Published in Crop Sci. 43:2284-2290 (2003).
© 2003 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
PLANT GENETIC RESOURCES
Development and Utilization of SSRs to Estimate the Degree of Genetic Relationships in a Collection of Pearl Millet Germplasm
H. Budak*,a,
F. Pedrazaa,
P. B. Creganb,
P. S. Baenzigera and
I. Dweikata
a 377 Plant Sci., Dep. of Agronomy and Horticulture, Univ. of Nebraska, Lincoln, NE 68583
b Soybean Genomics and Improvement Laboratory, USDA-ARS, Beltsville, MD 20705
* Corresponding author (hbudak3{at}unl.edu).
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ABSTRACT
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Pearl millet [Pennisetum glaucum (L.) R. Br.] cultivars derive from a narrow gene pool, and studies of genetic diversity in Pennisetum germplasm suggest promising opportunities for the use of undomesticated materials for improving pearl millet varieties. However, efficient utilization of wild germplasm will require effective DNA marker-based fingerprinting strategies for rapid assessment of genetic relationships. The present study aims at development and utilization of a collection of microsatellite (SSR, simple sequence repeat) markers for assessing the genetic diversity of 53 lines of millet. A small insert genomic library was screened with a (CT)15 oligonucleotide probe. A total of 34 (CT)n–containing clones were identified, and specific primers were designed for 18 of these. Of 18 new microsatellites developed as a part of this work, 11 were used to estimate the genetic diversity, along with 19 from other sources. Cluster analysis by the unweighted pair-group method with arithmetic averages (UPGMA) showed two major and eight minor clusters, suggesting that the millet germplasm could readily be distinguished by UPGMA. The coefficients of genetic distance among germplasm lines were high and averaged D = 0.60 (range 0.28–0.92). Genetic diversity averaged 0.38. These results demonstrated that genotypes with potential traits are maximally different from the cultivated gene pool and could readily be distinguished. Development and utilization of polymerase-chain-reaction-(PCR)-based markers such as SSRs is a valuable asset for estimating genetic diversity, the identification of unique genotypes as potentially important new sources of alleles for enhancing important characteristics, and analyzing the evolutionary and historical development of cultivars at the genomic level in pearl millet breeding programs.
Abbreviations: bp, base pair • EDTA, ethylenediamine tetraacetic acid • MAS, marker-assisted selection • PCR, polymerase chain reaction • PIC, polymorphism information content • SDS, sodium dodecyl sulfate • SSR, simple sequence repeat • UPGMA, unweighted pair-group method with arithmetic averages
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INTRODUCTION
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PEARL MILLET is a monocot species belonging to the Poaceae family and has a relatively small diploid genome (2n = 2x = 14) with a DNA content of 1C = 2.36 pg (Martel et al., 1997). Pearl millet is a highly cross-pollinated crop. It demonstrates the highest levels of tolerance to drought and heat found in domesticated cereals and, consequently, is grown on >26 million ha in the arid and semiarid regions of Africa and India (Food and Agriculture Organization, 2000). In addition to drought tolerance, millet has a relatively short growing season (60–90 d) that allows double cropping after wheat (Triticum aestivum L.) has been harvested. Pearl millet is an excellent forage and, because of its low hydrocyanic acid content, is the best annual grazing crop in the southern USA (Burton, 1995) and an important summer forage crop in Australia and South America as well (Hanna, 1996). The energy density of pearl millet is relatively high, arising from its higher oil content relative to maize, wheat or sorghum (Hill and Hanna, 1990). Pearl millet contains 27 to 32% more protein than maize, higher concentrations of essential amino acids, twice the ether extract, and higher gross energy than maize (Ejeta et al., 1987).
Some of the most threatening diseases of millet include downy mildew [Sclerospora graminicola (Sacc.) J. Schröt.], leaf rust (Puccinia substriata Ellis & Barth.), ergot [Claviceps purpurea (Fr.:Fr.) Tul.], and leaf spot [Bipolaris setariae (Sawada) Shoemaker]. Effective resistance to downy mildew and ergot are quantitatively inherited and are not yet well-characterized genetically (Andrews et al., 1985). The complex inheritance of these traits complicates their transfer to elite lines. The introgression of these resistances into elite germplasm would benefit from the availability of effective marker-assisted selection (MAS). Moreover, one of the most important concerns in the manipulation of millet germplasm is its relatively narrow genetic base. Pearl millet cultivars are generated from a narrow gene pool, and current breeding programs rarely use wild materials. Studies of genetic diversity in Pennisetum germplasm suggest promising opportunities for the use of undomesticated materials for improving pearl millet varieties. These efforts will require effective DNA marker-based fingerprinting strategies for rapid assessment of genetic relationships and MAS for trait introgressions.
Simple sequence repeats, or microsatellites, consist of 2 to 5 nucleotide sequences such as (GA)n, (ATT)n, or (ATGT)n that are tandemly repeated. Such repeated sequences are found throughout eukaryotic genomes and provide the basis for a PCR-based marker amplification strategy. Microsatellites can be isolated directly from total genomic DNA libraries, or from libraries enriched for specific microsatellites.
Microsatellites have proven informative to study genetic relationships among closely related plant species as well as among subpopulations of a single species (Bowcock et al., 1994) because of their exceptionally high level of polymorphism. In addition, microsatellites exhibit codominant inheritance and their detection can be readily automated (Hernandez et al., 2002). These features are essential for effective discrimination between closely related lines (Akkaya et al., 1992). Previous reports (Wu and Tanksley, 1993) indicate AT repeats appear to predominate in plant genomes. However, other studies (Chin et al., 1996; Ma et al., 1996) have suggested CT and/or TG repeats may also be highly prevalent. Simple sequence repeats serve as a tool for the identification of genotypes, tagging of important traits, and in population genetic studies (Gupta and Varshney, 2000). Plant SSRs are reported to exhibit high levels of polymorphism with as many as 37 alleles at individual loci in barley (Hordeum vulgare L.) (Saghai-Maroof et al., 1994) and 26 alleles in soybean (Rongwen et al., 1995). In most plant species, the level of detected polymorphism has been shown to be 10 times higher than with RFLP markers (Akkaya et al., 1992; Senior and Heun, 1993; Bell and Ecker, 1994).
To date, collections of 25 (Allouis et al., 2001) and 24 (Qi et al., 2001) SSR markers have been developed for pearl millet. Here, we add new SSR markers to these collections and estimate the utility of the markers for assessing pearl millet germplasm diversity.
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MATERIALS AND METHODS
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Plant Materials
Pedigree and origin of a collection of 53 pearl millet lines used in the present study are shown in Table 1. All plant introduction lines were kindly provided by the Plant Genetic Resources Conservation Unit (University of Georgia) and were chosen to represent variation in origin and diversity (Africa and India). The remaining lines were developed either at Nebraska, Georgia, or Kansas and represent cultivated pearl millet germplasm.
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Table 1. Pedigree and origin for the 53 pearl millet lines. Pedigree information was obtained from National Plant Germplasm System.
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Development of Microsatellites
Genomic DNA of pearl millet line IBMV 840 was extracted with the Qiagene DNAesy kit. Genomic DNA (40µg) was digested with the restriction enzyme SmaI and then size-separated on 1% agarose gel in 1 x TBE buffer for 3 h at 1.5 V cm-1. Following electrophoresis, fragments in the size range of 400 to 800 base pairs (bp) were extracted from the gel and the DNA purified using the Gene Clean II (Bio 101 Inc., LaJolla, CA).
Purified DNA fragments (0.7µg) were ligated to SmaI-digested pUC19 vector in the presence of SmaI. Following transformation of competent E. coli DH5 (Life Technology, Grand Island, NY) cells, the size selected library was screened by colony hybridization at 45°C with 32P-labeled poly (CT)15 in 6 x SSPE (1.08 M sodium chloride, 60 M sodium phosphate, and 6 mM ethylenediamine tetraacetic acid [EDTA]), 5 x Denhardt's solution, 1% sodium dodecyl sulfate (SDS), with three posthybridization washes in 0.5 x SSC (7.5 mM sodium citrate, 75 mM sodium chloride), 0.1% SDS. Putative positive colonies were replated and a second round of screening was performed to confirm the hybridization results. A total of 34 clones were identified using this procedure, and their inserts were sequenced. Primer pairs were designed to amplify the microsatellite from those clones containing >10 CT repeats using the OLIGO 6 (Molecular Biology Institute, Cascade, CO) program software. Simple sequence repeat marker names, sequences, repeat length, melting temperature, expected size, and nature of polymorphism used in this study are shown in Table 2.
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Table 2. Simple sequence repeat marker names developed, primer sequences, repeated type and length, melting temperature (Tm), expected size, and number of alleles with polymorphism information content (PIC) value.
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Polymerase Chain Reaction Conditions and Microsatellite Analysis
Genomic DNA of pearl millet lines (five plants per line) was extracted with the Qiagene DNAesy kit. A total of 65 SSR primers were assayed with 15 millet lines chosen to represent a range of diversity. Primers were excluded from the study if banding patterns were difficult to score or if the primers failed to amplify consistently in all lines. A final set of 30 SSR primers, 11 (CTM-8, CTM-9, CTM-10, CTM-12, CTM-21, CTM-26, CTM-55, CTM-56, CTM-58, CTM-59, CTM-60) out of 18 developed in this study CTM (Table 2), and 19 from previous studies PSMP (PSMP2201, PSMP2202, PSMP2203, PSMP2206, PSMP2207, PSMP2208, PSMP2209, PSMP2233, PSMP2249, PSMP2263, PSMP2271, PSMP2273, PSMP22012, PSMP22017, PSMP22018, PSMP22020, PSMP22023, PSMP22024, PSMP220218) (Allouis et al., 2001; Qi et al., 2001) were chosen for further analysis. The PCR reaction mixtures (25 µL total volume) consisted of 10 mM Tris-HCl, pH 8.8 at 25°C, 50 mM KCL, 2.0 mM MgCl2, nucleotides dATP, dTTP, dCTP, and dGTP (200 µM each), 0.2 µM primer, 30 ng template DNA, and 1.5 units of Taq DNA polymerase (Promega Corp., Madison, WI). Amplifications were performed in a MJ Research PTC-100 thermocycler programmed for 32 cycles of 1 min at 94°C, 1 min at 53°C, 1 min at 72°C, and ending with 5 min at 72°C.
Gel Electrophoresis
The PCR products (25 µL) were fractionated on 12% polyacrylamide on a Hoefer vertical-gel apparatus (SE600). Gels consisted of acrylamide (37.5:1 acrylamide: bisacrylamide) in TAE buffer (40 mM Tris-acetate, 20 mM sodium acetate, 1 mM EDTA; pH 7.7). Gels were 0.75 mm in thickness. Electrophoresis conditions were held at 300 V for 3 h at room temperature. To maintain the room temperature condition, circulating water bath temperature was set to 20°C. Gels were stained in ethidium bromide (1 µg mL-1) for 20 min, destained in deionized water for 1 h, and photographed under the Gel Doc 2000 (Bio-Rad).
Statistical Analysis
The distance matrix and dendogram were constructed with the Numerical Taxonomy Multivariate Analysis System (NTSYS-pc) version 2.1 software package (Exeter Software, Setauket, NY) software package (Rohlf, 2000). Genetic polymorphism (P = 0.05), Nei's gene diversity, and Shannon's information index were used to compute Nei's standard genetic distance coefficients (Nei and Li, 1979), and to construct a UPGMA dendogram (Sneath and Sokal, 1973). The FIND module (part of the NTSYS package) was used to identify all trees that could result from different choices of tied similarity or dissimilarity values. To test the robustness of tree topology, the trees were compiled by CONSEN (part of the NTSYS package).
To refer to the informativeness of microsatellites, we employed the term polymorphism information content (PIC). The PIC value was calculated according to the formula:
where pij is the frequency of the jth microsatellite allele for clone i. This value is referred to as heterozygosity and gene diversity (Weir, 1990; Anderson et al., 1993).
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RESULTS AND DISCUSSION
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The screening of a partial genomic library of P. glaucum, consisting of 
40000 clones with an average size of 400 bp, with a dinucleotide repeat oligonucleotide probe (CT)15 identified 80 positive clones. More than 50% (46 out of 80) of the positive clones were discarded as false positives during the second screening. Of the 34 remaining clones, sequence analysis determined that 18 contained microsatellites with at least 10 CT repeats and sufficient flanking sequence to design primers, while the remainder contained short repeats (2–9) and were discarded. The primers that contain the short repeats generally produced monomorphic PCR products or displayed very low PIC value (Qi et al., 2001).
Assuming an average insert size of 400 bp, the library contained 16000 kbp of genomic DNA. This gave an estimate of one microsatellite motif per 888 kbp. This estimate is roughly similar to those obtained from maize (Taramino and Tingey, 1996), but much lower than those reported in the genomes of wheat (Roder et al., 1995), and barley (Liu et al., 1996). On the basis of these findings, it appears that GA/CT repeats are abundant and easily detected in millet. Our results are consistent with reports from other plant species (Stallings, 1992; Lagercrantz et al., 1993; Wang et al., 1994). Although most of the primer pairs produced a maximum of two bands per genotype, two (CTM-26 and CTM-60) primer pairs produced more bands than expected according to the diploid constitution of this pearl millet, probably implying the duplication of some loci; in fact, Devos et al. (2000) reported that the pearl millet genome carries at least one, and probably two duplications between linkage groups 1 and 4.
All primer pairs (total of 65) that amplified microsatellite loci in line IBMV 840 were examined for polymorphisms among 15 lines of pearl millet (Fig. 1) to obtain an estimate of SSR length polymorphism associated with each locus. The number of alleles found for each of these loci varied from 1 to 9. The polymorphisms detected for CTM-9 and PSM 2202 are shown in Fig. 1. Of the 18 microsatellites developed as part of this work, only CTM-11 primer pairs detected a single allele (Table 2). The PIC values for the 18 CTM loci were calculated based on screening of 15 lines (Fig. 1). Polymorphism information content values of microsatellite markers varied from 0 to 0.88 with an average of 0.44 (Table 2).
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Fig. 1. Polymerase chain reaction amplification of pearl millet genomic DNA from 15 lines. Lanes: 1 = 81B, 2 = 94M59464B CBR, 3 = 293B, 4 = 51735B, 5 = 59025B, 6 = 086R1, 7 = Bmr eB, 8 = PI 164421, 9 = PI 286892, 10 = PI 536327, 11 = PI 307713, 12 = PI 511036, 13 = PI 561619, 14 = PI 583799, 15 = Tift 85DB, and lane M contains a 50-bp size marker (Promega Corp., Madison, WI). Two microsatellites, (A) PSM 2202 (Qi et al., 2001) and (B) CTM9, were assayed. The DNA samples were fractionated in 12% nondenaturing acrylamide gels stained with ethidum bromide.
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The degree of polymorphism detected by these primer pairs did not correlate with the number of repeats in the microsatellites. Although the relationship between the degree of polymorphism and the number of repeats has been reported in some species (Saghai-Maroof et al., 1994; Fisher et al., 1998), theoretically the number of repeats is correlated with the mutation rate, and not with the degree of polymorphism (Brinkmann et al., 1998; Xu et al., 2000). Polymorphism may correlate with the product of the mutation rate and the generation term of the locus. More recently evolved microsatellites would have fewer polymorphisms because of fewer occasions for mutation, even if they have longer repeats.
The 53 lines of millet used for diversity analysis were grouped into 10 clusters (Fig. 2). Of the 65 primer pairs tested, 30 pairs were used for analysis. All sample analyses were conducted twice to test for reproducibility and only the reproducible and unambiguous bands were used for the analysis. The 30 produced a total of 171 alleles. Of the 171 alleles, 77 were polymorphic (45%) and were shared among at least two individuals.
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Fig. 2. Dendrogram of 53 pearl millet lines based on the unweighted pair-group method with arithmetic averages analysis using the similarity matrix generated by the Nei and Li coefficient after amplification with 30 pairs of microsatellite primers.
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A dendogram was constructed using the UPGMA method. This approach successfully discriminated all the millet lines tested. Cluster analysis with SSR markers resulted in 10 cluster groups (Fig. 2). On the basis of polymorphism data, similarity index values were calculated that ranged from 0.45 to 0.92. PI 536327 (from India) and 81B (from ICRISAT) were found to span the extremes of the dendogram, with all other germplasm distributed in-between at a maximum genetic distance of 0.45 U (Fig. 2). All Tifton germplasm (Tift 85DB, Tift 23DB, Tift 23B1E1, and Tift 186) grouped with one introduction, PI 584509, from Faso and one line, 59025B, from Nebraska. This result was expected and served as an internal control for our analysis. PI 584509 was used in a backcross scheme with line Tift 383, and Nebraska line 59025B was derived from Tift 23DB and Tift 23DB1E1. Our results provided confidence that the analysis methods were reliable. Three Nebraska lines CBR, 293B, 77169vrbrst, and two plant introductions, PI 286849 and 286954 from Nigeria, grouped together. Two introductions from Nigeria (PI 287004 and PI 286844), one from Maryland (PI 300088) and one from India (PI 214330) were clustered with three other Nebraska lines (FS#1, 413B, and 16Rm1R) at a genetic distance value of 0.57. The introductions from Nigeria (PI 286839, PI 287064, PI 286845, PI 286892, PI 286945, and PI 286961), one introduction (PI 536320) from Burkina Faso, four lines (51735B, GRPF5, 086R1, and BmreB) from Nebraska, two lines (Yemen#4/E1R1 and PI 511036) from Yemen, and one introduction (PI 164421) from India were clustered together at a genetic value of 0.55. For the most part, indigenous lines originating from one location were clustered together.
However, the introduction from India PI 311274 did not cluster with other introductions from the same region. Likewise, line 81B and 3928B (both ICRISAT releases) did not group together. One cluster group consisted of two introductions from Burkina Faso (PI 536425 and PI 536398), one from India (PI 279664), and one from the USDA (PI 561619). One of the minor cluster groups contained one line from Nebraska (58012R1R4) and one from Nigeria (PI 286834). These observations are indicative of germplasm exchange among different geographical regions.
Coefficients of genetic distance D were calculated for pair-wise comparisons of the 53 millet lines (Nei and Li, 1979). The genetic distance for all 1378 pairs ranged from 0.28 to 0.92 and averaged 0.60. The lowest genetic distance (0.28) was obtained between the line PI 584509 (developed by introducing into Tift 383 through backcross and selection), and Tift 85DB. The highest genetic distance (0.92) observed in this study was between PI 279664 (India line) and PI 286977 (Nigeria line).
The materials used in this study are, for the most part, highly homogenous phenotypically. Likewise, extensive and routine intercontinental millet germplasm exchange has complicated the assessment of relatedness based on present day origin of the materials. It is our intention to devise a molecular marker-based strategy for estimation of diversity in parent selection for hybrid testing. In this regard, the outcome of this study is encouraging; the average genetic similarity among all Pennisetum lines analyzed was 60%. It is now our plan to test the utility of available Pennisetum SSR markers for predicting high performing crosses.
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NOTES
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Nebraska Agricultural Research Division, Journal Series No. 14049.
Received for publication May 27, 2003.
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ABSTRACT
INTRODUCTION
