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Genetic diversity in tropical legumes: cowpea (Vigna unguiculata (L.) Walp.) and lablab (Lablab purpureus (L.) Sweet)

Tefera Tolera1, Petr Karlovsky2 and Brigitte L. Maass3

1 University of Western Australia Field Research Station, 1 Underwood Avenue, Shenton Park, Western Australia 6008. Email teferatol@yahoo.com,
2
Georg-August-University Goettingen, Department of Crop Sciences, Molecular Phytopathology & Mycotoxin Research, Grisebachstr. 6, 37077 Goettingen, Germany. http://www.gwdg.de/~instphyt/ Email pkarlov@gwdg.de
3
Georg-August-University Goettingen, Department of Crop Sciences, Agronomy in the Tropics, Grisebachstr. 6, D-37077, Goettingen, Germany. http://www.tropical-resources.uni-goettingen.de/ Email bmaass@gwdg.de

Abstract

Several legume crops like cowpea (Vigna unguiculata Walp.) and lablab (Lablab purpureus (L.) Sweet) originated from Africa. Nevertheless, the production of lablab is impaired by the successful establishment of common bean (Phaseolus vulgaris) in African agriculture most likely resulting in genetic erosion of the former. On the other hand, cowpea, particularly the cultivar group unguiculata, has long been cultivated in sub-Saharan Africa where it is an important multi-purpose crop. However, recent studies demonstrated that genetic diversity in this crop is low. A project was initiated by The World Vegetable Center’s Regional Center for Africa to curb the problem of genetic erosion in these two indigenous legumes and other leafy vegetable crops by conservation and utilization. A prime objective of this project is to promote nutritional health in eastern and southern Africa. Sixty two cowpea and 33 lablab accessions were acquired from different sources and studied for their genetic diversity by Amplified Fragment Length Polymorphism (AFLP). Four different AFLP primer combinations were used for both crops. Our study revealed that 45% of 185 and 72% of 163 scored bands in cowpea and lablab, respectively, were polymorphic. The lowest Jaccard Coefficient of Genetic Similarity was 0.82 in cowpea and 0.45 in lablab. Lablab accessions from east Africa were distinct from the previously proposed core collection established on agro-morphological traits. The UPGMA cluster dendrogram largely grouped cowpea accessions according to their seed provenance signalling the potential effect of institutional management and research direction on cowpea genetic diversity. Although cowpea has received international attention in research, its genetic diversity was low compared to lablab.

Key Words

Legumes, accessions, genetic conservation, polymorphism, AFLP

Introduction

Cowpea (Vigna unguiculata (L.) Walp.) (Singh et al., 2003) and lablab (Lablab purpureus (L.) Sweet) are cultivated widely in the tropics and have multipurpose uses: as food for human beings, fodder for livestock and atmospheric nitrogen fixers (Pengelly and Maass, 2001). Despite a large number of cowpea accessions maintained at the International Institute of Tropical Agriculture (IITA), recent studies demonstrated that genetic diversity in cultivated cowpea is low (Li et al., 2001; Coulibaly et al., 2002; Tosti and Negri, 2005) though Nkongolo (2003) found a high level of RAPD marker diversity in landraces from Malawi.

Lablab has been neglected in research and development and consequently our understanding of its genetic diversity is limited (Pengelly and Maass, 2001; Maass et al., 2005). Studies by Liu (1996), Sultana et al. (2000), Maass et al. (2005), Venkatesha et al. (2007) and Wang et al. (2007) reported substantial diversity of different markers in lablab. Unfortunately, the diversity of lablab landraces is under threat by genetic erosion due to the expansion of crops of superior economic importance (Maass et al., 2008).

Identifying superior or genetically dissimilar lines from source populations for improvement or conservation requires a systematic and careful genetic study. Core collections can help in such endeavours. However, in cowpea (GRIN, 1998; Mahalakshmi et al., 2007) and lablab (Pengelly and Maass, 2001) they have mostly been identified on the basis of agro-morphological characteristics, whereas the importance of molecular markers in such studies has been generally recognized. Amplified Fragment Length Polymorphism (AFLP) (Vos et al., 1995) produces useful markers in cowpea (Coulibaly et al., 2002; Tosti and Negri, 2005; Fang et al., 2007) and lablab (Maass et al., 2005; Venkatesha et al., 2007).

This study was conducted within the project “Promotion of Neglected Indigenous Leafy and Legume Vegetable Crops for Nutritional Health in Eastern and Southern Africa (ProNIVA)” implemented by The World Vegetable Center’s Regional Center for Africa (AVRDC-RCA) in Arusha, Tanzania since 2003. One of the objectives of the project was to safeguard genetic diversity of legume vegetables. In line with this goal, our study was conducted to:

  • assess the level of genetic diversity in sets of cowpea and lablab accessions acquired from different sources in eastern Africa; and
  • compare the level of diversity in local lablab accessions in relation to the core collection.

Materials and Methods

When assembling a diverse cowpea collection from different African sources, emphasis was given to include mostly leafy or dual purpose types if known. The cowpea accessions used in the study included 21 IITA lines from the Ilonga Research Station of Tanzania; 14 gene bank accessions from the National Plant Genetic Resource Center of Tanzania; 12 cultivars from Tanzania, 8 research lines from the International Livestock Research Institute (ILRI), 4 landraces from Tanzania and 3 commercially purchased seed samples of different genotypes. The majority of lablab accessions belonged to the core collection developed by Pengelly and Maass (2001), while some local accessions from Tanzania and Uganda were included in our study.

Twelve seedlings per accession of both cowpea and lablab were raised in the greenhouse of Agronomy in the Tropics of the Department of Crop Sciences of University of Goettingen, Germany. Youngest leaves from each of six individual plants were selected, sliced and bulked to make up a weight of about 0.1 g to extract DNA with a modified CTAB method (Brandfass and Karlovsky, 2006). The AFLP protocol of Vos et al. (1995) adapted by Laurentin and Karlovsky (2006) was applied. Two sets of four different primer combinations each were used for cowpea and lablab separately. Detailed procedures of this study have been given by Tefera Tolera (2006).

Unambiguous bands were scored manually as present (1) or absent (0) using the adobe Photoshop and ImageReady programs. The proportion of polymorphism was used to describe diversity. Furthermore, the binary data were subjected to a cluster analysis based on Unweighted Pair Group Arithmetic Mean Method (UPGMA) algorithm from Jaccard Coefficient of Genetic Similarity (JCGS) using NTSYSpc software.

Results

The total number of unequivocally identified markers in cowpea was 185, of which 44% were polymorphic. In lablab 163 markers with 72% polymorphism were identified. In both crops, the scored markers were between 50-450 base pairs long. The level of marker polymorphism revealed by different primer combinations ranged from 20-45% in cowpea to 58-88% in lablab.

The lowest Jaccard Coefficient of Genetic Similarity (JCGS) observed was 0.82 in cowpea and 0.45 in lablab. In general, cluster formation of accessions in cowpea was mainly based on their provenance and also seemed to be dictated by seed colour and thousand-seed-weight (TSW). Cowpea accessions of Tanzanian landraces, gene bank materials and ILRI were grouped together, while those originating from IITA formed another major group. On average, accessions with very high Jaccard’s coefficient of genetic similarity (JCGS) had similar TSW. Also lablab accessions tended to be grouped according to their presumed origin. Local accessions of lablab were genetically different from the core collection developed based on agro-morphological traits.

Discussion

The number of AFLP markers revealed in cowpea (185) was in high agreement with a previous study (188) (Coulibaly et al., 2002) using the same primer combinations. However, the overall level of 44% polymorphism in our study was substantially lower than the 61% found by Coulibaly et al. (2002). Fang et al. (2007) detected 54% of polymorphism in a total of 382 markers when applying 6 different primer combinations. Tosti and Negri (2005) reported only 7% polymorphism in a total of 197 detected AFLP markers, again using different primer combinations. The level of diversity reflected by the overall proportion of polymorphic markers seems to be associated with the portion of the germplasm covered in a particular study (Coulibaly et al., 2002; Tosti and Negri, 2005; Fang et al., 2007). Coulibaly et al. (2002) covered geographically diverse domesticated and wild cowpea accessions from Africa, while Tosti and Negri (2005) studied three landraces from Lake Trasimeno of Italy representing early introductions of cowpea to Europe.

Compared to crops like maize, rice and soybean, cowpea lines also showed a low level of microsatellite marker polymorphism, indicating its narrow genetic diversity (Li et al., 2001), though Nkongolo (2003) demonstrated the opposite in Malawian landraces. In general, the phenogram of accessions and level of polymorphism detected in this study support the established view that genetic diversity in cultivated cowpea is low (Tefera Tolera, 2006). This was even more so, if regionally restricted germplasm is being investigated (Fang et al., 2007). However, a different diversity level from East African germplasm could be expected because cowpea’s use as a leafy vegetable is more popular and has relative higher importance over cowpea grain as a source of human food in this region (Madamba et al., 2006).

Considering lablab, most accessions have been exchanged between institutions inter- and/or intra-continentally, usually based on a very limited number of seeds. This could have undermined the level of genetic diversity due to genetic drift. However, the 72% polymorphism in 163 AFLP markers identified in this study, compared to 34% of 857 markers detected by GeneScan and GenoTyper software (Maass et al., 2005) for the same primer combinations shows high genetic diversity in lablab. Using RAPD for this species, Liu (1996) scored 273 markers, 69% being polymorphic in 40 genotypes. He indicated that the high level of genetic variation resulted mainly from the inclusion of wild accessions, a view confirmed by Maass et al. (2005); otherwise, diversity in all the cultivated accessions could only be regarded as moderate (Liu, 1996). Sultana et al. (2000) also used RAPD markers to study lablab diversity and scored a total of 101 markers, of which 95% were polymorphic. When Venkatesha et al. (2007) and Wang et al. (2007) applied microsatellites to different lablab germplasm collections, they also found that wild materials added considerable diversity, whereas cultivated accessions were less distinct. For example, landraces from southern India were shown to contain little diversity, especially when comparing them to African materials (Venkatesha et al., 2007). Nevertheless, lablab generally contains substantial genetic diversity.

Conclusion

Genetic diversity of cowpea accessions included in our study was low, corroborating studies conducted previously. Because the livelihood of numerous farmers around the globe depends on this crop, conserving genetic diversity in cowpea consequently deserves attention.

In lablab, accessions from East Africa included in our study were genetically distinct from the core collection, which was substantially more diverse than landraces in southern India. It is likely that this region of the world holds the most diverse germplasm. Therefore, we conclude that wide-scale collection and characterization of lablab is required to safeguard its genetic diversity as a prerequisite to exploit its economic potential. Research into the maintenance of diversity in lablab and promotion of its use in Africa may also help to tackle production constraints such as drought prevailing elsewhere.

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