Mapping of simple sequence repeat (SSR) DNA markers in diploid and tetraploid alfalfa

Cultivated alfalfa (Medicago sativa) is an autotetraploid. However, all three existing alfalfa genetic maps resulted from crosses of diploid alfalfa. The current study was undertaken to evaluate the use of Simple Sequence Repeat (SSR) DNA markers for mapping in diploid and tetraploid alfalfa. Ten SSR markers were incorporated into an existing F₂ diploid alfalfa RFLP map and also mapped in an F₂ tetraploid population. The tetraploid population had two to four alleles in each of the loci examined. The segregation of these alleles in the tetraploid mapping population generally was clear and easy to interpret. Because of the complexity of tetrasomic linkage analysis and a lack of computer software to accommodate it, linkage relationships at the tetraploid level were determined using a single-dose allele (SDA) analysis, where the presence or absence of each allele was scored independently of the other alleles at the same locus. The SDA diploid map was also constructed to compare mapping using SDA to the standard co-dominant method. Linkage groups were generally conserved among the tetraploid and the two diploid linkage maps, except for segments where severe segregation distortion was present. Segregation distortion, which was present in both tetraploid and diploid populations, probably resulted from inbreeding depression. The ease of analysis together with the abundance of SSR loci in the alfalfa genome indicated that SSR markers should be a useful tool for mapping tetraploid alfalfa. Received: 10 September 1999 / Accepted: 11 November 1999     

Two simple sequence repeat markers to select for soybean cyst nematode resistance coditioned by the rhg1 locus

The soybean cyst nematode (SCN) (Heterodera glycines Inchinoe) is the most economically significant soybean pest. The principal strategy to reduce or eliminate damage from this pest is the use of resistant cultivars. Identifying resistant segregants in a breeding program is a difficult and expensive process which is complicated by the oligogenic nature of the resistance and genetic variability in the pathogen. Fortunately, resistance at one SCN-resistance locus, rhg1, is generally accepted as a necessity for the development of resistant genotypes using any source of resistance and when challenged by any SCN race. Thus, the development of SCN resistant cultivars would be expedited if an effective and rapid system were available to identify breeding lines carrying a resistance allele at the rhg1 locus. In this study we report two simple sequence repeat (SSR) or microsatellite loci that cosegregate and map 0.4 cM from rhg1. Allelic variation at the first of these loci, BARC-Satt309, distinguished most, if not all, SCN-susceptible genotypes from those carrying resistance at rhg1 derived from the important SCN-resistance sources ’Peking’, PI 437654, and PI 90763. BARC-Satt309 was also effective in distinguishing SCN resistance sources PI 88788 and PI 209332 from many, but not all, susceptible genotypes. BARC-Satt309 cannot be used in marker-assisted selection in populations developed from typical southern US cultivars crossed with the important resistance sources PI 88788 or PI 209332 because these genotypes all carry the identical allele at the BARC-Satt309 locus. A second SSR locus, BARC-Sat_168, was developed from a bacterial artificial chromosome (BAC) clone that was identified using the primers to BARC-Satt309. BARC-Sat_168 distinguished PI 88788 and PI 209332 from southern US cultivars such as ’Lee’, ’Bragg’ and ’Essex’. Both BARC-Satt309 and BARC-Sat_168 were used to assay lines from SCN-susceptible×SCN-resistant crosses and proved to be highly effective in identifying lines carrying rhg1 resistance from those carrying the allele for SCN susceptibility at the rhg1 locus. Received: 5 November 1998 / Accepted: 3 February 1999     

Development and characterisation of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.)

Enrichment methods were optimised in order to isolate large numbers of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.), with the aim of developing a comprehensive set of loci for trait mapping and cultivar identification. Two libraries were constructed showing greater than 50% enrichment for a variety of SSR-motif types. Sequence characterisation of 1853 clones identified 859 SSR-containing clones, of which 718 were unique. Truncation of flanking sequences limited potential primer design to 366 clones. One-hundred selected SSR primer pairs were evaluated for amplification and genetic polymorphism across a panel of diverse genotypes. The efficiency of amplification was 81%. A relatively high level of SSR polymorphism was detected (67%), with a range of 2–7 alleles per locus. Mendelian segregation of alleles detected by selected SSR-locus primer pairs was demonstrated in the F₁ progeny of a pair cross. Cross-species amplification was detected in a number of related pasture and turfgrass species, with high levels of transfer to other Lolium species and members of the related genus Festuca. The identity of putative SSR ortholoci in these related species was confirmed by DNA sequence analysis. These loci constitute a valuable resource of ideal markers for the molecular breeding of ryegrasses and fescues. Received: 8 May 2000 / Accepted: 13 June 2000     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Utility of barley and wheat simple sequence repeat (SSR) markers for genetic analysis of Hordeum chilense and tritordeum

A selection of 36 wheat and 35 barley simple sequence repeat markers (SSRs) were studied for their utility in Hordeum chilense. Nineteen wheat and nineteen barley primer pairs amplified consistent H. chilense products. Nine wheat and two barley SSRs were polymorphic in a H. chilense mapping population, producing codominant markers that mapped to the expected homoeologous linkage groups in all but one case. Thirteen wheat and 10 barley primer pairs were suitable for studying the introgression of H. chilense into wheat because they amplified H. chilense products of distinct size. Analysis of wheat/H. chilense addition lines showed that the H. chilense products derived from the expected homoeologous linkage groups. The results showed that wheat and barley SSRs provide a valuable resource for the genetic characterization of H. chilense, tritordeums and derived introgression lines. Received: 20 November 2000 / Accepted: 12 April 2001     

Targeted isolation of simple sequence repeat markers through the use of bacterial artificial chromosomes

 Simple sequence repeats (SSRs) are versatile DNA markers that are readily assayed and highly informative. Unfortunately, non-targeted approaches to SSR development often leave large genomic regions without SSR markers. In some cases these same genomic regions are already populated by other types of DNA markers, especially restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), and amplified fragment length polymorphisms (AFLPs). To identify SSR markers in such regions, bacterial artificial chromosome (BAC) clones can be used as intermediaries. First, one or more BAC clones in a region of interest are identified through the use of an existing DNA marker. BAC clones uncovered in this initial step are then used to create a small insert DNA library that can be screened for the presence of SSR-containing clones. Because BAC inserts are often 100-kb pairs or more in size, most contain one or more SSRs. This strategy was applied to two regions of the soybean genome near genes that condition resistance to the soybean cyst nematode on molecular linkage groups G and A2. This targeted approach to identifying new DNA markers can readily be extended to other types of DNA markers, including single nucleotide polymorphisms. Received: 13 August 1998 / Accepted: 13 October 1998     

Identification of simple sequence repeats (SSRs) in olive ( Olea europaea L.)

A small insert genomic library of Olea europaea L., highly enriched in (GA/CT)n repeats, was obtained using the procedure of Kandpal et al. (1994). The sequencing of 103 clones randomly extracted from this library allowed the identification of 56 unique genomic inserts containing simple sequence repeat regions made by at least three single repeats. A sample of 20 primer pairs out of the 42 available were tested for functionality using the six olive varieties whose DNA served for library construction. All primer pairs succeeded in amplifying at least one product from the six DNA samples, and ten pairs detecting more than one allele were used for the genetic characterisation of a panel of 20 olive accessions belonging to 16 distinct varieties. A total of 57 alleles were detected among the 20 genotypes at the ten polymorphic SSR loci. The remaining primer pair allowed the amplification of a single SSR allele for all accessions plus a longer fragment for some genotypes. Considering the simple sequence repeat polymorphism, 5.7 alleles were scored on average for each of the ten SSR loci. A genetic dissimilarity matrix, based on the proportion of shared alleles among all the pair-wise combinations of genotypes, was constructed and used to disentangle the genetic relationships among varieties by means of the UPGMA clustering algorithm. Graphical representation of the results showed the presence of two distinct clusters of varieties. The first cluster grouped the varieties cultivated on the Ionian Sea coasts. The second cluster showed two subdivisions: the first sub-cluster agglomerated the varieties from some inland areas of Calabria; the second grouped the remaining varieties from Basilicata and Apulia cultivated in nearby areas. Results of cluster analysis showed a significant relationship between the multilocus genetic similarities and the geographic origin of the cultivars. Received: 2 February 2001 / Accepted: 1 June 2001     

Polymorphic simple sequence repeat markers in chloroplast genomes of Solanaceous plants

PCR-based markers were developed from mononucleotide simple-sequence repeats in the chloroplast genome of Nicotiana tabacum and applied to the analysis of genetic diversity. These markers were found to detect high levels of polymorphism at three taxonomic levels in Solanaceous plants. Of 36 chloroplast loci examined, 26 show some degree of polymorphism among potato accessions. Among a set of 30 tetraploid potato cultivars it is apparent that a single chloroplast haplotype is prevalent, presumably a result of the widespread use as a female parent of the imported US cultivar Rough Purple Chili in the latter half of the 19th century. Nonetheless, there is considerable chloroplast diversity in the cultivated potato, and it is clear that a large proportion of this variability has arisen through the use of wild or primitive cultivated species of potato in introgression programmes. This variability should be used in future breeding programmes. An examination of single accessions from 24 potato species, as well as representatives from tobacco and other members of the Solanaceae, reveals high levels of inter-specific chloroplast DNA variation. These data, and the ease of use and potential for multiplexing of these markers, suggest that cpSSRs will be of great utility in population genetics, germplasm management, evolutionary and phylogenetic studies as well as in, the analysis of material from introgression and somatic-fusion experiments. Interestingly, the polymorphism arising from one of the more-polymorphic chloroplast loci examined, does not originate solely from the SSR, and is due to variation in the copy number of two tandemly arrayed sequence elements. Received: 15 December 1998 / Accepted: 9 February 1999     

Simple sequence repeat (SSR) markers survey of the cassava (Manihot esculenta Crantz) genome: towards an SSR-based molecular genetic map of cassava

The development of PCR-based, easily automated molecular genetic markers, such as SSR markers, are required for realistic cost-effective marker-assisted selection schemes. This paper describes the development and characterization of 172 new SSR markers for the cassava genome. The placement of 36 of these markers on the existing RFLP framework map of cassava is also reported. Two similar enrichment methods were employed. The first method yielded 35 SSR loci, for which primers could be designed, out of 148 putative DNA clones. A total of 137 primer pairs could be designed from 544 putative clones sequenced for the second enrichment. Most of the SSRs (95%) were di-nucleotide repeats, and 21% were compound repeats. A major drawback of these methods of SSR discovery is the redundancy – 20% duplication; in addition, primers could not be designed for many SSR loci that were too close to the cloning site – 45% of the total. All 172 SSRs amplified the corresponding loci in the parents of the mapping progeny, with 66% of them revealing a unique allele in at least one of the parents, and 26% having unique alleles in both of the parents. Of the 36 SSRs that have been mapped, at least 1 was placed on 16 out of the 18 linkage groups of the framework map, indicating a broad coverage of the cassava genome. This preliminary mapping of the 36 markers has led to the joining of a few small groups and the creation of one new group. The abundance of allelic bridges as shown by these markers will lead to the development of a consensus map of the male- and female-derived linkage groups. In addition, the relatively higher number of these allelic bridges, 30% as against 10% for RFLPs in cassava, underscores SSR as the marker of choice for cassava. The 100% primer amplification obtained for this set of primers also confirms the appropriateness of SSR markers for use in cassava genome analysis and the transferability of the technology as a low-cost approach to increasing the efficiency of cassava breeding. Current efforts are geared towards the generation of more SSR markers to attain a goal of 200 SSR markers, or 1 SSR marker every 10 cM. Received: 15 November 1999 / Accepted: 14 April 2000     

Identification of DNA amplification fingerprinting (DAF) markers close to the symbiosis-ineffective sym31 mutation of pea (Pisum sativum L.)

 We demonstrate efficient genome mapping through a combination of bulked segregant analysis (BSA) with DNA amplification fingerprinting (DAF). Two sets of 64 octamer DAF primers, along with two PCR programs of low- and high-annealing temperatures (30°C and 55°C, respectively), appeared to be enough to locate molecular markers within 2–5 cM of a gene of interest. This approach allowed the rapid identification of four BSA markers linked to the pea (Pisum sativum L.) Sym31 gene, which is responsible for bacteroid and symbiosome differentiation. Three of these markers are shown to be tightly linked to the sym31 mutation. Two markers flanking the Sym31 gene, A21-310 and B1-277, cover a 4–5 cM interval of pea linkage group 3. Both markers were converted to sequence-characterized amplified regions (SCARs). The flanking markers may be potential tools for marker-assisted selection or for positional cloning of the Sym31 gene. Received: 2 July 1998 / Accepted: 8 October 1998     

Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

 Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)_n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats. Received: 15 September 1996 / Accepted: 25 October 1996     

Identification of different chromatin classes in wheat using in situ hybridization with simple sequence repeat oligonucleotides

Clusters of four simple sequence repeats (SSRs), AAC, AAG, AG and CAT, have been mapped physically to hexaploid wheat chromosomes; 15—24-bp synthetic oligonucleotides were labelled by random-primer labelling and used as probes for fluorescent in situ hybridization with standard formamide and low-salt conditions. AAC hybridized strongly to the pericentromeric regions and several intercalary sites of all seven chromosomes of the B-genome corresponding to N bands and enabling their identification. Most of the AAC sites also co-localize with AAG, although the strength of the AAC and AAG signal was often different at the same location. Not all heterochromatic bands showed AAC signals and a few AAC sites were detected that are neither AAG nor N band positive, revealing the complex and heterogeneous genome organization of wheat and identifying the four most frequent classes of banded chromatin. Clusters characterised by a high concentration of AG repeats were detected on chromosome arms 3BS, 4BL, 5BS and 5BL, adjacent to AAG sites. The only detectable CAT cluster was found on chromosome arm 3BL, making this oligonucleotide valuable in identifying this particular chromosome. SSR in situ hybridization is useful as a diagnostic tool in cytogenetics and for understanding genome organization in wheat. Received: 21 September 1999 / Accepted: 19 March 2000     

Evaluating the potential of SSR flanking regions for examining taxonomic relationships in the Vitaceae

Three EST-derived microsatellite loci from Vitis vinifera were amplified and sequenced across eight species of Vitaceae from four different genera. Phylogenetic analysis of the microsatellite’s flanking regions produced informative results in congruence with previous studies. Generic relationships were respected and the data produced sufficient inter-specific variation to distinguish between Cayratia acris and Cayratia saponaria, two very closely related species. Overall, the sequence alignments showed that priming sites were conserved, whereas microsatellite repeats were present in most cases but structurally variable. The sequence data provided information on the evolutionary patterns of various microsatellite repeats and their correlation to evolutionary relationships among taxa. Received: 15 December 2000 / Accepted: 12 April 2001     

Study of simple sequence repeat (SSR) markers from wheat expressed sequence tags (ESTs)

The increasing availability of expressed sequence tags (ESTs) in wheat (Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. We examined 170,746 wheat ESTs from the public (International Triticeae EST Cooperative) and Génoplante databases, previously clustered in contigs, for the presence of di- to hexanucleotide simple sequence repeats (SSRs). Analysis of 46,510 contigs identified 3,530 SSRs, which represented 7.5% of the total number of contigs. Only 74% of the sequences allowed primer pairs to be designed, 70% led to an amplification product, mainly of a high quality (68%), and 53% exhibited polymorphism for at least one cultivar among the eight tested. Even though dinucleotide SSRs were less represented than trinucleotide SSRs (15.5% versus 66.5%, respectively), the former showed a much higher polymorphism level (83% versus 46%). The effect of the number and type of repeats is also discussed. The development of new EST-SSRs markers will have important implications for the genetic analysis and exploitation of the genetic resources of wheat and related species and will provide a more direct estimate of functional diversity.     

Characterisation of resistance to turnip mosaic virus in oilseed rape (Brassica napus) and genetic mapping of TuRB01

Turnip mosaic virus (TuMV) is the major virus infecting Brassica crops. A dominant gene, TuRB01, that confers extreme resistance to some isolates of TuMV on Brassica napus (oilseed rape), has been mapped genetically. The mapping employed a set of doubled-haploid lines extracted from a population used previously to develop a reference RFLP map of the B. napus genome. The positioning of TuRB01 on linkage group N6 of the B. napus A–genome indicated that the gene probably originated from Brassica rapa. Resistance phenotypes were confirmed by indirect plate-trapped antigen ELISA using a monoclonal antibody raised against TuMV. The specificity of TuRB01 was determined using a wide range of TuMV isolates, including representatives of the European and American/Taiwanese pathotyping systems. Some isolates of TuMV that did not normally infect B. napus plants possessing TuRB01 produced mutant viruses able to overcome the action of the resistance gene. TuRB01 is the first gene for host resistance to TuMV to be mapped in a Brassica crop. A second locus, TuRB02, that appeared to control the degree of susceptibility to the TuMV isolate CHN 1 in a quantitative manner, was identified on the C-genome linkage group N14. The mapping of other complementary genes and the selective combining of such genes, using marker-assisted breeding, will make durable resistance to TuMV a realisable breeding objective. Received: 14 December 1998 / Accepted: 10 April 1999     

Identification of inter simple sequence repeat (ISSR) markers associated with seed size in wheat

The feasibility of identifying inter-simple sequence repeat markers associated with seed weight in hexaploid wheat was tested using 113 recombinant inbred lines developed by the single-seed descent method, from a cross between Rye selection111, an Indian genetic stock obtained through the introgression of genes for bold seed size from rye, and Chinese Spring having small seed size. Three markers were associated with low seed size with gene effects of 14.8%, 9.5%, and 6%, while four markers with contributions of 8%, 4.66%, 2.92% and 2.61% were found to be linked to high seed size, together contributing 31% of the phenotypic variance in seed size. Nulli-tetrasomic and di-telosomic analysis revealed the presence of three low seed size QTL-associated markers on three chromosomes, 6BL, 2DL, and 1DS respectively. This study clearly demonstrates that ISSRs are highly useful for finding markers associated with major and minor genes controlling agronomically important traits in wheat. Received: 24 February 2000 / Accepted: 31 March 2000     

Genetic variance, coefficient of parentage, and genetic distance of six soybean populations

Plant breeders would like to predict which biparental populations will have the largest genetic variance. If the population genetic variance could be predicted using coefficient of parentage or genetic distance estimates based on molecular marker data, breeders could choose parents that produced segregating populations with a large genetic variance. Three biparental soybean {Glycine max (L.) Merr.} populations were developed by crossing parents that were closely related, based on pedigree relationships. Three additional biparental populations were developed by crossing parents that were assumed to be unrelated. The genetic variance of each population was estimated for yield, lodging, physiological maturity, and plant height. Coefficient of parentage was calculated for each pair of parents used to develop the segregating populations. Genetic distance was determined, based on the number of random amplified polymorphic markers (RAPD) that were polymorphic for each pair of parents. Genetic distance was not associated with the coefficient of parentage or the magnitude of the genetic variance. The genetic variance pooled across the three closely related populations was smaller than the genetic variance pooled across the three populations derived from crossing unrelated parents for all four traits that were evaluated. Received: 24 April 1996 / Accepted: 17 May 1996     

Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.)

 Inter-simple sequence repeat (ISSR) amplification was used to analyze microsatellite motif frequency in the rice genome and to evaluate genetic diversity among rice cultivars. A total of 32 primers, containing different simple sequence repeat (SSR) motifs, were tested for amplification on a panel of 59 varieties, representative of the diversity of cultivated rice (Oryza sativa L.). The ISSR analysis provided insights into the organization, frequency and levels of polymorphism of different simple sequence repeats in rice. The more common dinucleotide motifs were more amenable to ISSR analysis than the more infrequent tri-, tetra- and penta-nucleotide motifs. The ISSR results suggested that within the dinucleotide class, the poly(GA) motif was more common than the poly(GT) motif and that the frequency and clustering of specific tri- and tetra-nucleotide simple sequence repeats was variable and motif-specific. Furthermore, trinucleotide ISSR markers were found to be less polymorphic than either dinucleotide or certain tetranucleotide ISSR markers, suggesting which motifs would be better targets for microsatellite marker development. The ISSR amplification pattern was used to group the rice genotypes by cluster analysis. These results were compared to surveys of the same varieties for amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) and isozyme markers. The ISSR fingerprint could be used to differentiate the genotypes belonging to either Japonica or Indica sub species of cultivated rice and to dissect finer levels of diversity within each subspecies. A higher percentage of polymorphic bands was produced with the ISSR technique than the AFLP method, based on a similar PCR reaction. Therefore, ISSR amplification proved to be a valuable method for determining genetic variability among rice varieties and for rapidly identifying cultivars. This efficient genetic fingerprinting technique would be useful for characterizing the large numbers of rice accessions held in national and international germplasm centers. Received: 25 May 1998 / Accepted: 17 September 1998     

Hybrid performance and genetic distance as revealed by the (GATA)4 microsatellite and RAPD markers in pearl millet

 Genetic diversity in five cytoplasmic male-sterile and seven restorer lines of pearl millet was determined by DNA fingerprinting using a (GATA)₄ microsatellite and randomly amplified polymorphic DNAs (RAPDs). A total of 160 polymorphic loci were generated and, based on the polymorphism data, similarity index values ranged from 0.81 to 0.50. Cluster analysis was performed and relationships among these lines revealed that they were not in agreement with the available pedigree data. The per se performance of parents and hybrids was analyzed for days-to-50% flowering, plant height, productive tillers, ear length, ear width, 1000-grain weight and grain yield per plot. Path co-efficient analysis revealed that productive tillers, ear width and days-to-50% flowering had a relatively large positive effect. The correlation values were mostly not significant with respect to genetic distance, except for days-to-50% flowering, ear length and ear width. Our results have indicated that genetic-distance measures based on the (GATA)₄ microsatellite and RAPDs may be useful for the grouping of parents, but not for predicting heterotic combinations, in pearl millet. Received: 3 January 1998 / Accepted: 25 February 1998     

The effect of habitat fragmentation on dispersal patterns, mating behavior, and genetic variation in a pika (Ochotona princeps) metapopulation

 Habitat fragmentation is becoming increasingly common, yet, the effect of habitat spatial structure on population dynamics remains undetermined for most species. Populations of a single species found in fragmented and nonfragmented habitat present a rare opportunity to examine the effect of habitat spatial structure on population dynamics. This study investigates the impact of highly fragmented habitat on dispersal patterns, mating behavior, and genetic variation in a pika (Ochotona princeps) population with a mainland-island spatial structure. Juvenile dispersal patterns in fragmented habitat revealed that individuals tended to disperse to neighboring habitat patches. However, within-patch band-sharing scores from multilocus DNA fingerprints did not differ from what would be expected if individuals were assorting randomly among habitat patches each year. Multiple, short-distance dispersal targets for juveniles and occasional long-distance dispersal events suggest that habitat fragmentation on this scale has not resulted in restricted dispersal and a genetically subdivided population. Although pikas tended to mate with the closest available partner, DNA fingerprinting band-sharing scores between mated pairs were consistent with a random mating hypothesis. Random mating in this population appears to be an incidental effect of dispersal in a fragmented habitat. This pattern is distinct from that found in nonfragmented habitat (large talus patches) where mating was non-random and consistent with mating between individuals of intermediate relatedness. DNA fingerprinting data revealed within-species variation in the mating habits of the pika directly attributable to habitat spatial structure. Received: 4 November 1996 / Accepted: 30 June 1997