Crop Science 41:189-197 (2001)
© 2001 Crop Science Society of America
CELL BIOLOGY & MOLECULAR GENETICS
Determining Genetic Similarities and Relationships among Cowpea Breeding Lines and Cultivars by Microsatellite Markers
Cheng-Dao Lia,
Christian A. Fatokunb,
Benjamin Ubib,
Bir B. Singhb and
Graham J. Scolesa
a Dep. of Plant Sciences, Univ. of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
b International Institute Of Tropical Agriculture, Oyo Road, PMB 5320, Ibadan, Nigeria
Corresponding author (graham.scoles{at}usask.ca)
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ABSTRACT
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Cowpea [Vigna unguiculata (L.) Walp] is an important grain legume crop grown for its protein rich grains. It is an inexpensive source of protein in the diets of people in sub-Saharan Africa. The International Institute of Tropical Agriculture (IITA) has been working on the improvement of cowpea for more than 30 yr. Over 60 countries receive cowpea cultivars improved by IITA for testing and adoption where needed. Many of these cultivars have identical parentage but look very different morphologically when grown in the field. Forty-six microsatellite DNA markers were used to evaluate genetic similarities among 90 cowpea breeding lines developed at IITA. Twenty-seven primer pairs could amplify polymorphic single-locus microsatellites from all of these materials. Two to seven alleles per primer were detected with a polymorphic information content varying from 0.02 to 0.73. By means of only five polymorphic microsatellite primers, 88 of the 90 cowpea lines could be distinguished. A dendrogram based on the microsatellite polymorphisms generally agreed with the pedigree of the cowpea lines.
Abbreviations: AFLP, amplified fragment length polymorphism • IITA, International Institute of Tropical Agriculture • PCR, polymerase chain reaction • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeats
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INTRODUCTION
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COWPEA is one of the world's important legume food crops. At least 12.5 million hectares of cowpea are cultivated with an annual production of over 3 million metric tons worldwide (Singh et al., 1997). Development of new cultivars with early maturity, acceptable grain quality, and resistance to some important diseases and pests has significantly increased the yield and cultivated area (Ehlers and Hall, 1997). For example, cowpea production increased 341% from 1961 to 1995 in Nigeria (Reeves, 1997). The International Institute of Tropical Agriculture (IITA) in Nigeria has developed many cultivars which are grown widely in over 60 countries (Reeves, 1997).
The overall effect of plant breeding on genetic diversity has been a long-standing concern in the evolutionary biology of crop plants (Simmonds, 1962). The loss of genetic diversity, in part due to the conventional breeding programs associated with modern agricultural practices, has been dramatic for many cultivated species (Wilkes, 1983). In consequence, the narrow genetic base of the elite germplasm has increased the potential vulnerability to pests and abiotic stress. Better knowledge of the genetic similarity of breeding materials could help to maintain genetic diversity and sustain long-term selection gain. Furthermore, monitoring the genetic variability within the gene pool of elite breeding material could make crop improvement more efficient by the directed accumulation of favored alleles thus decreasing the amount of material to be screened.
The genetic similarity in cultivated cowpea has been assessed on the basis of morphological and physiological traits (Ehlers and Hall, 1996; Fery, 1985), allozymes (Panella and Gepts, 1992; Pasquet, 1993, 1999; Vaillancourt et al., 1993), seed storage proteins (Fotso et al., 1994), chloroplast DNA polymorphism (Vaillancourt and Weeden, 1992), restriction fragment length polymorphisms (RFLP) (Fatokun et al., 1993), amplified fragment length polymorphisms (AFLP) (Fatokun et al., 1997), and random amplified polymorphic DNA (RAPD) (Mignouna et al., 1998).
Microsatellites or simple sequence repeats (SSR) are DNA sequences with repeat lengths of a few base pairs. Variation in the number of repeats can be detected with PCR by developing primers for the conserved DNA sequence flanking the SSR. As molecular markers, SSR combine many desirable marker properties including high levels of polymorphism and information content, unambiguous designation of alleles, even dispersal, selective neutrality, high reproducibility, codominance, and rapid and simple genotyping assays. Microsatellites have become the molecular markers of choice for a wide range of applications in genetic mapping and genome analysis (Chen et al., 1997; Li et al., 2000), genotype identification and variety protection (Senior et al., 1998), seed purity evaluation and germplasm conservation (Brown et al., 1996), diversity studies (Xiao et al., 1996), paternity determination and pedigree analysis (Ayres et al., 1997; Bowers et al., 1999; van de Ven and McNicol, 1996), gene and quantitative trait locus analysis (Blair and McCouch, 1997; Koh et al., 1996), and marker-assisted breeding (Ayres et al., 1997; Weising et al., 1998). For measuring genetic diversity, assigning lines to heterotic groups and genetic fingerprinting, microsatellites provide power of discrimination equal to or greater than that of RFLP in a more cost effective manner (Smith et al., 1997; Senior et al., 1998).
Recently, we constructed two microsatellite-enriched libraries of cowpea, and more than 100 microsatellite sequences were isolated (Li and Scoles, unpublished data). In the present study, we demonstrate the application of cowpea microsatellites for the differentiation and estimation of genetic relationships of 90 cowpea breeding lines.
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MATERIALS AND METHODS
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Ninety cowpea breeding lines from the International Institute of Tropical Agriculture were used with one cowpea wild relative (Vigna unguiculata ssp. dekindtiana var. pubescens) as control. The inbred lines and their pedigrees are listed in Table 1. DNA of each line was extracted by the modified CTAB method (Rogers and Bendich, 1988).
Forty-six microsatellite primer pairs were used. Their names, repeat types, predicted fragment length, and PCR reaction conditions are listed in Table 2. VM21 and VM22 were designed based on the sequences of the 1-amino-cycloprane-1-carboxylate oxidase cDNA of mung bean [Vigna radiata (L.) R. Wilczek] and the protein kinase cDNA of mothbean [Vigna aconitifolia (Jacq.) Maréchal], respectively (Taek and Fa, 1994; Hong et al., 1993). The other 44 primer sets were isolated from cowpea microsatellite-enriched libraries constructed from the DNA of the breeding line IT84S-2264-2 (Li and Scoles, unpublished data).
PCR reactions were carried out in a Thermolyne Amplitron II
thermocycler (Barnstead–Thermolyne, Dubuque, IA). Each
25-µL reaction contains 50 ng of genomic DNA, 1x reaction buffer
(GIBCO–BRL), 1.5 mM MgCl2, 200 µM of
each dNTP, 10 pmol of each primer and 1 unit of Taq
polymerase. Depending on the Tm of the primers used,
amplification was performed by means of one of the following two
"Touchdown" PCR profiles (Don et al., 1991).
- A PCR profile consisting of 18 cycles of 94°C for 1 min (denaturing) and 72°C for 1 min (extension). Annealing temperatures (30 s) were progressively decreased by 0.5°C every cycle from 64 to 55°C. The PCR reaction continued for 30 additional cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The reaction ended with a 10 min extension at 72°C.
- Similar PCR conditions to those in (1) except that the annealing temperature was decreased from 67 to 58°C over 18 cycles, and the reaction was continued for 20 additional cycles at 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min.
PCR products were separated on a sequencing gel containing 6% (w/v) polyacrylamide, 7 M urea and 1x TBE at 85 W constant power for 3 h (BioRad sequencing system, RioRad, Richmond, CA). The gel was fixed, stained and dried by a DNA silver staining kit (Promega Corp., Madison, WI).
The polymorphism information content (PIC) of each microsatellite was determined as described by Weir (1996). 
, where Pi is the frequency of the ith allele in the examined test lines. NTSYSpc (Version 2.0) was used to calculate the genetic similarity (Jaccard's coefficient), principal coordinate, and cluster analyses (Unweighted Paired Group Method Using Arithmetic Averages).
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RESULTS
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Polymorphism of Microsatellites in Cowpea Breeding Lines
Forty-six primer pairs were used to amplify the DNA of cowpea (Table 2). Forty-one primer pairs produced a single band within the predicted size range and four primer pairs (VM23, VM25, VM33, and VM34) amplified multiple fragments while VM7 failed to amplify DNA from cowpea. The two primer pairs (VM21 and VM22) designed from the DNA sequences of mung bean and mothbean amplified a single DNA fragment from cowpea with predicted size in the original sequences (data not shown).
Thirty-one of the 46 primer pairs (67%) produced polymorphisms among the 90 cowpea lines and one wild relative. One example is shown in Fig. 1
. Ten primer pairs (VM1, VM9, VM10, VM15, VM16, VM18, VM20, VM21, VM24, VM29, VM32, VM69, VM72, and VM73) were monomorphic. Four primer pairs (VM2, VM4, VM6, and VM8) which were polymorphic but produced complex "stutter" bands on the polyacrylamide gels were not used for further analyses.
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Fig. 1. Polymorphism of the microsatellite amplified by VM35 in 90 cowpea lines and one wild relative. Numbers of the lines is listed in Table 1 and the 91 is the wild relative
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Twenty-seven primer pairs which amplified clear polymorphic bands on the polyacrylamide gels were used to analyze the 90 cowpea lines and one wild relative. These primers and their repeat type, repeat number, allele number, and polymorphism information content are listed in Table 3. The allele number per primer varied from two to seven with an average of 4.7. Primer pairs VM5, VM31, VM35, VM36, and VM39, all amplified seven alleles, the maximum number. The polymorphism information content varied from 0.02 to 0.73 with an average of 0.45. No significant correlation was detected between the repeat number and the allele number 
or polymorphism information content 
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Genetic Diversity of the Cowpea Lines
Except for Lines 27, 52, 57, and 62, the pedigrees of the other 86 lines used in the present study were clear (Table 1). As most of the lines were from the IITA breeding program, it is to be expected that the lines shared common parents. The pedigrees (Table 1) show that IT84S-2246-4 was most frequently used as a parent. Thirty lines (35%) had IT84S-2246-4 in their pedigree. If the parents of IT84S-2246-4 are considered, 51 lines (59.3%) had at least one common parent.
In total, 128 alleles were detected among the 90 lines and one wild relative using the 27 microsatellite primer pairs. The microsatellite primers were able to distinguish all the 90 lines, including the lines with same name (IT91K-118-20: Lines 13 and 35; IT84S-2246-4: Lines 6 and 90). In fact, the five most polymorphic primer pairs (VM5, VM31, VM35, VM36, and VM39) could distinguish all lines except Lines 32 and 33 (data not shown). A two-dimension principal coordinate analysis (Fig. 2)
failed to detect significant subgrouping among the 90 breeding lines. However, the wild cowpea relative (Line 91) was independent from all the other breeding lines (Fig. 2).
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Fig. 2. Two-dimension principle coordinate analysis of the 90 cowpea lines and one wild relative. Numbers of the lines is listed in Table 1 and the 91 is the wild relative. Line numbers followed by an S contain the line IT84S-2246-4 in their pedigree
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A dendrogram of the 90 cowpea lines was constructed by the Unweighted Paired Group Method Using Arithmetic Averages on the basis of the genetic similarity (Jaccard's coefficient) (Fig. 3)
. The dendrogram generally agreed with the pedigree of the lines (Table 1). Lines 81, 82, 83, and 87 were the most diverse among the inbred lines, the first three lines originating from the same cross. This result is consistent with the AFLP analysis (Fatokun et al., 1997). The 90 cowpea lines shared an average of 44% similarity. A large group of 47 cowpea lines shared over 45% similarity on the dendrogram. Most lines (87%) of this group contained either IT84S-2246-4 or its parents in their pedigree (Fig. 3). A small group including the Lines 9, 11, 12, 27, and 68 interrupted this large group. Except for Line 27 whose pedigree was not available, the other four lines were selected from a common cross (Table 1).
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Fig. 3. Phylogenetic relationship of the 90 cowpea lines constructed using 27 microsatellite polymorphisms. Numbers of the lines as listed in Table 1. Line numbers followed by an S contain the line IT84S-2246-4 in their pedigree
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DISCUSSION
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Microsatellite markers have shown high levels of polymorphism in many important crops including rice (Oryza sativa L., Chen et al., 1997), wheat (Triticum aestivum L., Devos et al., 1995; Roder et al., 1995), barley (Hordeum vulgare L., Liu et al., 1996), oat (Avena sativa L., Li et al., 2000), maize (Zea mays L., Senior et al., 1998), sorghum [Sorghum bicolor (L.) Moench, Brown et al., 1996], soybean [Glycine max (L.) Merr., Akkaya et al., 1992], beans (Phaseolus and Vigna, Yu et al., 1999), Brassica species (Szewc-McFadden et al., 1996), alfalfa (Medicago spp., Diwan et al., 1997), sunflower (Helianthus annuus L., Brunel, 1994), and tomato (Lycopersicon esculentum Mill., Smulders et al., 1997). The present study showed that microsatellite markers were also highly polymorphic in cowpea. They could distinguish cowpea breeding lines and could be used to determine pedigrees to a certain extent. In fact, five polymorphic microsatellites were able to distinguish 88 of the 90 breeding lines. All the microsatellite primer pairs of cowpea could successfully amplify DNA from the related wild relative in the present study. Furthermore, two microsatellite primer sets designed from the sequences of mothbean and mung bean were able to amplify DNA of cowpea with the predicted size. Therefore, microsatellite markers of cowpea could be used in germplasm conservation and analysis, not only for breeding lines and cultivars but also for the wild cowpea species and other Vigna species. In addition, these microsatellite primers could be used for comparative genome analysis between the different Vigna species.
Microsatellite markers have been used to investigate genetic diversity of a large number of cultivars in rice (Yang et al., 1994), soybean (Rongwen et al., 1995), wheat (Plaschke et al., 1995), and maize (Senior et al., 1998). The number of alleles amplified per primer pair was from 3 to 25 for rice, 11 to 26 for soybean, 3 to 16 for wheat, and 2 to 23 for maize. In the present study, only two to seven alleles per primer pair were amplified from the 91 cowpea lines. Thus the level of microsatellite polymorphism in cowpea, although relatively high, is much lower than other crops. One possible reason is that the materials used in the present study were all from the IITA breeding program and thus had a relatively narrow genetic base. In a study of genetic diversity in soybean, 11 to 26 alleles per microsatellite primer pair were amplified from 96 soybean genotypes while this number was reduced to five to 10 alleles per primer pair in 26 cultivars from North American breeding programs (Rongwen et al., 1995). At IITA, when crosses are made between improved cowpea breeding lines and land races with desirable attributes the common practice is to backcross the F1 to the breeding line. When a desirable trait is detected in the new line, backcross is used to transfer this trait to a good genetic background (C.A. Fatokun, 2000, personal communication). This practice may further reduce genetic diversity of the cowpea breeding lines in the IITA program.
An other possible reason for the low level of microsatellite polymorphism is that the cultivated cowpea is relatively low in genetic diversity compared with other crops. Genetic diversity of cultivated cowpea and its wild species have been extensively investigated by means of isozyme markers (Panella and Gepts, 1992; Pasquet, 1993, 1999; Vaillancourt et al., 1993) and seed storage proteins (Fotso et al., 1994). The cultivated cowpea had lower genetic diversity than many other crops (Doebley, 1989), especially legume crops (Pasquet, 1993, 1999). It has been suggested that cowpea was only domesticated once (Pasquet, 1999), unlike P. vulgaris (Singh et al., 1991) or rice (Second, 1985). The low genetic diversity in cultivated cowpea may be a result of this narrow genetic base.
The low level of genetic diversity at the DNA level among cowpea breeding lines and cultivars could be increased by using its wild relatives to broaden the genetic base. The present study has demonstrated that microsatellite markers are conserved among Vigna species. Hence microsatellite markers could provide a simple approach to assaying the introduction of such genetic material.
Groupings of the 90 lines based on microsatellite polymorphisms generally agreed with the pedigree of these lines (Fig. 3). Several discrepancies were present, such as the subgrouping of the Lines 9, 11, 12 and 68 in the large group with the parent IT84S-2246-4. Such incongruities were also observed in the other studies (Plaschke et al., 1995; Senior et al., 1998). One or more of several factors outlined by Mumm and Dudley (1994) may explain the incongruities.
Comparison of the dendrogram produced by the present study with that constructed by AFLP (Fatokun, 1999, unpublished data) showed consistency only in the large group. This lack of consistency between different marker techniques was also observed in soybean, especially for inbred lines (Powell et al., 1996). This may be due to the fact that different marker systems detected different components of DNA variation, subject to different evolutionary mechanisms.
Weber (1990) reported that there was a significant relationship between the repeat length and the degree of polymorphism in human microsatellites. This relationship has also been demonstrated in plants in several studies (Bryan et al., 1997; Grist et al., 1993; Saghai Maroof et al., 1994; Smulders et al., 1997; Thomas and Scott, 1993). In contrast, no significant correlation was detected between the repeat length and the degree of polymorphism in the present study. However, this has also been the case in several different plant species (Bell and Ecker, 1994; Li et al., 2000; Plaschke et al., 1995; Roder et al., 1995; Rongwen et al., 1995; Sun et al., 1998; Szewc-McFadden et al., 1996).
In conclusion, microsatellite markers are polymorphic in cowpea. They can be used to distinguish breeding lines of cowpea. A dendrogram constructed based on microsatellite polymorphism generally agreed with pedigree records of the cowpea lines. The degree of the polymorphism is relatively low in cowpea compared with other crops. No significant correlation was detected between the repeat length and the degree of polymorphism.
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ACKNOWLEDGMENTS
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This project was supported by a grant from CIDA under the CGIAR-Canadian Linkage Fund. Many thanks to Minyuan Lu for technical assistance.
Received for publication March 10, 2000.
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