Association Analysis of SSR Markers with Phenology, Grain, and Stover-Yield Related Traits in Pearl Millet (Pennisetum glaucum (L.) R. Br.)

Pearl millet is a staple food crop for millions of people living in the arid and semi-arid tropics. Molecular markers have been used to identify genomic regions linked to traits of interest by conventional QTL mapping and association analysis. Phenotypic recurrent selection is known to increase frequencies of favorable alleles and decrease those unfavorable for the traits under selection. This study was undertaken (i) to quantify the response to recurrent selection for phenotypic traits during breeding of the pearl millet open-pollinated cultivar “CO (Cu) 9” and its four immediate progenitor populations and (ii) to assess the ability of simple sequence repeat (SSR) marker alleles to identify genomic regions linked to grain and stover yield-related traits in these populations by association analysis. A total of 159 SSR alleles were detected across 34 selected single-copy SSR loci. SSR marker data revealed presence of subpopulations. Association analysis identified genomic regions associated with flowering time located on linkage group (LG) 6 and plant height on LG4, LG6, and LG7. Marker alleles on LG6 were associated with stover yield, and those on LG7 were associated with grain yield. Findings of this study would give an opportunity to develop marker-assisted recurrent selection (MARS) or marker-assisted population improvement (MAPI) strategies to increase the rate of gain for pearl millet populations undergoing recurrent selection.

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title: Genetic diversity among pearl millet maintainers using microsatellite markers
abstract: Genetic diversity among 70 maintainers and two pollinators of sub-Saharan and Indian origin was studied for simple sequence repeat (SSR) loci using 34 primer pairs. A total of 213 alleles were detected with an average of 6.26 alleles per locus. Polymorphic information content (PIC) ranged from 0.05 to 0.96 with a mean of 0.58 for the SSR loci. Mean PIC across the linkage groups and number of alleles in dinucleotide motifs varied significantly. The lowest PIC (0.239) for linkage group 6 indicated comparatively conserved nature of this linkage group. Genetic similarity estimates ranged from 0.05 to 0.73 with an average value of 0.29. This indicated sufficient diversity among the maintainer and pollinator lines. The 72 lines fell in five clusters, and the clustering pattern corroborated with their pedigree and characteristic traits. Pollinator ICMR 356 was more diverse from the maintainer lines analysed, and can be a potential parent for pearl millet hybrid development.

title: Genetic Diversity in Maintainer and Restorer Lines of Pearl Millet
abstract: Molecular markers facilitate rapid and environment-neutral characterization of the pattern of genetic diversity. The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) at Patancheru, India, has developed a large number and diverse range of maintainer lines (B-lines) and restorer lines (R-lines) of pearl millet [Pennisetum glaucum (L.) R. Br.] in last three decades. In the present study, 213 lines (98 B-lines and 115 R-lines) were genotyped using high throughput assay and 38 polymorphic simple sequence repeat (SSR) markers, which detected 308 alleles averaging 8.1 alleles per locus. Restorer lines were genetically more diverse than B-lines, as higher average gene diversity was detected among R-lines (0.62) than among B-lines (0.50). A neighbor-joining tree based on simple matching dissimilarity distance matrix clearly differentiated B-lines from R-lines into two clusters, with further subdivision of B-lines into four subclusters and R-lines into five subclusters. Seven B-lines clustered with R-lines, and only two R-lines clustered with B-lines. Few unique alleles (1 to 3) were detected in 14 B- and 30 R-lines, which could be used to distinguish them from each other as well as from the remaining lines used in this study. The SSR alleles of each of the parental lines along with distinctness, uniformity, and stability (DUS) characterization may enable ICRISAT to protect these lines from any kind of infringement by the multiple users who have received these lines for hybrid development.

title: Assessment of genetic diversity within and between pearl millet landraces
abstract: A minimum core subset of pearl millet [Pennisetum glaucum (L.) R. Br.], which comprised 504 landrace accessions, was recently established from the global pearl millet germplasm collection of ICRISAT. The accessions for this core were selected by a random proportional sampling strategy following stratification of the entire landrace collection (about 16,000 accessions) according to their geographic origin and morpho-agronomic traits. In this study RFLP probes were used to quantify the genetic diversity within and between landrace accessions of this minimum core using a subset comprising ten accessions of Indian origin. Twenty five plants per accession were assayed with EcoRI, EcoRV, HindIII and DraI restriction enzymes, and 16 highly polymorphic RFLP probes, nine associated with a quantitative trait loci (QTLs) for downy mildew resistance, and five associated with a QTL for drought tolerance. A total of 51 alleles were detected using 16 different probe-enzyme combinations. The partitioning of variance components based on the analysis of molecular variance (AMOVA) for diversity analysis revealed high within-accession variability (30.9%), but the variability between accessions was significantly higher (69.1%) than that within the accessions. A dendrogram based on the dissimilarity matrix obtained using Ward's algorithm further delineated the 250 plants into ten major clusters, each comprised of plants from a single accession (with the exception of two single plants). A similar result was found in an earlier study using morpho-agronomic traits and geographic origin. This study demonstrated the utility of RFLP markers in detecting polymorphism and estimating genetic diversity in a highly cross-pollinated species such as pearl millet. When less-tedious marker systems are available, this method could be further extended to assess the genetic diversity between and within the remaining accessions in the pearl millet core subset.

title: Diversity of wild and cultivated pearl millet accessions (Pennisetum glaucum [L.] R. Br.) in Niger assessed by microsatellite markers
abstract: Genetic diversity of crop species in sub-Sahelian Africa is still poorly documented. Among such crops, pearl millet is one of the most important staple species. In Niger, pearl millet covers more than 65% of the total cultivated area. Analyzing pearl millet genetic diversity, its origin and its dynamics is important for in situ and ex situ germplasm conservation and to increase knowledge useful for breeding programs. We developed new genetic markers and a high-throughput technique for the genetic analysis of pearl millet. Using 25 microsatellite markers, we analyzed genetic diversity in 46 wild and 421 cultivated accessions of pearl millet in Niger. We showed a significantly lower number of alleles and lower gene diversity in cultivated pearl millet accessions than in wild accessions. This result contrasts with a previous study using iso-enzyme markers showing similar genetic diversity between cultivated and wild pearl millet populations. We found a strong differentiation between the cultivated and wild groups in Niger. Analyses of introgressions between cultivated and wild accessions showed modest but statistically supported evidence of introgressions. Wild accessions in the central region of Niger showed introgressions of cultivated alleles. Accessions of cultivated pearl millet showed introgressions of wild alleles in the western, central, and eastern parts of Niger.

title: Integration of gene-based markers in a pearl millet genetic map for identification of candidate genes underlying drought tolerance quantitative trait loci
abstract: BackgroundIdentification of genes underlying drought tolerance (DT) quantitative trait loci (QTLs) will facilitate understanding of molecular mechanisms of drought tolerance, and also will accelerate genetic improvement of pearl millet through marker-assisted selection. We report a map based on genes with assigned functional roles in plant adaptation to drought and other abiotic stresses and demonstrate its use in identifying candidate genes underlying a major DT-QTL.ResultsSeventy five single nucleotide polymorphism (SNP) and conserved intron spanning primer (CISP) markers were developed from available expressed sequence tags (ESTs) using four genotypes, H 77/833-2, PRLT 2/89-33, ICMR 01029 and ICMR 01004, representing parents of two mapping populations. A total of 228 SNPs were obtained from 30.5 kb sequenced region resulting in a SNP frequency of 1/134 bp. The positions of major pearl millet linkage group (LG) 2 DT-QTLs (reported from crosses H 77/833-2 × PRLT 2/89-33 and 841B × 863B) were added to the present consensus function map which identified 18 genes, coding for PSI reaction center subunit III, PHYC, actin, alanine glyoxylate aminotransferase, uridylate kinase, acyl-CoA oxidase, dipeptidyl peptidase IV, MADS-box, serine/threonine protein kinase, ubiquitin conjugating enzyme, zinc finger C- × 8-C × 5-C × 3-H type, Hd3, acetyl CoA carboxylase, chlorophyll a/b binding protein, photolyase, protein phosphatase1 regulatory subunit SDS22 and two hypothetical proteins, co-mapping in this DT-QTL interval. Many of these candidate genes were found to have significant association with QTLs of grain yield, flowering time and leaf rolling under drought stress conditions.ConclusionsWe have exploited available pearl millet EST sequences to generate a mapped resource of seventy five new gene-based markers for pearl millet and demonstrated its use in identifying candidate genes underlying a major DT-QTL in this species. The reported gene-based markers represent an important resource for identification of candidate genes for other mapped abiotic stress QTLs in pearl millet. They also provide a resource for initiating association studies using candidate genes and also for comparing the structure and function of distantly related plant genomes such as other Poaceae members.

title: Construction of a BAC library of pearl millet, Pennisetum glaucum
abstract: A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from pearl millet (Pennisetum glaucum), and used as a resource for the isolation of microsatellite sequences. The library contains a total of 159,100 clones with an average insert size of 90 kb, and corresponds to 5.8 haploid genome equivalents. The BAC library was pooled for screening by the polymerase chain reaction (PCR) as well as robotically gridded on high-density filters. PCR-based screening of a subset of the library (4.7 haploid genome equivalents) using five sequence-tagged site (STS) and six microsatellite markers identified between 2 and 11 positives superpools (5.4 on average). The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be less than 1% by hybridisation with a rice chloroplast probe.

title: Development of simple sequence repeat markers from bacterial artificial chromosomes without subcloning.
abstract: Simple sequence repeats (SSRs) were isolated from pearl millet bacterial artificial clones (BACs) without any subcloning steps. SSR sequences were targeted using 3' end-anchored SSR primers. Flanking sequences were isolated by suppression PCR. In this pilot study, 25 SSR markers have been developed from 40 BAC pools, comprising a total of 384 clones. This novel way to develop new markers has the added advantage that mapping the SSR markers will anchor individual BACs to the genetic maps and, thus, facilitate the construction of BAC contigs.

title: An integrated genetic map and a new set of simple sequence repeat markers for pearl millet, Pennisetum glaucum
abstract: Over the past 10 years, resources have been established for the genetic analysis of pearl millet, Pennisetum glaucum (L.) R. Br., an important staple crop of the semi-arid regions of India and Africa. Among these resources are detailed genetic maps containing both homologous and heterologous restriction fragment length polymorphism (RFLP) markers, and simple sequence repeats (SSRs). Genetic maps produced in four different crosses have been integrated to develop a consensus map of 353 RFLP and 65 SSR markers. Some 85% of the markers are clustered and occupy less than a third of the total map length. This phenomenon is independent of the cross. Our data suggest that extreme localization of recombination toward the chromosome ends, resulting in gaps on the genetic map of 30 cM or more in the distal regions, is typical for pearl millet. The unequal distribution of recombination has consequences for the transfer of genes controlling important agronomic traits from donor to elite pearl millet germplasm. The paper also describes the generation of 44 SSR markers from a (CA)n-enriched small-insert genomic library. Previously, pearl millet SSRs had been generated from BAC clones, and the relative merits of both methodologies are discussed.

title: Development and Utilization of SSRs to Estimate the Degree of Genetic Relationships in a Collection of Pearl Millet Germplasm
abstract: 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.

title: Development and mapping of Simple Sequence Repeat markers for pearl millet from data mining of Expressed Sequence Tags
abstract: BackgroundPearl millet [Pennisetum glaucum (L.) R. Br.] is a staple food and fodder crop of marginal agricultural lands of sub-Saharan Africa and the Indian subcontinent. It is also a summer forage crop in the southern USA, Australia and Latin America, and is the preferred mulch in Brazilian no-till soybean production systems. Use of molecular marker technology for pearl millet genetic improvement has been limited. Progress is hampered by insufficient numbers of PCR-compatible co-dominant markers that can be used readily in applied breeding programmes. Therefore, we sought to develop additional SSR markers for the pearl millet research community.ResultsA set of new pearl millet SSR markers were developed using available sequence information from 3520 expressed sequence tags (ESTs). After clustering, unigene sequences (2175 singlets and 317 contigs) were searched for the presence of SSRs. We detected 164 sequences containing SSRs (at least 14 bases in length), with a density of one per 1.75 kb of EST sequence. Di-nucleotide repeats were the most abundant followed by tri-nucleotide repeats. Ninety primer pairs were designed and tested for their ability to detect polymorphism across a panel of 11 pairs of pearl millet mapping population parental lines. Clear amplification products were obtained for 58 primer pairs. Of these, 15 were monomorphic across the panel. A subset of 21 polymorphic EST-SSRs and 6 recently developed genomic SSR markers were mapped using existing mapping populations. Linkage map positions of these EST-SSR were compared by homology search with mapped rice genomic sequences on the basis of pearl millet-rice synteny. Most new EST-SSR markers mapped to distal regions of linkage groups, often to previous gaps in these linkage maps. These new EST-SSRs are now are used by ICRISAT in pearl millet diversity assessment and marker-aided breeding programs.ConclusionThis study has demonstrated the potential of EST-derived SSR primer pairs in pearl millet. As reported for other crops, EST-derived SSRs provide a cost-saving marker development option in pearl millet. Resources developed in this study have added a sizeable number of useful SSRs to the existing repertoire of circa 100 genomic SSRs that were previously available to pearl millet researchers.

title: SSCP-SNP in pearl millet—a new marker system for comparative genetics
abstract: A considerable array of genomic resources are in place in pearl millet, and marker-aided selection is already in use in the public breeding programme at ICRISAT. This paper describes experiments to extend these publicly available resources to a single nucleotide polymorphism (SNP)-based marker system. A new marker system, single-strand conformational polymorphism (SSCP)-SNP, was developed using annotated rice genomic sequences to initially predict the intron-exon borders in millet expressed sequence tags (ESTs) and then to design primers that would amplify across the introns. An adequate supply of millet ESTs was available for us to identify 299 homologues of single-copy rice genes in which the intron positions could be precisely predicted. PCR primers were then designed to amplify approximately 500-bp genomic fragments containing introns. Analysis of these fragments on SSCP gels revealed considerable polymorphism. A detailed DNA sequence analysis of variation at four of the SSCP-SNP loci over a panel of eight inbred genotypes showed complex patterns of variation, with about one SNP or indel (insertion-deletion) every 59 bp in the introns, but considerably fewer in the exons. About two-thirds of the variation was derived from SNPs and one-third from indels. Most haplotypes were detected by SSCP. As a marker system, SSCP-SNP has lower development costs than simple sequence repeats (SSRs), because much of the work is in silico, and similar deployment costs and through-put potential. The rates of polymorphism were lower but useable, with a mean PIC of 0.49 relative to 0.72 for SSRs in our eight inbred genotype panel screen. The major advantage of the system is in comparative applications. Syntenic information can be used to target SSCP-SNP markers to specific chromosomal regions or, conversely, SSCP-SNP markers can be used to unravel detailed syntenic relationships in specific parts of the genome. Finally, a preliminary analysis showed that the millet SSCP-SNP primers amplified in other cereals with a success rate of about 50%. There is also considerable potential to promote SSCP-SNP to a COS (conserved orthologous set) marker system for application across species by more specifically designing primers to precisely match the model genome sequence.

title: Development of a molecular linkage map of pearl millet integrating DArT and SSR markers
abstract: Pearl millet is an important component of food security in the semi-arid tropics and is assuming greater importance in the context of changing climate and increasing demand for highly nutritious food and feed. Molecular tools have been developed and applied for pearl millet on a limited scale. However, the existing tool kit needs to be strengthened further for its routine use in applied breeding programs. Here, we report enrichment of the pearl millet molecular linkage map by exploiting low-cost and high-throughput Diversity Arrays Technology (DArT) markers. Genomic representation from 95 diverse genotypes was used to develop a DArT array with circa 7,000 clones following PstI/BanII complexity reduction. This array was used to genotype a set of 24 diverse pearl millet inbreds and 574 polymorphic DArT markers were identified. The genetic relationships among the inbred lines as revealed by DArT genotyping were in complete agreement with the available pedigree data. Further, a mapping population of 140 F7 Recombinant Inbred Lines (RILs) from cross H 77/833-2 × PRLT 2/89-33 was genotyped and an improved linkage map was constructed by integrating DArT and SSR marker data. This map contains 321 loci (258 DArTs and 63 SSRs) and spans 1148 cM with an average adjacent-marker interval length of 3.7 cM. The length of individual linkage groups (LGs) ranged from 78 cM (LG 3) to 370 cM (LG 2). This better-saturated map provides improved genome coverage and will be useful for genetic analyses of important quantitative traits. This DArT platform will also permit cost-effective background selection in marker-assisted backcrossing programs as well as facilitate comparative genomics and genome organization studies once DNA sequences of polymorphic DArT clones are available.

title: Mapping and characterisation of QTL × E interactions for traits determining grain and stover yield in pearl millet
abstract: A mapping population of 104 F3 lines of pearl millet, derived from a cross between two inbred lines H 77/833-2 × PRLT 2/89-33, was evaluated, as testcrosses on a common tester, for traits determining grain and stover yield in seven different field trials, distributed over 3 years and two seasons. The total genetic variation was partitioned into effects due to season (S), genotype (G), genotype × season interaction (G × S), and genotype × environment-within-season interaction [G × E(S)]. QTLs were determined for traits for their G, G × S, and G × E(S) effects, to assess the magnitude and the nature (cross over/non-crossover) of environmental interaction effects on individual QTLs. QTLs for some traits were associated with G effects only, while others were associated with the effects of both G and G × S and/or G, G × S and G × E(S) effects. The major G × S QTLs detected were for flowering time (on LG 4 and LG 6), and mapped to the same intervals as G × S QTLs for several other traits (including stover yield, harvest index, biomass yield and panicle number m–2). All three QTLs detected for grain yield were unaffected by G × S interaction however. All three QTLs for stover yield (mapping on LG 2, LG 4 and LG 6) and one of the three QTLs for grain yield (mapping on LG 4) were also free of QTL × E(S) interactions. The grain yield QTLs that were affected by QTL × E(S) interactions (mapping on LG 2 and LG 6), appeared to be linked to parallel QTL × E(S) interactions of the QTLs for panicle number m–2 on (LG 2) and of QTLs for both panicle number m–2 and harvest index (LG 6). In general, QTL × E(S) interactions were more frequently observed for component traits of grain and stover yield, than for grain or stover yield per se.

title: Maize association population: a high-resolution platform for quantitative trait locus dissection.
abstract: Crop improvement and the dissection of complex genetic traits require germplasm diversity. Although this necessary phenotypic variability exists in diverse maize, most research is conducted using a small subset of inbred lines. An association population of 302 lines is now available--a valuable research tool that captures a large proportion of the alleles in cultivated maize. Provided that appropriate statistical models correcting for population structure are included, this tool can be used in association analyses to provide high-resolution evaluation of multiple alleles. This study describes the population structure of the 302 lines, and investigates the relationship between population structure and various measures of phenotypic and breeding value. On average, our estimates of population structure account for 9.3% of phenotypic variation, roughly equivalent to a major quantitative trait locus (QTL), with a high of 35%. Inclusion of population structure in association models is critical to meaningful analyses. This new association population has the potential to identify QTL with small effects, which will aid in dissecting complex traits and in planning future projects to exploit the rich diversity present in maize.

title: Plant molecular diversity and applications to genomics.
abstract: Surveys of nucleotide diversity are beginning to show how genomes have been shaped by evolution. Nucleotide diversity is also being used to discover the function of genes through the mapping of quantitative trait loci (QTL) in structured populations, the positional cloning of strong QTL, and association mapping.

title: Applications of single nucleotide polymorphisms in crop genetics.
abstract: The discovery of single nucleotide polymorphisms (SNPs) and insertions/deletions, which are the basis of most differences between alleles, has been simplified by recent developments in sequencing technology. SNP discovery in many crop species, such as corn and soybean, is relatively straightforward because of their high level of intraspecific nucleotide diversity, and the availability of many gene and expressed sequence tag (EST) sequences. For these species, direct readout of SNP haplotypes is possible. Haplotype-based analysis is more informative than analysis based on individual SNPs, and has more power in analyzing association with phenotypes. The elite germplasm of some crops may have been subjected to bottlenecks relatively recently, increasing the amount of linkage disequilibrium (LD) present and facilitating the association of SNP haplotypes at candidate gene loci with phenotypes. Whole-genome scans may help identify genome regions that are associated with interesting phenotypes if sufficient LD is present. Technological improvements make the use of SNP and indel markers attractive for high-throughput use in marker-assisted breeding, EST mapping and the integration of genetic and physical maps.

title: Genetic basis of pearl millet adaptation along an environmental gradient investigated by a combination of genome scan and association mapping
abstract: Identifying the molecular bases of adaptation is a key issue in evolutionary biology. Genome scan is an efficient approach for identifying important molecular variation involved in adaptation. Association mapping also offers an opportunity to gain insight into genotype–phenotype relationships. Using these two approaches coupled with environmental data should help to come up with a refined picture of the evolutionary process underlying adaptation. In this study, we first conducted a selection scan analysis on a transcription factor gene family. We focused on the MADS‐box gene family, a gene family which plays a crucial role in vegetative and flower development. Twenty‐one pearl millet populations were sampled along an environmental gradient in West Africa. We identified one gene, i.e. PgMADS11, using Bayesian analysis to detect selection signatures. Polymorphism at this gene was also associated with flowering time variation in an association mapping framework. Finally, we found that PgMADS11 allele frequencies were closely associated with annual rainfall. Overall, we determined an efficient way to detect functional polymorphisms associated with climate variation in non‐model plants by combining genome scan and association mapping. These results should help monitor the impact of recent climatic changes on plant adaptation.

title: Optimizing parental selection for genetic linkage maps.
abstract: Genetic linkage maps based on restriction fragment length polymorphisms are useful for many purposes; however, different populations are required to fulfill different objectives. Clones from the linkage map(s) are subsequently probed onto populations developed for special purposes such as gene tagging. Therefore, clones contained on the initial map(s) must be polymorphic on a wide range of genotypes to have maximum utility. The objectives of this research were to (i) calculate polymorphism information content values of 51 low-copy DNA clones and (ii) use the resulting values to choose potential mapping parents. Polymorphism information content was calculated using gene diversity by classifying restriction fragment patterns on a diverse set of 18 wheat genotypes. Combinations of potential parents were then compared by examining both the proportion of polymorphic clones and the likelihood that those mapped clones would give a polymorphism when used on other populations. Genotype pairs were identified that would map more highly informative DNA clones compared with a population derived from the most polymorphic potential parents. The methodologies used to characterize clones and rank potential parents should be applicable to other species and types of markers as well.

title: GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update
abstract: Genetic Analysis in Excel is a cross-platform package for population genetic analyses that runs within Microsoft Excel. GenAlEx offers analysis of diploid codominant, haploid and binary genetic loci and DNA sequences. Both frequency-based (F-statistics, heterozygosity, HWE, population assignment, relatedness) and distance-based (AMOVA, PCoA, Mantel tests, multivariate spatial autocorrelation) analyses are provided. New features include calculation of new estimators of population structure: G′ST, G′′ST, Jost’s Dest and F′ST through AMOVA, Shannon Information analysis, linkage disequilibrium analysis for biallelic data and novel heterogeneity tests for spatial autocorrelation analysis. Export to more than 30 other data formats is provided. Teaching tutorials and expanded step-by-step output options are included. The comprehensive guide has been fully revised. Availability and implementation: GenAlEx is written in VBA and provided as a Microsoft Excel Add-in (compatible with Excel 2003, 2007, 2010 on PC; Excel 2004, 2011 on Macintosh). GenAlEx, and supporting documentation and tutorials are freely available at: http://biology.anu.edu.au/GenAlEx.

title: Inference of population structure using multilocus genotype data.
abstract: We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci-e.g. , seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from http://www.stats.ox.ac.uk/ approximately pritch/home. html.

title: Detecting the number of clusters of individuals using the software structure: a simulation study
abstract: The identification of genetically homogeneous groups of individuals is a long standing issue in population genetics. A recent Bayesian algorithm implemented in the software structure allows the identification of such groups. However, the ability of this algorithm to detect the true number of clusters (K) in a sample of individuals when patterns of dispersal among populations are not homogeneous has not been tested. The goal of this study is to carry out such tests, using various dispersal scenarios from data generated with an individual‐based model. We found that in most cases the estimated ‘log probability of data’ does not provide a correct estimation of the number of clusters, K. However, using an ad hoc statistic ΔK based on the rate of change in the log probability of data between successive K values, we found that structure accurately detects the uppermost hierarchical level of structure for the scenarios we tested. As might be expected, the results are sensitive to the type of genetic marker used (AFLP vs. microsatellite), the number of loci scored, the number of populations sampled, and the number of individuals typed in each sample.

title: Association Mapping of Kernel Size and Milling Quality in Wheat (Triticum aestivum L.) Cultivars
abstract: Association mapping is a method for detection of gene effects based on linkage disequilibrium (LD) that complements QTL analysis in the development of tools for molecular plant breeding. In this study, association mapping was performed on a selected sample of 95 cultivars of soft winter wheat. Population structure was estimated on the basis of 36 unlinked simple-sequence repeat (SSR) markers. The extent of LD was estimated on chromosomes 2D and part of 5A, relative to the LD observed among unlinked markers. Consistent LD on chromosome 2D was <1 cM, whereas in the centromeric region of 5A, LD extended for ∼5 cM. Association of 62 SSR loci on chromosomes 2D, 5A, and 5B with kernel morphology and milling quality was analyzed through a mixed-effects model, where subpopulation was considered as a random factor and the marker tested was considered as a fixed factor. Permutations were used to adjust the threshold of significance for multiple testing within chromosomes. In agreement with previous QTL analysis, significant markers for kernel size were detected on the three chromosomes tested, and alleles potentially useful for selection were identified. Our results demonstrated that association mapping could complement and enhance previous QTL information for marker-assisted selection.

title: Genome-wide association studies for agronomical traits in a world wide spring barley collection
abstract: BackgroundGenome-wide association studies (GWAS) based on linkage disequilibrium (LD) provide a promising tool for the detection and fine mapping of quantitative trait loci (QTL) underlying complex agronomic traits. In this study we explored the genetic basis of variation for the traits heading date, plant height, thousand grain weight, starch content and crude protein content in a diverse collection of 224 spring barleys of worldwide origin. The whole panel was genotyped with a customized oligonucleotide pool assay containing 1536 SNPs using Illumina's GoldenGate technology resulting in 957 successful SNPs covering all chromosomes. The morphological trait "row type" (two-rowed spike vs. six-rowed spike) was used to confirm the high level of selectivity and sensitivity of the approach. This study describes the detection of QTL for the above mentioned agronomic traits by GWAS.ResultsPopulation structure in the panel was investigated by various methods and six subgroups that are mainly based on their spike morphology and region of origin. We explored the patterns of linkage disequilibrium (LD) among the whole panel for all seven barley chromosomes. Average LD was observed to decay below a critical level (r2-value 0.2) within a map distance of 5-10 cM. Phenotypic variation within the panel was reasonably large for all the traits. The heritabilities calculated for each trait over multi-environment experiments ranged between 0.90-0.95. Different statistical models were tested to control spurious LD caused by population structure and to calculate the P-value of marker-trait associations. Using a mixed linear model with kinship for controlling spurious LD effects, we found a total of 171 significant marker trait associations, which delineate into 107 QTL regions. Across all traits these can be grouped into 57 novel QTL and 50 QTL that are congruent with previously mapped QTL positions.ConclusionsOur results demonstrate that the described diverse barley panel can be efficiently used for GWAS of various quantitative traits, provided that population structure is appropriately taken into account. The observed significant marker trait associations provide a refined insight into the genetic architecture of important agronomic traits in barley. However, individual QTL account only for a small portion of phenotypic variation, which may be due to insufficient marker coverage and/or the elimination of rare alleles prior to analysis. The fact that the combined SNP effects fall short of explaining the complete phenotypic variance may support the hypothesis that the expression of a quantitative trait is caused by a large number of very small effects that escape detection. Notwithstanding these limitations, the integration of GWAS with biparental linkage mapping and an ever increasing body of genomic sequence information will facilitate the systematic isolation of agronomically important genes and subsequent analysis of their allelic diversity.

title: Genetic analysis and characterization of a new maize association mapping panel for quantitative trait loci dissection
abstract: Association mapping based on the linkage disequilibrium provides a promising tool to identify genes responsible for quantitative variations underlying complex traits. Presented here is a maize association mapping panel consisting of 155 inbred lines with mainly temperate germplasm, which was phenotyped for 34 traits and genotyped using 82 SSRs and 1,536 SNPs. Abundant phenotypic and genetic diversities were observed within the panel based on the phenotypic and genotypic analysis. A model-based analysis using 82 SSRs assigned all inbred lines to two groups with eight subgroups. The relative kinship matrix was calculated using 884 SNPs with minor allele frequency ≥20% indicating that no or weak relationships were identified for most individual pairs. Three traits (total tocopherol content in maize kernel, plant height and kernel length) and 1,414 SNPs with missing data <20% were used to evaluate the performance of four models for association mapping analysis. For all traits, the model controlling relative kinship (K) performed better than the model controlling population structure (Q), and similarly to the model controlling both population structure and relative kinship (Q + K) in this panel. Our results suggest this maize panel can be used for association mapping analysis targeting multiple agronomic and quality traits with optimal association model.

title: Association genetics of chilling injury susceptibility in peach (Prunus persica (L.) Batsch) across multiple years
abstract: Peach and nectarine (Prunus persica L.) are highly perishable; they ripen and deteriorate quickly at ambient temperature. Storage at low temperature (0–5 °C) is a common strategy used to slow the ripening processes and to extend shelf life. However, if susceptible varieties are held too long at a low temperature, they will not ripen properly and will develop chilling injury (CI) symptoms like mealiness (M), flesh browning (FB), and flesh bleeding (FBL). Understanding the genetic control of these traits to produce CI resistant cultivars will greatly benefit producers, shippers and consumers. In this study, we evaluated a population of 51 individuals from Pop-DG across 4 years with CI traits observed in one or two time points to detect molecular marker association with selected 960 single-nucleotide polymorphisms (SNPs) from 1,536 SNPs chip. Genotypic and phenotypic data were analyzed by general linear model and mixed linear model to see comparative results from both analyses. Among 960 SNPs used, 22 SNPs were found associated with CI susceptibility traits like M, FB, and FBL. Many SNP markers were located in or close to previously reported quantitative trait loci mapped by linkage analysis.

title: A unified mixed-model method for association mapping that accounts for multiple levels of relatedness
abstract: As population structure can result in spurious associations, it has constrained the use of association studies in human and plant genetics. Association mapping, however, holds great promise if true signals of functional association can be separated from the vast number of false signals generated by population structure. We have developed a unified mixed-model approach to account for multiple levels of relatedness simultaneously as detected by random genetic markers. We applied this new approach to two samples: a family-based sample of 14 human families, for quantitative gene expression dissection, and a sample of 277 diverse maize inbred lines with complex familial relationships and population structure, for quantitative trait dissection. Our method demonstrates improved control of both type I and type II error rates over other methods. As this new method crosses the boundary between family-based and structured association samples, it provides a powerful complement to currently available methods for association mapping.

title: Identification of single nucleotide polymorphisms and haplotypes associated with yield and yield components in soybean (Glycine max) landraces across multiple environments
abstract: Genome-wide association analysis is a powerful approach to identify the causal genetic polymorphisms underlying complex traits. In this study, we evaluated a population of 191 soybean landraces in five environments to detect molecular markers associated with soybean yield and its components using 1,536 single-nucleotide polymorphisms (SNPs) and 209 haplotypes. The analysis revealed that abundant phenotypic and genetic diversity existed in the studied population. This soybean population could be divided into two subpopulations and no or weak relatedness was detected between pair-wise landraces. The level of intra-chromosomal linkage disequilibrium was about 500 kb. Genome-wide association analysis based on the unified mixed model identified 19 SNPs and 5 haplotypes associated with soybean yield and yield components in three or more environments. Nine markers were found co-associated with two or more traits. Many markers were located in or close to previously reported quantitative trait loci mapped by linkage analysis. The SNPs and haplotypes identified in this study will help to further understand the genetic basis of soybean yield and its components, and may facilitate future high-yield breeding by marker-assisted selection in soybean.

title: SSR allele frequency changes in response to recurrent selection for pearl millet grain yield and other agronomic traits
abstract: Allele frequency changes resulting from conventional recurrent selection were monitored at 34 SSR loci distributed across all 7 linkage groups using full-sibprogenies (50 each) from released pearl millet openpollinated variety CO (Cu) 9 and its four immediate progenitor populations (ICMP 87750, ICMP 91751,ICMP 93752 and UCC 23). Agronomic performance of these 250 full-sibs was assessed in two seasons of replicated field trials at ICRISAT, Patancheru, India, which demonstrated significant selection responses for flowering time and grain, stover and biomass yields, and their components. These selection responses were accompanied by significant, but non-linear, frequency changes for common alleles at many monitored SSR loci.Substantial genetic variability remains within CO (Cu) 9,suggesting further selection to improve its grain and stover yield performance should be effective.

title: Quantitative trait loci associated with traits determining grain and stover yield in pearl millet under terminal drought-stress conditions
abstract: Drought stress during the reproductive stage is one of the most important environmental factors reducing the grain yield and yield stability of pearl millet. A QTL mapping approach has been used in this study to understand the genetic and physiological basis of drought tolerance in pearl millet and to provide a more-targeted approach to improving the drought tolerance and yield of this crop in water-limited environments. The aim was to identify specific genomic regions associated with the enhanced tolerance of pearl millet to drought stress during the flowering and grain-filling stages. Testcrosses of a set of mapping-population progenies, derived from a cross of two inbred pollinators that differed in their response to drought, were evaluated in a range of managed terminal drought-stress environments. A number of genomic regions were associated with drought tolerance in terms of both grain yield and its components. For example, a QTL associated with grain yield per se and for the drought tolerance of grain yield mapped on linkage group 2 and explained up to 23% of the phenotypic variation. Some of these QTLs were common across stress environments whereas others were specific to only a particular stress environment. All the QTLs that contributed to increased drought tolerance did so either through better than average maintenance (compared to non-stress environments) of harvest index, or harvest index and biomass productivity. It is concluded that there is considerable potential for marker-assisted backcross transfer of selected QTLs to the elite parent of the mapping population and for their general use in the improvement of pearl millet productivity in water-limited environments.

title: Assessment of Drought Resistance in Pearl Millet (Pennisetum americanum (L.) Leeke). I Factors Affecting Yields under Stress
abstract: Yield trials of advanced lines of pearl millet were grown under midseason (panicle initiation to flowering) and terminal (flowering to maturity) drought stresses in the dry seasons of 1981, 1982, and 1983. Grain yield and its components were severely reduced by the terminal stress, but were little affected by the midseason drought, as there was compensation by later tillers for yield lost on the earlier shoots. The major factor determining grain yield of a genotype in both stress treatments was its time to flowering. Because of this, yield-yield component relationships under stress were a result of yield component-phenology relationships. Yield potential (measured in an irrigated treatment in the same field) was generally positively related to yield under stress, but accounted for a much smaller fraction of yield variation than time to flowering. These two factors together accounted for more than 50% of the total variation in grain yield under stress, suggesting that genotype drought response (drought resistance/susceptibility) was a secondary rather than a primary factor in differences in yield under stress among the genotypes tested.

title: Effect of timing of water deficit on pearl millet (Pennisetum americanum)
abstract: The susceptibility of pearl millet to soil water deficits at different times of growth was determined in field experiments conducted over 2 years. Grain yields and yield components were expressed as ratios of stressed to non-stressed treatments. Where water deficit was imposed earlier in crop growth and then relieved, the time of termination of stress from flowering determined the extent of grain yield loss. Water stress relieved at anthesis or early grain filling had little adverse effect on the grain yield because additional tillers produced panicles. This response by tillers was less evident and grain yields were more severely reduced when water stress was not relieved until after flowering. When stress was imposed late in crop growth and not relieved, the time of initiation of stress was directly related to the extent of loss in grain yield. Thus susceptibility to mid-season drought stress was related to the time stress was terminated and in late-season drought to the time stress was initiated.

title: Structure of variation among morphological and physiological traits in three pearl millet composites
abstract: SummaryThe plant breeder's task of improving and stabilizing many plant traits simultaneously is complicated by interrelationships that occur among the traits. Factor analyses were conducted on three phenotypically diverse pearl millet [Pennisetum glaucum (L.) R. Br.] composites to describe the structure of relationships among yield, morphological, and physiological traits. Approximately 1000 S0 spaced-plants from each composite were evaluated for 20 traits, and random samples of 289 S1 progenies from each composite were evaluated for 18 of these traits. Factors extracted in S0 and S1 populations identified unique sets of traits that were interrelated along axes of (a) biological yield, (b) panicle size, (c) dry matter partitioning and (d) compensation between number and size of seeds. Several plant traits had large loading coefficients on the ‘Biological Yield’ and also, but with opposite signs, on the ‘Dry Matter Partitioning’ factor. The traits having large loading on these two factors differed between space-planted and normal-density stands, showing that environmental conditions contributed to the associations observed among traits. Correlations of S1 with parental S0 factor scores for the ‘Biological Yield’, ‘Panicle Size’ and ‘Seed Paramters’ factors produced significant correlation coefficients, indicating that these trait complexes had a genetic basis. The implications of these results for millet breeding are discussed.

title: Exploring the genetic diversity of Ethiopian grass pea (Lathyrus sativus L.) using EST-SSR markers
abstract: Expressed sequence tags (ESTs) in public databases and cross-species transferable markers are considered to be a cost-effective means for developing sequence-based markers for less-studied species. In this study, EST-simple sequence repeat (SSR) markers developed from Lathyrus sativus L. EST sequences and cross-transferable EST-SSRs derived from Medicago truncatula L. were utilized to investigate the genetic diversity among grass pea populations from Ethiopia. A total of 45 alleles were detected using eleven EST-SSRs with an average of four alleles per locus. The average polymorphism information content for all primers was 0.416. The average gene diversity was 0.477, ranging from 0.205 for marker Ls942 to 0.804 for MtBA32F05. FST values estimated by analysis of molecular variance were 0.01, 0.15, and 0.84 for among regions, among accessions and within accessions respectively, indicating that most of the variation (84%) resides within accessions. Model-based cluster analysis grouped the accessions into three clusters, grouping accessions irrespective of their collection regions. Among the regions, high levels of diversity were observed in Gojam, Gonder, Shewa and Welo regions, with Gonder region showing a higher number of different alleles. From breeding and conservation aspects, conducting a close study on a specific population would be advisable for genetic improvement in the crop, and it would be appropriate if future collection and conservation plans give due attention to under-represented regions.

title: A first insight into population structure and linkage disequilibrium in the US peanut minicore collection
abstract: Knowledge of genetic diversity, population structure, and degree of linkage disequilibrium (LD) in target association mapping populations is of great importance and is a prerequisite for LD-based mapping. In the present study, 96 genotypes comprising 92 accessions of the US peanut minicore collection, a component line of the tetraploid variety Florunner, diploid progenitors A. duranensis (AA) and A. ipaënsis (BB), and synthetic amphidiploid accession TxAG-6 were investigated with 392 simple sequence repeat (SSR) marker bands amplified using 32 highly-polymorphic SSR primer pairs. Both distance- and model-based (Bayesian) cluster analysis revealed the presence of structured diversity. In general, the wild-species accessions and the synthetic amphidiploid grouped separately from most minicore accessions except for COC155, and were eliminated from most subsequent analyses. UPGMA analysis divided the population into four subgroups, two major subgroups representing subspecies fastigiata and hypogaea, a third group containing individuals from each subspecies or possibly of mixed ancestry, and a fourth group, either consisting of COC155 alone if wild species were excluded, or of COC155, the diploid species, and the synthetic amphidiploid. Model-based clustering identified four subgroups- one each for fastigiata and hypogaea subspecies, a third consisting of individuals of both subspecies or of mixed ancestry predominantly from Africa or Asia, and a fourth group, consisting of individuals predominantly of var fastigiata,peruviana, and aequatoriana accessions from South America, including COC155. Analysis of molecular variance (AMOVA) revealed statistically-significant (P < 0.0001) genetic variance of 16.87% among subgroups. A total of 4.85% of SSR marker pairs revealed significant LD (at r2 ≥ 0.1). Of the syntenic marker pairs separated by distances < 10 cM, 11–20 cM, 21–50 cM, and > 50 cM, 19.33, 5.19, 6.25 and 5.29% of marker pairs were found in strong LD (P ≤ 0.01), in accord with LD extending to great distances in self pollinated crops. A threshold value of r2 > 0.035 was found to distinguish mean r2 values of linkage distance groups statistically from the mean r2 values of unlinked markers; LD was found to extend to 10 cM over the entire minicore collection by this criterion. However, there were large differences in r2 values among marker pairs even among tightly-linked markers. The implications of these findings with regard to the possibility of using association mapping for detection of genome-wide SSR marker-phenotype association are discussed.

title: Genetic variation, population structure and linkage disequilibrium in peach commercial varieties
abstract: BackgroundPeach [Prunus persica (L.) Batsch] is one of the most economically important fruit crops that, due to its genetic and biological characteristics (small genome size, taxonomic proximity to other important species and short juvenile period), has become a model plant in genomic studies of fruit trees. Our aim was an in-depth study of the extent, distribution and structure of peach genetic variation in North American and European commercial varieties as well as old Spanish varieties and several founders used in the early USA peach breeding programmes. For this we genotyped 224 peach cultivars using 50 SSRs evenly distributed along the 8 linkage groups of the Prunus reference map.ResultsGenetic distance analysis based on SSRs divided the peach cultivars in three main groups based mainly on their fruit characteristics: melting flesh peaches, melting flesh nectarines and non-melting varieties. Whereas non-melting flesh peaches had a higher number of alleles than melting peaches and nectarines, they were more homozygous. With some exceptions ('Admiral Dewey', 'Early Crawford' and 'Chinese Cling'), the founder US cultivars clustered together with the commercial melting peaches, indicating that their germplasm is well represented in modern cultivars. Population structure analysis showed a similar subdivision of the sample into subpopulations. Linkage disequilibrium (LD) analysis in three unstructured, or barely structured, subpopulations revealed a high level of LD conservation in peach extending up to 13-15 cM.ConclusionsUsing a much larger set of SSRs, our results confirm previous observations on peach variability and population structure and provide additional tools for breeding and breeders' rights enforcement. SSR data are also used for the estimation of marker mutation rates and allow pedigree inferences, particularly with founder genotypes of the currently grown cultivars, which are useful to understand the evolution of peach as a crop. Results on LD conservation can be explained by the self-pollinating nature of peach cultivated germplasm and by a bottleneck that occurred at the beginning of modern breeding practices. High LD suggests that the development of whole-genome scanning approaches is suitable for genetic studies of agronomically important traits in peach.

title: Genetic diversity, linkage disequilibrium, and association mapping analyses of peach (Prunus persica) landraces in China
abstract: The genetic diversity, population structure, and linkage disequilibrium (LD) of peaches are greatly important in genome-wide association mapping. In the current study, 104 peach landrace accessions from six Chinese geographical regions were evaluated for fruit and phenological period. The accessions were genotyped with 53 genome-wide simple sequence repeat (SSR) markers. All SSR markers were highly polymorphic across the accessions, and a total of 340 alleles were detected, including 59 private alleles. Of the six regions studied, the northern part of China as well as the middle and lower reaches of the Changjiang River were found to be the most highly diverse genetically. Based on population structure analysis, the peaches were divided into five groups, which well agreed with the geographical distribution. Of the SSR pairs in these accessions, 18.07% (P < 0.05) were in LD. The mean r2 value for all intrachromosomal loci pairs was 0.0149, and LD decayed at 6.01 cM. The general linear model was used to calculate the genome-wide marker-trait associations of 10 complex traits. The traits include flesh color around the stone, red pigment in the flesh, flesh texture, flesh adhesion, flesh firmness, fruit weight, chilling requirement, flowering time, ripening time, and fruit development period. These traits were estimated by analyzing the 104 landraces. Many of the associated markers were located in regions where quantitative trait loci (QTLs) were previously identified. Peach association mapping is an effective approach for identifying QTLs and may be an alternative to QTL mapping based on crosses between different lines.

title: Downy mildew resistance QTLs from a seedling heat tolerance mapping population.
abstract: An RFLP based map for a pearl millet (Pennisetum glaucum) population derived from a cross between H77/833-2 and ICMP451 was evaluated for its ability to detect quantitative trait loci by combining it with field data on downy mildew (Sclerospora graminicola) incidence, flowering time and 1000-grain mass from a trial involving the F4 generation derived from 94 of the mapped F2 plants in 1994 at Patancheru. Late flowering of ICMP451 was tightly linked to downy mildew quantitative trait loci in linkage group 1.

title: Opportunities for marker-assisted selection (MAS) to improve the feed quality of crop residues in pearl millet and sorghum
abstract: Cereal crop residues (straw, chaff, etc.) are important components of maintenance rations for ruminant livestock in many parts of the world. They are especially important in small-holder crop-livestock production systems in the sub-humid, semi-arid, and arid tropics and subtropics where most of the world’s poorest livestock producers and consumers are found. Taking as examples tropically adapted cereals in the crop improvement mandate of the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), namely pearl millet [Pennisetum glaucum (L.) R. Br.] and sorghum [Sorghum bicolor (L.) Moench], this paper explores opportunities for using marker-assisted crop breeding methods to improve the quality and quantity of cereal crop residues for use as ruminant livestock feedstuffs. In the case of pearl millet, ICRISAT has been heavily involved with several UK-based collaborating research institutes, in development and initial application of the molecular genetic tools for marker-assisted breeding. We have obtained some useful experience in quantitative trait loci (QTL) mapping and marker-assisted selection (MAS) for stover yield, foliar disease resistance, and in vitro estimates of the nutritive value of various stover fractions for ruminants. In sorghum, ICRISAT has focused on initiating a large-scale high-throughput marker-assisted backcrossing program for the stay-green component of terminal drought tolerance—a trait that is likely to be associated not only with more stable grain and stover yield, but which is also expected to contribute to maintenance of ruminant nutritional value of stover produced under drought stress conditions. Conventional and marker-assisted breeding for foliar disease resistance is recommended for dual-purpose cereal improvement, or indeed for improvement of the nutritional value of residues for any crop in which these are used as feedstuffs for ruminant livestock. Practical problems faced and proposed ways of dealing with these are discussed.

title: Genomic regions associated with grain yield and aspects of post-flowering drought tolerance in pearl millet across stress environments and tester background
abstract: A pearl millet mapping population from a cross between ICMB841 and 863B was studied for DNA polymorphism to construct a genetic linkage map, and to map genomic regions associated with grain and stover yield, and aspects of drought tolerance. To identify genomic regions associated with these traits, mapping population testcrosses of 79 F3 progenies were evaluated under post-flowering drought stress conditions over 2 years and in the background of two elite testers. A significant genotype × drought stress treatment interaction was evident in the expression of grain and stover yield in drought environments and in the background of testers over the 2 years. As a result of this, genomic regions associated with grain and stover yield and the aspects of drought tolerance were also affected: some regions were more affected by the changes in the environments (i.e. severity and duration of drought stress) while others were commonly identified across the drought stress environments and tester background used. In most instances, both harvest index and panicle harvest index co-mapped with grain yield suggesting that increased drought tolerance and yield of pearl millet that mapped to these regions was achieved by increased partitioning of dry matter from stover to the grains. Drought stress treatments, years and testers interactions on genomic regions associated with grain and stover yield of pearl millet are discussed, particularly, in reference to genetic improvement of drought tolerance of this crop using marker-assisted selection.

title: Mapping the d1 and d2 dwarfing genes and the purple foliage color locus P in pearl millet.
abstract: The d(1) and d(2) dwarfing genes and the P purple foliage color gene were placed on the restriction fragment length polymorphism (RFLP)-based molecular marker linkage map of pearl millet [Pennisetum glaucum (L.) R. Br.] using a mapping population based on a cross of inbred lines IP 18293 (D(1)/D(1), d(2)/d(2), P/P) and Tift 238D1 (d(1)/d(1) D(2)/D(2) p/p). A skeleton genetic linkage map of 562 cM (Haldane function) was constructed using 33 RFLP markers and these three morphological markers. The D(1)/d(1) plant height locus mapped to pearl millet linkage group 1, while the D(2)/d(2) plant height locus and the P/p foliage color locus mapped to pearl millet linkage group 4. Loose genetic linkage was observed between the D(2)/d(2) and P/p loci, with 42% repulsion-phase recombination corresponding to 92 cM (Haldane). This loose linkage of morphological marker loci detected on pearl millet LG4 can likely find use in applied pearl millet breeding programs, as host plant resistances to both downy mildew and rust have previously been identified in this genomic region. Such exploitation of these morphological markers in an applied disease resistance breeding program would require development of appropriate genetic stocks, but the relatively loose genetic linkage between d(2) and P suggests that this should not be difficult.

title: Quantitative trait loci for grain yield in pearl millet under variable postflowering moisture conditions
abstract: Bidinger, F. R., Nepolean, T., Hash, C. T., Yadav, R. S., Howarth, C. J., (2007) Quantitative trait loci for grain yield in pearl millet under variable postflowering moisture conditions. Crop Science 47, 969–980.  BLUP, best linear unbiased predictor; GRMA, individual grain mass; GRNO, grain number; GRYLD, grain yield; HI, harvest index; LG, linkage group; LOD, logarithm of odds; MAS, marker-assisted selection; PNHI, panicle harvest index; QTL, quantitative trait locus; RFLP, restriction fragment length polymorphism; SSR, simple sequence repeat.  This document is an output from projects (Plant Sciences Research Programme R7375 and R8183) funded by the UK Dep. for International Development (DFID)

title: Genetic Mapping of Quantitative Trait Loci in Maize in Stress and Nonstress Environments: I. Grain Yield and Yield Components
abstract: The utility of restriction fragment length polymorphisms (RFLPs) in detecting quantitative trait loci (QTL) in maize (Zea mays L.) populations has been established. Conflicting results have been reported on the ability to consistently detect the same QTL across environments. Grain yield is considered to be highly polygenic and strongly influenced by the environment. QTL mapping studies conducted in contrasting environments might increase our understanding genotype x environment interactions and improve selection response in maize breeding. Our objectives were to compare the QTL for grain yield and yield components detected in two diverse environments in an elite, adapted maize population and to investigate the relationship among the genetic factors controlling these traits. A population of 150 F 2:3 lines was produced from the cross of elite inbreds Mo17 and H99. The lines per se were evaluated for 2 yr at the same location. The first year had relatively average growing conditions, but the second year with cool, wet growing conditions provided a stress environment. Grain yield and yield components were measured on replicated progeny each year. By using a linkage map of 111 loci, QTL determinations were made in the two environments and in the mean of the two environments. About 50% of all QTL detected across environments were detected in both environments. These QTL were consistent in the magnitude of their effects and parental contribution. Determinations of QTL in the mean of the two environments were most informative in that 74% of all QTL and 82% of the QTL detected in each of the individual environments were identified in the mean environment. Most QTL identified regions associated with two or more yield components in a manner consistent with correlations among traits and parents phenotypes.

title: Pearl millet (Pennisetum glaucum)-a cereal of the Sahel
abstract: D E WET, J , hi. J., BIDIKGER, F. R. and PEACOCK,  1991. Pcarl rnillct (Pmnirr fum gloumm) a ccrcal of thc Sahcl. I n considering thc rolc of grasscs i n thc conrainmcnt and rctricval or dcscrts i r is clcar that pcarl rnillct has bccn sclcctcd by rarmcrs on thc fringcr or thc Sahara for its adaptability to drought and high tcmpcraturc. As a conscqucncc, Iandraccs arc charactcrircd by r*ild-type adapratio~lr such as the production of largc numbcrs of small rccds, long sccd viabi l~ty and sccd dormancy undcr conditions or low humidity and high tcrnpcraturcs, rapid sccd grrrninarion and gro,vth whcn sufficicnr moirturc bccomcs available, sccdli~ig sunrival undcr high tcmprrarurcs, complcrion of a lirc cyclc in as l inlc as 60 days, and thc ability to fill grain undcr cxtrcmc hcat and drought strcss, all or which allow for sur\,i\al i n this harsh cn~iranmcxit. Man). plant brccdcrr ivould arguc that rhcsc rathcr conscn,ativc ccolugical traits should be sclcctcd against. This is not to say *c should nor dcvclop millcts that arc niorc resistant to insects and dircases and arc highcr yiclding that1 thc traditional varictics, but the cmphasis must be on thc retrr,tlon or thc high rtrbi l i ty of yield undcr low inputs. \\'c bclicvc chat this can bc donc, cnsuring that pcarl rnillct rcmains onc o f the most important crop spccics i n rtic dcscrt regions of the rropics.

title: Structure of linkage disequilibrium and phenotypic associations in the maize genome
abstract: Association studies based on linkage disequilibrium (LD) can provide high resolution for identifying genes that may contribute to phenotypic variation. We report patterns of local and genome-wide LD in 102 maize inbred lines representing much of the worldwide genetic diversity used in maize breeding, and address its implications for association studies in maize. In a survey of six genes, we found that intragenic LD generally declined rapidly with distance (r2 < 0.1 within 1500 bp), but rates of decline were highly variable among genes. This rapid decline probably reflects large effective population sizes in maize during its evolution and high levels of recombination within genes. A set of 47 simple sequence repeat (SSR) loci showed stronger evidence of genome-wide LD than did single-nucleotide polymorphisms (SNPs) in candidate genes. LD was greatly reduced but not eliminated by grouping lines into three empirically determined subpopulations. SSR data also supplied evidence that divergent artificial selection on flowering time may have played a role in generating population structure. Provided the effects of population structure are effectively controlled, this research suggests that association studies show great promise for identifying the genetic basis of important traits in maize with very high resolution.

title: Association Analysis as a Strategy for Improvement of Quantitative Traits in Plants
abstract: Association analysis is a method potentially useful for detection of marker-trait associations based on linkage disequilibrium, but little information is available on the application of this technique to plant breeding populations. With appropriate statistical methods, valid association analysis can be done in plant breeding populations; however, the most significant marker may not be closest to the functional gene. Bias can arise from (i) covariance among markers and QTL, frequently related to population structure or intense selection and (ii) differences in initial frequencies of marker alleles in the population, such that exclusive alleles tend to be in higher association. The potentials and limitations of germplasm bank collections, synthetic populations, and elite germplasm are compared, as experimental materials for association analysis integrated with plant breeding practice. Synthetics offer a favorable balance of power and precision for association analysis and would allow mapping of quantitative traits with increasing resolution through cycles of intermating. A model to describe the association between markers and genes as conditional probabilities in synthetic populations under recurrent selection is proposed, which can be computed on the basis of assumptions related to the history of the population. This model is useful for predicting the potential of different populations for association analysis and forecasting the response to marker-assisted selection.

title: Strategies of Marker‐Aided Recurrent Selection
abstract: The choice of a marker-aided recurrent selection (MARS) strategy depends on a number of factors. An experimental comparison of these factors can be costly or infeasible, but a theoretical comparison can provide some guidelines. This study was conducted to evaluate the genetic aspects of expected responses to various MARS methods and to compare the efficiencies of MARS relative to traditional phenotypic recurrent selection (PRS) and to marker-aided mass selection (MS). The MARS methods examined here are MS, half-sib family (HSF), full-sib family (FSF), S 1 family (S 1 F), testcross progeny (TC), combined FSF selection with within-family MS (CSFS), and combined HSF selection with within-family MS (CSHS). If a large fraction of the additive genetic variance (p ) in a character can be explained by marker loci, the efficiencies of MARS relative to corresponding PRS can be great. This is most pronounced when family size (n) is small and decreases as n increases. An exception to this is for CSHS, where MARS is superior to PRS across all family sizes. Similarly, CSFS performs better than FSF selection under a wider range of family sizes (1 < n < 50). With a constant rate selected and a relatively large family size, S 1 F, CSFS and CSHS selection are superior to MS, whereas HSF selection is inferior to MS. With constraints of the same effective size selected for each method, MS, which takes advantage of the greatest selection intensity, is the most efficient method. Nevertheless, MARS among small families or individual plants can produce substantial increases in selection responses.

title: Marker-assisted recurrent selection for cumulating additive and interactive QTLs in recombinant inbred lines
abstract: A computer program has been designed to manage marker information in recombinant inbred-line populations. The objective is to select pairs of inbred lines (either recombinant-inbred or doubled-haploid) to be intercrossed, in order to accumulate all or most favourable alleles, either with additive effects or with interactive effects. The population size required to have a 95% chance of obtaining the best line from a given cross is computed, taking into account the number of QTLs and the probability that no recombination event occurs in any of the QTL confidence intervals. It is shown that the accuracy of QTL location greatly affects selection efficiency and that a recurrent selection scheme is highly preferable for pyramiding many QTLs. An application to the bread-making quality improvement of wheat is presented.