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The yield of pearl millet, a resilient cereal crop crucial for African food security, is severely impacted by the root parasitic weed Striga hermonthica, which requires host-released hormones, called strigolactones (SLs), for seed germination. Herein, we identify four SLs present in the Striga-susceptible line SOSAT-C88-P10 (P10) but absent in the resistant 29Aw (Aw). We generate chromosome-scale genome assemblies, including four gapless chromosomes for each line. The Striga-resistant Aw lacks a 0.7 Mb genome segment containing two putative CARLACTONOIC ACID METHYLTRANSFERASE1 (CLAMT1) genes, which may contribute to SL biosynthesis. Functional assays show that P10CLAMT1b produces the SL-biosynthesis intermediate methyl carlactonoate (MeCLA) and that MeCLA is the precursor of P10-specific SLs. Screening a diverse pearl millet panel confirms the pivotal role of the CLAMT1 section for SL diversity and Striga susceptibility. Our results reveal a reason for Striga susceptibility in pearl millet and pave the way for generating resistant lines through marker-assisted breeding or direct genetic modification., (© 2024. The Author(s).)

Main Conclusion: Pearl millet wild relatives harbour novel alleles which could be utilized to broaden genetic base of cultivated species. Genomics-informed pre-breeding is needed to speed up introgression from wild to cultivated gene pool in pearl millet. Rising episodes of intense biotic and abiotic stresses challenge pearl millet production globally. Wild relatives provide a wide spectrum of novel alleles which could address challenges posed by climate change. Pre-breeding holds potential to introgress novel diversity in genetically narrow cultivated Pennisetum glaucum from diverse gene pool. Practical utilization of gene pool diversity remained elusive due to genetic intricacies. Harnessing promising traits from wild pennisetum is limited by lack of information on underlying candidate genes/QTLs. Next-Generation Omics provide vast scope to speed up pre-breeding in pearl millet. Genomic resources generated out of draft genome sequence and improved genome assemblies can be employed to utilize gene bank accessions effectively. The article highlights genetic richness in pearl millet and its utilization with a focus on harnessing next-generation Omics to empower pre-breeding., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In addition to their roles in developmental and metabolic processes, MYB transcription factors play crucial roles in plant defense mechanisms and stress responses. A comprehensive analysis of six pearl millet genomes revealed the presence of 1133 MYB genes, which can be classified into four phylogenetically distinct subgroups. The duplication pattern of MYB genes across the pearl millet genomes demonstrates their conserved and similar evolutionary history. Overall, MYB genes were observed to be involved in drought and heat stress responses, with stronger differential expressed observed in root tissues. Multiple analyses indicated that MYB genes mediate abiotic stress responses by modulating abscisic acid-related pathways, circadian rhythms, and histone modification processes. A substantial number of duplicated genes were determined to exhibit differential expression under abiotic stress. The consistent positive expression trend observed in duplicated gene pairs, such as PMA5G04432.1 and PMA2G00728.1, across various abiotic stresses suggests that duplicated MYB genes plays a key role in the evolution of adaptive responses of pearl millet to abiotic stresses., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Pearl millet is an important staple food crop of poor people and excels all other cereals due to its unique features of resilience to adverse climatic conditions. It is rich in micronutrients like iron and zinc and amenable for focused breeding for these micronutrients along with high yield. Hence, this is a key to alleviate malnutrition and ensure nutritional security. This study was conducted to identify and validate candidate genes governing grain iron and zinc content enabling the desired modifications in the genotypes. Transcriptome sequencing using ION S5 Next Generation Sequencer generated 43.5 million sequence reads resulting in 83,721 transcripts with N 50 of 597 bp and 84.35% of transcripts matched with the pearl millet genome assembly. The genotypes having high iron and zinc showed differential gene expression during different stages. Of which, 155 were up-regulated and 251 were down-regulated while during flowering stage and milking stage 349 and 378 transcripts were differentially expressed, respectively. Gene annotation and GO term showed the presence of transcripts involved in metabolic activities associated with uptake and transport of iron and zinc. Information generated will help in gaining insights into iron and zinc metabolism and develop genotypes with high yield, grain iron and zinc content., (© 2022. The Author(s).)

As efforts are made to increase food security, millets are gaining increasing importance due to their excellent nutritional credentials. Among the millets, pearl millet is the predominant species possessing several health benefiting nutritional traits in its grain that are helpful in mitigating chronic illnesses such as type-2 diabetes and obesity. In this paper, we conducted metabolomic fingerprinting of 197 pearl millet inbred lines drawn randomly from within the world collection of pearl millet germplasm and report the extent of genetic variation for health benefitting metabolites in these genotypes. Metabolites were extracted from seeds and assessed using flow infusion high-resolution mass spectrometry (FIE-HRMS). Metabolite features ( m / z ), whose levels significantly differed among the germplasm inbred lines, were identified by ANOVA corrected for FDR and subjected to functional pathway analysis. A number of health-benefiting metabolites linked to dietary starch, antioxidants, vitamins, and lipid metabolism-related compounds were identified. Metabolic genome-wide association analysis (mGWAS) performed using the 396 m / z as phenotypic traits and the 76 K SNP as genotypic variants identified a total of 897 SNPs associated with health benefiting nutritional metabolite at the -log p -value ≤ 4.0. From these associations, 738 probable candidate genes were predicted to have an important role in starch, antioxidants, vitamins, and lipid metabolism. The mGWAS analysis focused on genes involved in starch branching (α-amylase, β-amylase), vitamin-K reductase, UDP-glucuronosyl, and UDP-glucosyl transferase (UGTs), L-ascorbate oxidase, and isoflavone 2'-monooxygenase genes, which are known to be linked to increases in human health benefiting metabolites. We demonstrate how metabolomic, genomic, and statistical approaches can be utilized to pinpoint genetic variations and their functions linked to key nutritional properties in pearl millet, which in turn can be bred into millets and other cereals crops using plant breeding methods.

Background: The characteristics of elephant grass, especially its stem lignocellulose, are of great significance for its quality as feed or other industrial raw materials. However, the research on lignocellulose biosynthesis pathway and key genes is limited because the genome of elephant grass has not been deciphered., Results: In this study, RNA sequencing (RNA-seq) combined with lignocellulose content analysis and cell wall morphology observation using elephant grass stems from different development stages as materials were applied to reveal the genes that regulate the synthesis of cellulose and lignin. A total of 3852 differentially expressed genes (DEGs) were identified in three periods of T1, T2, and T3 through RNA-seq analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of all DEGs showed that the two most abundant metabolic pathways were phenylpropane metabolism, starch and sucrose metabolism, which were closely related to cell wall development, hemicellulose, lignin and cellulose synthesis. Through weighted gene co-expression network analysis (WGCNA) of DEGs, a 'blue' module highly associated with cellulose synthesis and a 'turquoise' module highly correlated with lignin synthesis were exhibited. A total of 43 candidate genes were screened, of which 17 had function annotations in other species. Besides, by analyzing the content of lignocellulose in the stem tissues of elephant grass at different developmental stages and the expression levels of genes such as CesA, PAL, CAD, C4H, COMT, CCoAMT, F5H and CCR, it was found that the content of lignocellulose was related to the expression level of these structural genes., Conclusions: This study provides a basis for further understanding the molecular mechanisms of cellulose and lignin synthesis pathways of elephant grass, and offers a unique and extensive list of candidate genes for future specialized functional studies which may promote the development of high-quality elephant grass varieties with high cellulose and low lignin content.

Pearl millet hybrids biofortified with iron (Fe) and zinc (Zn) promise to be part of a long-term strategy to combat micronutrient malnutrition in the arid and semi-arid tropical (SAT) regions of the world. Biofortification through molecular breeding is the way forward to achieving a rapid trait-based breeding strategy. This genome-wide association study (GWAS) was conducted to identify significant marker-trait associations (MTAs) for Fe, Zn, and protein content (PC) for enhanced biofortification breeding. A diverse panel of 281 advanced inbred lines was evaluated for Fe, Zn, and PC over two seasons. Phenotypic evaluation revealed high variability (Fe: 32-120 mg kg -1 , Zn: 19-87 mg kg -1 , PC: 8-16%), heritability (h bs 2  ≥ 90%) and significantly positive correlation among Fe, Zn and PC (P = 0.01), implying concurrent improvement. Based on the Diversity Arrays Technology (DArT) seq assay, 58,719 highly informative SNPs were filtered for association mapping. Population structure analysis showed six major genetic groups (K = 6). A total of 78 MTAs were identified, of which 18 were associated with Fe, 43 with Zn, and 17 with PC. Four SNPs viz., Pgl04_64673688, Pgl05_135500493, Pgl05_144482656, and Pgl07_101483782 located on chromosomes Pgl04 (1), Pgl05 (2) and Pgl07 (1), respectively were co-segregated for Fe and Zn. Promising genes, 'Late embryogenesis abundant protein', 'Myb domain', 'pentatricopeptide repeat', and 'iron ion binding' coded by 8 SNPs were identified. The SNPs/genes identified in the present study presents prospects for genomics assisted biofortification breeding in pearl millet.

Napiergrass (Cenchrus purpureus Schumach) is a tropical forage grass and a promising lignocellulosic biofuel feedstock due to its high biomass yield, persistence, and nutritive value. However, its utilization for breeding has lagged behind other crops due to limited genetic and genomic resources. In this study, next-generation sequencing was first used to survey the genome of napiergrass. Napiergrass sequences displayed high synteny to the pearl millet genome and showed expansions in the pearl millet genome along with genomic rearrangements between the two genomes. An average repeat content of 27.5% was observed in napiergrass including 5,339 simple sequence repeats (SSRs). Furthermore, to construct a high-density genetic map of napiergrass, genotyping-by-sequencing (GBS) was employed in a bi-parental population of 185 F 1 hybrids. A total of 512 million high quality reads were generated and 287,093 SNPs were called by using multiple de-novo and reference-based SNP callers. Single dose SNPs were used to construct the first high-density linkage map that resulted in 1,913 SNPs mapped to 14 linkage groups, spanning a length of 1,410 cM and a density of 1 marker per 0.73 cM. This map can be used for many further genetic and genomic studies in napiergrass and related species.

There have been intense debates over the geographic origin of African crops and agriculture. Here, we used whole-genome sequencing data to infer the domestication origin of pearl millet (Cenchrus americanus). Our results supported an origin in western Sahara, and we dated the onset of cultivated pearl millet expansion in Africa to 4,900 years ago. We provided evidence that wild-to-crop gene flow increased cultivated genetic diversity leading to diversity hotspots in western and eastern Sahel and adaptive introgression of 15 genomic regions. Our study reconciled genetic and archaeological data for one of the oldest African crops.

Pearl millet is a non-model grain and fodder crop adapted to extremely hot and dry environments globally. In India, a great deal of public and private sectors' investment has focused on developing pearl millet single cross hybrids based on the cytoplasmic-genetic male sterility (CMS) system, while in Africa most pearl millet production relies on open pollinated varieties. Pearl millet lines were phenotyped for both the inbred parents and hybrids stage. Many breeding efforts focus on phenotypic selection of inbred parents to generate improved parental lines and hybrids. This study evaluated two genotyping techniques and four genomic selection schemes in pearl millet. Despite the fact that 6× more sequencing data were generated per sample for RAD-seq than for tGBS, tGBS yielded more than 2× as many informative SNPs (defined as those having MAF > 0.05) than RAD-seq. A genomic prediction scheme utilizing only data from hybrids generated prediction accuracies (median) ranging from 0.73-0.74 (1000-grain weight), 0.87-0.89 (days to flowering time), 0.48-0.51 (grain yield) and 0.72-0.73 (plant height). For traits with little to no heterosis, hybrid only and hybrid/inbred prediction schemes performed almost equivalently. For traits with significant mid-parent heterosis, the direct inclusion of phenotypic data from inbred lines significantly ( P < 0.05) reduced prediction accuracy when all lines were analyzed together. However, when inbreds and hybrid trait values were both scored relative to the mean trait values for the respective populations, the inclusion of inbred phenotypic datasets moderately improved genomic predictions of the hybrid genomic estimated breeding values. Here we show that modern approaches to genotyping by sequencing can enable genomic selection in pearl millet. While historical pearl millet breeding records include a wealth of phenotypic data from inbred lines, we demonstrate that the naive incorporation of this data into a hybrid breeding program can reduce prediction accuracy, while controlling for the effects of heterosis per se allowed inbred genotype and trait data to improve the accuracy of genomic estimated breeding values for pearl millet hybrids., (Copyright © 2018 Liang et al.)

Pearl millet, (Pennisetum glaucum L.), an efficient (C4) crop of arid/semi-arid regions is known for hardiness. Crop is valuable for bio-fortification combating malnutrition and diabetes, higher caloric value and wider climatic resilience. Limited studies are done in pot-based experiments for drought response at gene-expression level, but field-based experiment mimicking drought by withdrawal of irrigation is still warranted. We report de novo assembly-based transcriptomic signature of drought response induced by irrigation withdrawal in pearl millet. We found 19983 differentially expressed genes, 7595 transcription factors, gene regulatory network having 45 hub genes controlling drought response. We report 34652 putative markers (4192 simple sequence repeats, 12111 SNPs and 6249 InDels). Study reveals role of purine and tryptophan metabolism in ABA accumulation mediating abiotic response in which MAPK acts as major intracellular signal sensing drought. Results were validated by qPCR of 13 randomly selected genes. We report the first web-based genomic resource ( http://webtom.cabgrid.res.in/pmdtdb/ ) which can be used for candidate genes-based SNP discovery programs and trait-based association studies. Looking at climatic change, nutritional and pharmaceutical importance of this crop, present investigation has immense value in understanding drought response in field condition. This is important in germplasm management and improvement in endeavour of pearl millet productivity.

Pearl millet [Cenchrus americanus (L.) Morrone] is a staple food for more than 90 million farmers in arid and semi-arid regions of sub-Saharan Africa, India and South Asia. We report the ∼1.79 Gb draft whole genome sequence of reference genotype Tift 23D 2 B 1 -P1-P5, which contains an estimated 38,579 genes. We highlight the substantial enrichment for wax biosynthesis genes, which may contribute to heat and drought tolerance in this crop. We resequenced and analyzed 994 pearl millet lines, enabling insights into population structure, genetic diversity and domestication. We use these resequencing data to establish marker trait associations for genomic selection, to define heterotic pools, and to predict hybrid performance. We believe that these resources should empower researchers and breeders to improve this important staple crop.

The diploid C 4 plant foxtail millet (Setaria italica L. Beauv.) is an important crop in many parts of Africa and Asia for the vast consumption of its grain and ability to grow in harsh environments, but remains understudied in terms of complete genomic architecture. To date, there have been only two genome assembly and annotation efforts with neither assembly reaching over 86% of the estimated genome size. We have combined de novo assembly with custom reference-guided improvements on a popular cultivar of foxtail millet and have achieved a genome assembly of 477 Mbp in length, which represents over 97% of the estimated 490 Mbp. The assembly anchors over 98% of the predicted genes to the nine assembled nuclear chromosomes and contains more functional annotation gene models than previous assemblies. Our annotation has identified a large number of unique gene ontology terms related to metabolic activities, a region of chromosome 9 with several growth factor proteins, and regions syntenic with pearl millet or maize genomic regions that have been previously shown to affect growth. The new assembly and annotation for this important species can be used for detailed investigation and future innovations in growth for millet and other grains.

A pearl millet inbred germplasm association panel (PMiGAP) comprising 250 inbred lines, representative of cultivated germplasm from Africa and Asia, elite improved open-pollinated cultivars, hybrid parental inbreds and inbred mapping population parents, was recently established. This study presents the first report of genetic diversity in PMiGAP and its exploitation for association mapping of drought tolerance traits. For diversity and genetic structure analysis, PMiGAP was genotyped with 37 SSR and CISP markers representing all seven linkage groups. For association analysis, it was phenotyped for yield and yield components and morpho-physiological traits under both well-watered and drought conditions, and genotyped with SNPs and InDels from seventeen genes underlying a major validated drought tolerance (DT) QTL. The average gene diversity in PMiGAP was 0.54. The STRUCTURE analysis revealed six subpopulations within PMiGAP. Significant associations were obtained for 22 SNPs and 3 InDels from 13 genes under different treatments. Seven SNPs associations from 5 genes were common under irrigated and one of the drought stress treatments. Most significantly, an important SNP in putative acetyl CoA carboxylase gene showed constitutive association with grain yield, grain harvest index and panicle yield under all treatments. An InDel in putative chlorophyll a/b binding protein gene was significantly associated with both stay-green and grain yield traits under drought stress. This can be used as a functional marker for selecting high yielding genotypes with 'stay green' phenotype under drought stress. The present study identified useful marker-trait associations of important agronomics traits under irrigated and drought stress conditions with genes underlying a major validated DT-QTL in pearl millet. Results suggest that PMiGAP is a useful panel for association mapping. Expression patterns of genes also shed light on some physiological mechanisms underlying pearl millet drought tolerance.

The availability of well-validated informative co-dominant microsatellite markers and saturated genetic linkage map has been limited in foxtail millet (Setaria italica L.). In view of this, we conducted a genome-wide analysis and identified 28 342 microsatellite repeat-motifs spanning 405.3 Mb of foxtail millet genome. The trinucleotide repeats (∼48%) was prevalent when compared with dinucleotide repeats (∼46%). Of the 28 342 microsatellites, 21 294 (∼75%) primer pairs were successfully designed, and a total of 15 573 markers were physically mapped on 9 chromosomes of foxtail millet. About 159 markers were validated successfully in 8 accessions of Setaria sp. with ∼67% polymorphic potential. The high percentage (89.3%) of cross-genera transferability across millet and non-millet species with higher transferability percentage in bioenergy grasses (∼79%, Switchgrass and ∼93%, Pearl millet) signifies their importance in studying the bioenergy grasses. In silico comparative mapping of 15 573 foxtail millet microsatellite markers against the mapping data of sorghum (16.9%), maize (14.5%) and rice (6.4%) indicated syntenic relationships among the chromosomes of foxtail millet and target species. The results, thus, demonstrate the immense applicability of developed microsatellite markers in germplasm characterization, phylogenetics, construction of genetic linkage map for gene/quantitative trait loci discovery, comparative mapping in foxtail millet, including other millets and bioenergy grass species.

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., (© 2010 Blackwell Publishing Ltd.)

The compelling elegance of using genome-wide scans to detect the signature of selection is difficult to resist, but is countered by the low demonstrated efficacy of pinpointing the actual genes and traits that are the targets of selection in nonmodel species. While the difficulty of going from a suggestive signature to a functional nucleotide polymorphism should not prevent researchers from using genome scans, it does lessen their long-term utility within and across study systems. In a new study published in this issue of Molecular Ecology (Mariac et al. 2011), researchers have gone a long way towards increasing the relevance of genome-wide scans for selection via two approaches: (i) they tailored the markers used in the scan to target a family of developmental genes that were good candidates for controlling a trait of interest and (ii) they used an independent mapping population to confirm the association of the gene with polymorphism in the trait of interest. All of this was completed in the nonmodel system of pearl millet (Pennisetum glaucum) and may provide a road map for other researchers hoping to pin down solid candidate genes for selected traits in natural or cultivated systems. Outside of these broad methodological innovations, the paper specifically focuses on a trait (flowering time) that varies across an environmental gradient (rainfall). This environmental gradient potentially serves as a model for environmental change over time, and allele frequencies at the gene can therefore be used to track how populations of pearl millet will adapt to future climate shifts at the genetic level., (© 2010 Blackwell Publishing Ltd.)

Pennisetum squamulatum reproduces by apomixis, a type of asexual reproduction through seeds. Apomixis in P. squamulatum is transmitted as a dominant Mendelian trait, and a genomic region, the apospory-specific genomic region (ASGR), is sufficient for inheritance of the trait. The ASGR is physically large (>50 Mb), highly heterochromatic, hemizygous, and recombinationally suppressed. These characteristics have hindered high-resolution genetic mapping and map-based cloning of apomixis genes. In this study, the long terminal repeat (LTR) regions of ASGR-abundant retrotransposons in the genome of P. squamulatum and ASGR-linked bacterial artificial chromosome clones were identified and sequenced for designing LTR-specific primers. Two hundred and ninety single-dose sequence specific amplified polymorphism (SSAP) markers were generated from 38 primer combinations. The SSAP markers combined with two previous ASGR-mapped markers were used for genetic linkage analysis and construction of a genetic map resulting in the formation of 27 linkage groups at LOD 10, one of which contained >60% of the SSAP markers. After removing identical markers (identical band scoring) on the largest linkage group, 46 markers were finally used for genetic mapping at LOD 10. The markers distributed across 10 different loci covering 19 cM; however, 45 markers were distributed within 9 cM. Six markers were recovered and sequenced. Five markers were successfully converted into sequence characterized amplified regions (SCARs). Segregation of SCAR markers was not always consistent with the SSAP markers of origin suggesting a greater level of error in the SSAP map resulting in an inflated map distance for the ASGR. One SCAR marker (Pst 56-1205-400) detected expression of an ASGR retrotransposon in root, anther, leaf and ovary of P. squamulatum, although sequencing of the RT-PCR product failed to find a functional open reading frame for the transcript.

We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5' ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5' end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5' flanking regions/promoters of selected plant genes.

In gametophytic apomicts of the aposporous type, each cell of the embryo sac is genetically identical to somatic cells of the ovule because they are products of mitosis, not of meiosis. The egg of the aposporous embryo sac follows parthenogenetic development into an embryo; therefore, uniform progeny result even from heterozygous plants, a trait that would be valuable for many crop species. Attempts to introgress apomixis from wild relatives into major crops through traditional breeding have been hindered by low or no recombination within the chromosomal region governing this trait (the apospory-specific genomic region or ASGR). The lack of recombination also has been a major obstacle to positional cloning of key genes. To further delineate and characterize the nonrecombinant ASGR, we have identified eight new ASGR-linked, AFLP-based molecular markers, only one of which showed recombination with the trait for aposporous embryo sac development. Bacterial artificial chromosome (BAC) clones identified with the ASGR-linked AFLPs or previously mapped markers, when mapped by fluorescence in situ hybridization in Pennisetum squamulatum and Cenchrus ciliaris, showed almost complete macrosynteny between the two apomictic grasses throughout the ASGR, although with an inverted order. A BAC identified with the recombinant AFLP marker mapped most proximal to the centromere of the ASGR-carrier chromosome in P. squamulatum but was not located on the ASGR-carrier chromosome in C. ciliaris. Exceptional regions where synteny was disrupted probably are nonessential for expression of the aposporous trait. The ASGR appears to be maintained as a haplotype even though its position in the genome can be variable.

This study describes the first detailed linkage maps of two bermudagrass species, Cynodon dactylon (T89) and Cynodon transvaalensis (T574), based on single-dose restriction fragments (SDRFs). The mapping population consisted of 113 F1 progeny of a cross between the two parents. Loci were generated using 179 bermudagrass genomic clones and 50 heterologous cDNAs from Pennisetum and rice. The map of T89 is based on 155 SDRFs and 17 double-dose restriction fragments on 35 linkage groups, with an average marker spacing of 15.3 cM. The map of T574 is based on 77 SDRF loci on 18 linkage groups with an average marker spacing of 16.5 cM. About 16 T89 linkage groups were arranged into four complete and eight into four incomplete homologous sets, while 15 T574 linkage groups were arranged into seven complete homologous sets, all on the basis of multi-locus probes and repulsion linkages. Eleven T89 and three T574 linkage groups remain unassigned. In each parent consensus maps were built based on alignments of homologous linkage groups. Four ancestral chromosomes were inferred after aligning T89 and T574 parental consensus maps using multi-locus probes. The inferred ancestral marker orders were used in comparisons to a detailed Sorghum linkage map using 40 common probes, and to the rice genome sequence using 98 significant BLAST hits, to find regions of colinearity. Using these maps we have estimated the recombinational length of the T89 and T574 genomes at 3,012 and 1,569 cM, respectively, which are 61 and 62% covered by our maps.

Gametophytic apomixis is asexual reproduction as a consequence of parthenogenetic development of a chromosomally unreduced egg. The trait leads to the production of embryos with a maternal genotype, i.e. progeny are clones of the maternal plant. The application of the trait in agriculture could be a tremendous tool for crop improvement through conventional and nonconventional breeding methods. Unfortunately, there are no major crops that reproduce by apomixis, and interspecific hybridization with wild relatives has not yet resulted in commercially viable germplasm. Pennisetum squamulatum is an aposporous apomict from which the gene(s) for apomixis has been transferred to sexual pearl millet by backcrossing. Twelve molecular markers that are linked with apomixis coexist in a tight linkage block called the apospory-specific genomic region (ASGR), and several of these markers have been shown to be hemizygous in the polyploid genome of P. squamulatum. High resolution genetic mapping of these markers has not been possible because of low recombination in this region of the genome. We now show the physical arrangement of bacterial artificial chromosomes containing apomixis-linked molecular markers by high resolution fluorescence in situ hybridization on pachytene chromosomes. The size of the ASGR, currently defined as the entire hemizygous region that hybridizes with apomixis-linked bacterial artificial chromosomes, was estimated on pachytene and mitotic chromosomes to be approximately 50 Mbp (a quarter of the chromosome). The ASGR includes highly repetitive sequences from an Opie-2-like retrotransposon family that are particularly abundant in this region of the genome.

Apomixis is defined as asexual reproduction through seeds, although this outcome can be achieved by multiple pathways. Since little is known about the molecular control of these pathways, how they might intersect is also a mystery. Two of these pathways in the grass family, diplospory and apospory, are receiving attention from molecular biologists. Apospory in Pennisetum/Cenchrus, two genera of panicoid grasses, results in the formation of four-nucleate embryo sacs that lack antipodals. Sexual reproduction frequently aborts so that the resulting seed is composed of (1) a parthenogenetically derived embryo that is genetically identical to the mother and (2) endosperm formed through pseudogamy. The transmission of apomixis is associated with the transfer of a linkage block on a single chromosome. This linkage block contains repetitive sequences as well as hemizygous, low-copy DNA sequences. Fluorescence in situ hybridization has demonstrated that these DNA regions occur on only a single chromosome, but not its homologs, in the polyploid apomicts studied. Features of the apomixis-associated region resemble those of other chromosomal segments isolated from recombination and replete with "selfish" DNAs.

Linkage analyses increasingly complement cytological and traditional plant breeding techniques by providing valuable information regarding genome organization and transmission genetics of complex polyploid species. This study reports a genome map of buffelgrass (Pennisetum ciliare (L.) Link syn. Cenchrus ciliaris L.). Maternal and paternal maps were constructed with restriction fragment length polymorphisms (RFLPs) segregating in 87 F1 progeny from an intraspecific cross between two heterozygous genotypes. A survey of 862 heterologous cDNAs and gDNAs from across the Poaceae, as well as 443 buffelgrass cDNAs, yielded 100 and 360 polymorphic probes, respectively. The maternal map included 322 RFLPs, 47 linkage groups, and 3464 cM, whereas the paternal map contained 245 RFLPs, 42 linkage groups, and 2757 cM. Approximately 70 to 80% of the buffelgrass genome was covered, and the average marker spacing was 10.8 and 11.3 cM on the respective maps. Preferential pairing was indicated between many linkage groups, which supports cytological reports that buffelgrass is a segmental allotetraploid. More preferential pairing (disomy) was found in the maternal than paternal parent across linkage groups (55 vs. 38%) and loci (48 vs. 15%). Comparison of interval lengths in 15 allelic bridges indicated significantly less meiotic recombination in paternal gametes. Allelic interactions were detected in four regions of the maternal map and were absent in the paternal map.