middle East. Acknowledgments The authors wish to express their gratefulness and gratitude’s to National Science and Technology Plan (NSTP), project no. 09-BIO680-02 for financial support for this manuscript.

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