Genome-wide analysis of the WRKY gene family and their response to low-temperature stress in elephant grass.

BACKGROUD: Elephant grass (Cenchrus purpureus) is a perennial forage grass characterized by tall plants, high biomass and wide adaptability. Low-temperature stress severely limits elephant grass biomass and geographic distribution. WRKY is one of the largest families of plant-specific transcription factors and plays important roles in plant resistance to low-temperature. However, the understanding of the WRKY family in grasses is limited. In this study, we conducted a genome-wide characterization of WRKY proteins in elephant grass, including gene structure, phylogeny, expression, conserved motif organization, and functional annotation, to identify key CpWRKY candidates involved in cold tolerance. RESULTS: In this study, a total of 176 WRKY genes were identified in elephant grass. It was found that 172 were unevenly distributed across its 14 chromosomes, while the remaining 4 genes were not anchored to any chromosome. The genes were classified into three groups based on their WRKY conserved domains and zinc finger motifs. There were 12, 8, 19, 27, 12, 18 and 80 CpWRKYs belonging to group I, group IIa, group IIb, group IIc, group IId, group IIe and group III, respectively. We hypothesized that the ancient subgroup IIc WRKY gene is the ancestor of all WRKY genes in elephant grass. Most CpWRKYs in the same group have similar structure and motif composition. A total of 169 duplicate gene pairs were identified, suggesting that segmental duplication might have contributed to the expansion of the CpWRKY gene family. Ka/Ks analysis revealed that most of the CpWRKYs were subjected to purifying selection during the evolution. It was also found that six genes (CpWRKY51, CpWRKY81, CpWRKY100, CpWRKY101, CpWRKY140 and CpWRKY143) exhibited higher expression in roots compare to leaves, and were significantly induced by low temperature stress. Among them, CpWRKY81 had the highest expression under low-temperature stress, and its over-expression significantly enhanced the cold tolerance in yeast. CONLUSIONS: In this study, we characterized WRKY genes in elephant grass and further investigated their physicochemical properties, evolution, and expression patterns under low-temperature stress. This research provides valuable resources for identifying key CpWRKY genes that contribute to cold tolerance in elephant grass.

Genome-wide characterization of the NBLRR gene family provides evolutionary and functional insights into blast resistance in pearl millet (Cenchrus americanus (L.) Morrone).

MAIN CONCLUSION: The investigation is the first report on genome-wide identification and characterization of NBLRR genes in pearl millet. We have shown the role of gene loss and purifying selection in the divergence of NBLRRs in Poaceae lineage and candidate CaNBLRR genes for resistance to Magnaporthe grisea infection. Plants have evolved multiple integral mechanisms to counteract the pathogens' infection, among which plant immunity through NBLRR (nucleotide-binding site, leucine-rich repeat) genes is at the forefront. The genome-wide mining in pearl millet (Cenchrus americanus (L.) Morrone) revealed 146 CaNBLRRs. The variation in the branch length of NBLRRs showed the dynamic nature of NBLRRs in response to evolving pathogen races. The orthology of NBLRRs showed a predominance of many-to-one orthologs, indicating the divergence of NBLRRs in the pearl millet lineage mainly through gene loss events followed by gene gain through single-copy duplications. Further, the purifying selection (Ka/Ks < 1) shaped the expansion of NBLRRs within the lineage of pear millet and other members of Poaceae. Presence of cis-acting elements, viz. TCA element, G-box, MYB, SARE, ABRE and conserved motifs annotated with P-loop, kinase 2, RNBS-A, RNBS-D, GLPL, MHD, Rx-CC and LRR suggests their putative role in disease resistance and stress regulation. The qRT-PCR analysis in pearl millet lines showing contrasting responses to Magnaporthe grisea infection identified CaNBLRR20, CaNBLRR33, CaNBLRR46 CaNBLRR51, CaNBLRR78 and CaNBLRR146 as putative candidates. Molecular docking showed the involvement of three and two amino acid residues of LRR domains forming hydrogen bonds (histidine, arginine and threonine) and salt bridges (arginine and lysine) with effectors. Whereas 14 and 20 amino acid residues of CaNBLRR78 and CaNBLRR20 showed hydrophobic interactions with 11 and 9 amino acid residues of effectors, Mg.00g064570.m01 and Mg.00g006570.m01, respectively. The present investigation gives a comprehensive overview of CaNBLRRs and paves the foundation for their utility in pearl millet resistance breeding through understanding of host-pathogen interactions.

Microbial colonization on the leaf surfaces of different genotypes of Napier grass.

To address correlations between population sizes of microbes on the leaf surfaces and leaf morphological and physicochemical properties, various leaf morphological and physicochemical features as possible predictors of microbial population sizes on the leaf surfaces of four Napier grass cultivars were assessed. Results indicated microbes except for lactic acid bacteria (LAB) preferred to colonize the leaf surfaces bearing trichomes, and their population sizes were significantly correlated with trichomes, especially for yeasts. The population sizes of microbes were positively correlated with soluble sugar content (p < 0.05). Furthermore, no significant correlation was found between population sizes of microbes and wax content, except for yeasts. The multivariate regression trees (MRT) analysis showed different genotypes of leaf-microbe system could be characterized by four-leaf attributes with soluble sugar of leaf tissues being the primary explanatory attribute. Leaves with soluble sugar content below 9.72 mg g-1 fresh weight (FW) were rarely colonized. For leaves with soluble sugar content above 9.72 mg g-1 FW, water content was the next explanatory leaf attribute, followed by wax content on the leaf surfaces. Leaves with higher water content (> 73%) were more colonized, and small microbial population was associated with higher wax content (> 10.66 mg g-1 dry matter). In conclusion, leaf chemical attributes have a higher contribution than morphological structure properties in determining population sizes of microbes on the leaf surfaces. The exuded soluble sugar and protein promote the development of microbial populations. For different genotypes of leaf-microbe system, the relationship between microbial abundance on their leaf surfaces and leaf morphological structure or physicochemical properties may be predicted by the MRT. Population sizes of microbes are primarily influenced by soluble sugar content under the water-rich conditions.

Unraveling candidate genes underlying biomass digestibility in elephant grass (Cenchrus purpureus).

BACKGROUND: Elephant grass [Cenchrus purpureus (Schumach.) Morrone] is used for bioenergy and animal feed. In order to identify candidate genes that could be exploited for marker-assisted selection in elephant grass, this study aimed to investigate changes in predictive accuracy using genomic relationship information and simple sequence repeats for eight traits (height, green biomass, dry biomass, acid and neutral detergent fiber, lignin content, biomass digestibility, and dry matter concentration) linked to bioenergetics and animal feeding. RESULTS: We used single-step, genome-based best linear unbiased prediction and genome association methods to investigate changes in predictive accuracy and find candidate genes using genomic relationship information. Genetic variability (p < 0.05) was detected for most of the traits evaluated. In general, the overall means for the traits varied widely over the cuttings, which was corroborated by a significant genotype by cutting interaction. Knowing the genomic relationships increased the predictive accuracy of the biomass quality traits. We found that one marker (M28_161) was significantly associated with high values of biomass digestibility. The marker had moderate linkage disequilibrium with another marker (M35_202) that, in general, was detected in genotypes with low values of biomass digestibility. In silico analysis revealed that both markers have orthologous regions in other C4 grasses such as Setaria viridis, Panicum hallii, and Panicum virgatum, and these regions are located close to candidate genes involved in the biosynthesis of cell wall molecules (xyloglucan and lignin), which support their association with biomass digestibility. CONCLUSIONS: The markers and candidate genes identified here are useful for breeding programs aimed at changing biomass digestibility in elephant grass. These markers can be used in marker-assisted selection to grow elephant grass cultivars for different uses, e.g., bioenergy production, bio-based products, co-products, bioactive compounds, and animal feed.

Molecular understandings on 'the never thirsty' and apomictic Cenchrus grass.

The genus Cenchrus comprises around 25 species of 'bristle clade' grasses. Cenchrus ciliaris (buffel grass) is a hardy, perennial range grass that survives in poor sandy soils and limiting soil moisture conditions and, due to the very same reasons, this grass is one of the most prevalent fodder grasses of the arid and semi-arid regions. Most of the germplasms of Cenchrus produce seeds asexually through the process of apomeiosis. Therefore, the lack of sufficient sexual lines has hindered the crop improvement efforts in Cenchrus being confined to simple selection methods. Many attempts have been initiated in buffel grass to investigate the various molecular aspects such as genomic signatures of different species and genotypes, molecular basis of abiotic stress tolerance and reproductive performance. Even though it is an important fodder crop, molecular investigations in Cenchrus lack focus and the molecular information available on this grass is scanty. Cenchrus is a very good gene source for abiotic stress tolerance and apomixis studies. Biotechnological interventions in Cenchrus can help in crop improvement in Cenchrus as well as other crops through transgenic technology or marker assisted selection. To date no consolidated review on biotechnological interventions in Cenchrus grass has been published. Therefore we provide a thorough and in depth review on molecular research in Cenchrus focusing on molecular signatures of evolution, tolerance to abiotic stress and apomictic reproductive mechanism.

Assessing genotypes of buffel grass (Cenchrus ciliaris) as an alternative to maize silage for sheep nutrition.

Nutritive value of five Cenchrus ciliaris (buffel grass) genotypes (IG96-50, IG96-96, IG96-358, IG96-401 and IG96-403) weredetermined. Their sugar contents (>70 mg/g of dry matter) and ensiling potential were evaluated using in vitro batch culture and in vivo studies. Research indicated significant differences (P < 0.05) in the dry matter, organic matter, ether extract, neutral detergent fiber, acid detergent fiber, cellulose and lignin contents of the C. ciliaris genotypes tested. Genotypes also differed (P < 0.05) in total carbohydrates, structural carbohydrates, non-structural carbohydrates and protein fractions. Genotype IG96-96 had the lowest total digestible nutrients, digestible energy and metabolizable energy contents (377.2 g/kg, 6.95 and 5.71 MJ/kg of dry matter, respectively), and net energy values for lactation, maintenance and growth. After 45 days of ensiling, C. ciliaris silages differed (P < 0.05) in dry matter, pH, and lactic acid contents, and their values ranged between 255-339, 4.06-5.17 g/kg of dry matter and 10.8-28.0 g/kg of dry matter, respectively. Maize silage had higher (P < 0.05) Organic Matter (919.5g/kg of dry matter), ether extract (20.4g/kg of dry matter) and hemi-cellulose (272.3 g/kg of dry matter) than IG96-401 and IG96-96 silages. The total carbohydrates and non-structural carbohydrates of maize silage were higher (P < 0.05), while structural carbohydrates were comparable (P < 0.05) with C. ciliaris silages. Sheep on maize silage had (P < 0.05) higher metabolizable energy, lower crude protein, and digestible crude protein intake (g/kg of dry matter) than those on C. ciliaris silage diets. Nitrogen intake and urinary-N excretion were higher (P < 0.05) on genotype IG96-96 silage diet. Overall, this study suggested that certain C. ciliaris genotypes, notably IG96-401 and IG96-96, exhibited nutritive values comparable to maize silage in sheep studies, offering a promising avenue for future exploration as potential alternatives in diversified and sustainable livestock nutrition programs.

Recombination within the apospory specific genomic region leads to the uncoupling of apomixis components in Cenchrus ciliaris.

Apomixis enables the clonal propagation of maternal genotypes through seed. If apomixis could be harnessed via genetic engineering or introgression, it would have a major economic impact for agricultural crops. In the grass species Pennisetum squamulatum and Cenchrus ciliaris (syn. P. ciliare), apomixis is controlled by a single dominant "locus", the apospory-specific genomic region (ASGR). For P. squamulatum, 18 published sequenced characterized amplified region (SCAR) markers have been identified which always co-segregate with apospory. Six of these markers are conserved SCARs in the closely related species, C. ciliaris and co-segregate with the trait. A screen of progeny from a cross of sexual × apomictic C. ciliaris genotypes identified a plant, A8, retaining two of the six ASGR-linked SCAR markers. Additional and newly identified ASGR-linked markers were generated to help identify the extent of recombination within the ASGR. Based on analysis of missing markers, the A8 recombinant plant has lost a significant portion of the ASGR but continues to form aposporous embryo sacs. Seedlings produced from aposporous embryo sacs are 6× in ploidy level and hence the A8 recombinant does not express parthenogenesis. The recombinant A8 plant represents a step forward in reducing the complexity of the ASGR locus to determine the factor(s) required for aposporous embryo sac formation and documents the separation of expression of the two components of apomixis in C. ciliaris.

Root microbiome diversity and structure of the Sonoran desert buffelgrass (Pennisetum ciliare L.).

Buffelgrass (Pennisetum ciliare) is an invasive plant introduced into Mexico's Sonoran desert for cattle grazing and has converted large areas of native thorn scrub. One of the invasion mechanisms buffelgrass uses to invade is allelopathy, which consists of the production and secretion of allelochemicals that exert adverse effects on other plants' growth. The plant microbiome also plays a vital role in establishing invasive plants and host growth and development. However, little is known about the buffelgrass root-associated bacteria and the effects of allelochemicals on the microbiome. We used 16S rRNA gene amplicon sequencing to obtain the microbiome of buffelgrass and compare it between samples treated with root exacknudates and aqueous leachates as allelochemical exposure and samples without allelopathic exposure in two different periods. The Shannon diversity values were between H' = 5.1811-5.5709, with 2,164 reported bacterial Amplicon Sequence Variants (ASVs). A total of 24 phyla were found in the buffelgrass microbiome, predominantly Actinobacteria, Proteobacteria, and Acidobacteria. At the genus level, 30 different genera comprised the buffelgrass core microbiome. Our results show that buffelgrass recruits microorganisms capable of thriving under allelochemical conditions and may be able to metabolize them (e.g., Planctomicrobium, Aurantimonas, and Tellurimicrobium). We also found that the community composition of the microbiome changes depending on the developmental state of buffelgrass (p = 0.0366; ANOSIM). These findings provide new insights into the role of the microbiome in the establishment of invasive plant species and offer potential targets for developing strategies to control buffelgrass invasion.

Chromosome-scale genome assembly provides insights into speciation of allotetraploid and massive biomass accumulation of elephant grass (Pennisetum purpureum Schum.).

Elephant grass (Pennisetum purpureum Schum) is an important forage, biofuels and industrial plant widely distributed in tropical and subtropical areas globally. It is characterized with robust growth and high biomass. We sequenced its allopolyploid genome and assembled 2.07 Gb into A' and B subgenomes of 14 chromosomes with scaffold N50 of 8.47 Mb, yielding a total of 77,139 genes. The allotetraploid speciation occurred approximately 15 Ma after the divergence between Setaria italica and Pennisetum glaucum, according to a phylogenetic analysis of Pennisetum species. Double whole-genome duplication (WGD) and polyploidization events resulted in large-scale gene expansion, especially in the key steps of growth and biomass accumulation. Integrated transcriptome profiling revealed the functional divergence between subgenomes A' and B. A' subgenome mainly contributed to plant growth, development and photosynthesis, whereas the B subgenome was primarily responsible for effective transportation and resistance to stimulation. Some key gene families related to cellulose biosynthesis were expanded and highly expressed in stems, which could explain the high cellulose content in elephant grass. Our findings provide deep insights into genetic evolution of elephant grass and will aid future biological research and breeding, even for other grasses in the family Poaceae.

The elephant grass (Cenchrus purpureus) genome provides insights into anthocyanidin accumulation and fast growth.

Elephant grass (2n = 4x = 28; Cenchrus purpureus Schumach.), also known as Napier grass, is an important forage grass and potential energy crop in tropical and subtropical regions of Asia, Africa and America. However, no study has yet reported a genome assembly for elephant grass at the chromosome scale. Here, we report a high-quality chromosome-scale genome of elephant grass with a total size of 1.97 Gb and a 1.5% heterozygosity rate, obtained using short-read sequencing, single-molecule long-read sequencing and Hi-C chromosome conformation capture. Evolutionary analysis showed that subgenome A' of elephant grass and pearl millet may have originated from a common ancestor more than 3.22 million years ago (MYA). Further, allotetraploid formation occurred at approximately 6.61 MYA. Syntenic analyses within elephant grass and with other grass species indicated that elephant grass has experienced chromosomal rearrangements. We found that some key enzyme-encoding gene families related to the biosynthesis of anthocyanidins and flavonoids were expanded and highly expressed in leaves, which probably drives the production of these major anthocyanidin compounds and explains why this elephant grass cultivar has a high anthocyanidin content. In addition, we found a high copy number and transcript levels of genes involved in C4 photosynthesis and hormone signal transduction pathways that may contribute to the fast growth of elephant grass. The availability of elephant grass genome data advances our knowledge of the genetic evolution of elephant grass and will contribute to further biological research and breeding as well as for other polyploid plants in the genus Cenchrus.

Phylogenetic studies favour the unification of Pennisetum, Cenchrus and Odontelytrum (Poaceae): a combined nuclear, plastid and morphological analysis, and nomenclatural combinations in Cenchrus.

BACKGROUNDS AND AIMS: Twenty-five genera having sterile inflorescence branches were recognized as the bristle clade within the x = 9 Paniceae (Panicoideae). Within the bristle clade, taxonomic circumscription of Cenchrus (20-25 species), Pennisetum (80-140) and the monotypic Odontelytrum is still unclear. Several criteria have been applied to characterize Cenchrus and Pennisetum, but none of these has proved satisfactory as the diagnostic characters, such as fusion of bristles in the inflorescences, show continuous variation. METHODS: A phylogenetic analysis based on morphological, plastid (trnL-F, ndhF) and nuclear (knotted) data is presented for a representative species sampling of the genera. All analyses were conducted under parsimony, using heuristic searches with TBR branch swapping. Branch support was assessed with parsimony jackknifing. KEY RESULTS: Based on plastid and morphological data, Pennisetum, Cenchrus and Odontelytrum were supported as a monophyletic group: the PCO clade. Only one section of Pennisetum (Brevivalvula) was supported as monophyletic. The position of P. lanatum differed among data partitions, although the combined plastid and morphology and nuclear analyses showed this species to be a member of the PCO clade. The basic chromosome number x = 9 was found to be plesiomorphic, and x = 5, 7, 8, 10 and 17 were derived states. The nuclear phylogenetic analysis revealed a reticulate pattern of relationships among Pennisetum and Cenchrus, suggesting that there are at least three different genomes. Because apomixis can be transferred among species through hybridization, its history most likely reflects crossing relationships, rather than multiple independent appearances. CONCLUSIONS: Due to the consistency between the present results and different phylogenetic hypotheses (including morphological, developmental and multilocus approaches), and the high support found for the PCO clade, also including the type species of the three genera, we propose unification of Pennisetum, Cenchrus and Odontelytrum. Species of Pennisetum and Odontelytrum are here transferred into Cenchrus, which has priority. Sixty-six new combinations are made here.

Structural Characterization of ABCB1, the Gene Underlying the d2 Dwarf Phenotype in Pearl Millet, Cenchrus Americanus (L.) Morrone.

Pearl millet is an important food crop in arid and semi-arid regions of South Asia and sub-Saharan Africa and is grown in Australia and the United States as a summer fodder crop. The d2 dwarf germplasm has been widely used in the last half-century to develop high-performing pearl millet hybrids. We previously mapped the d2 phenotype to a 1.6 cM region in linkage group (LG) 4 and identified the ABCB1 gene as a candidate underlying the trait. Here, we report the sequence, structure and expression of ABCB1 in tall (D2D2) and d2 dwarf (d2d2) germplasm. The ABCB1 allele in d2 dwarfs differs from that in tall inbreds by the presence of two different high copy transposable elements, one in the coding region and the second located 664 bp upstream of the ATG start codon. These transposons were present in all d2 dwarfs tested that were reported to be of independent origin and absent in the analyzed wild-type tall germplasm. We also compared the expression profile of this gene in different organs of multiple tall and d2 dwarf inbreds, including the near-isogenic inbreds at the d2 locus, Tift 23B (D2D2) and Tift 23DB (d2d2). Heterologous transformation of the tall (Ca_ABCB1) and the d2 dwarf (Ca_abcb1) pearl millet alleles in the Arabidopsis double mutant abcb1abcb19 showed that the pearl millet D2 but not the d2 allele complements the Arabidopsis abcb1 mutation. Our studies also show the importance of the COOH-terminal 22 amino acids of the ABCB1 protein in either protein function or stability.

Development of EST-SSR markers in Cenchrus ciliaris and their applicability in studying the genetic diversity and cross-species transferability.

Most of the grasses of the genus Cenchrus (20-25 species) and Pennisetum (80-140 species) are distributed throughout the tropical and subtropical regions of the world and reproduce both by sexual and apomictic modes. However, the relationships among the Cenchrus-Pennisetum species are not very clear yet. Molecular markers like expressed sequence tag-simple sequence repeats (EST-SSRs) have been reported to be a better choice for resolving the phylogenetic relationships and to estimate the genetic diversity. The present study describes the identification of EST-SSR markers based on the transcriptome data of Cenchrus ciliaris inflorescence and illustrates the genetic diversity and phylogenetic relationships among these species. Of the 378 primer pairs used across 33 accessions of 21 Cenchrus, Pennisetum, and related grass (Bothriochloa, Dichanthium and Panicum) species, 116 EST-SSR markers were found to be polymorphic with an average polymorphism information content (PIC) of 0.49. Fifty-one EST-SSR loci and 520 alleles showed that where the PIC value is >0.5 there the GAG repeat motif was highly polymorphic. Two EST-SSR markers, CcSSR_80 and CcSSR_102, are polymorphic among the Cenchrus species, while they are absent in Pennisetum and the allied species. Five SSR markers (CcSSR_75, CcSSR_85, CcSSR_87, CcSSR_88 and CcSSR_114) showed 100% cross-transferability among the 21 Cenchrus-Pennisetum species. Species-specific alleles could also be detected for seven species of Cenchrus, Pennisetum and Panicum across 10 SSR markers. Assay of polymorphism across these agamic complexes showed that the three SSR markers (CcSSR_26, CcSSR_97 and CcSSR_109) were associated with Cenchrus-Pennisetum complex, and one (CcSSR_47) with Bothriochloa-Dichanthium complex. Markers with high discriminating power, namely CcSSR_4, CcSSR_38, CcSSR_48, CcSSR_66, CcSSR_67 and CcSSR_70, can be used to estimate the allelic sequence divergence across the sexual and apomictic lineages. Genetic diversity analysis using neighbour-joining (NJ) and principal co-ordinate analysis (PCoA) based approaches showed six and five clusters for the 33 accessions, respectively, having congruence in the pattern of clustering. These accessions were grouped according to their mode of reproduction. Cenchrus and Pennisetum species were grouped separately within the same clade, implying monophyletic group within a 'bristle clade'. Thus, this study showed high discrimination power of microsatellite (EST-SSR) markers to resolve the phylogenetic relationships.

Surveying the genome and constructing a high-density genetic map of napiergrass (Cenchrus purpureus Schumach).

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 F1 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.

Antinutritional factors in pearl millet grains: Phytate and goitrogens content variability and molecular characterization of genes involved in their pathways.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is an important "orphan" cereal and the most widely grown of all the millet species worldwide. It is also the sixth most important cereal in the world after wheat, rice, maize, barley, and sorghum, being largely grown and used in West Africa as well as in India and Pakistan. The present study was carried out in the frame of a program designed to increase benefits and reduce potential health problems deriving from the consumption of pearl millet. The specific goal was to provide a database of information on the variability existing in pearl millet germplasm as to the amounts of phytate, the most relevant antinutrient compound, and the goitrogenic compounds C-glycosylflavones (C-GFs) accumulated in the grain.Results we obtained clearly show that, as indicated by the range in values, a substantial variability subsists across the investigated pearl millet inbred lines as regards the grain level of phytic acid phosphate, while the amount of C-GFs shows a very high variation. Suitable potential parents to be used in breeding programs can be therefore chosen from the surveyed material in order to create new germplasm with increased nutritional quality and food safety. Moreover, we report novel molecular data showing which genes are more relevant for phytic acid biosynthesis in the seeds as well as a preliminary analysis of a pearl millet orthologous gene for C-GFs biosynthesis. These results open the way to dissect the genetic determinants controlling key seed nutritional phenotypes and to the characterization of their impact on grain nutritional value in pearl millet.

Comparative physical mapping of the apospory-specific genomic region in two apomictic grasses: Pennisetum squamulatum and Cenchrus ciliaris.

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.

Genotypic variation in response to salinity in a new sexual germplasm of Cenchrus ciliaris L.

As part of a breeding program for new salt-tolerant sexual genotypes of Cenchrus ciliaris L., here we evaluated the salt-stress response of two new sexual hybrids, obtained by controlled crosses, at seedling and germination stages. A seedling hydroponic experiment with 300 mM NaCl was performed and physiological variables and growth components were evaluated. While salt-treated sexual material did not show a decrease in productivity with respect to control plants, a differential response in some physiological characteristics was observed. Sexual hybrid 1-9-1 did not suffer oxidative damage and its proline content did not differ from that of control treatment. By contrast, sexual hybrid 1-7-11 suffered oxidative damage and accumulated proline, maintaining its growth under saline stress. At the germination stage, sexual hybrid 1-9-1 presented the highest Germination Rate Index at the maximum NaCl concentration assayed, suggesting an ecological advantage in this genotype. These new sexual resources are promising maternal parental with differential response to salt and could be incorporated in a breeding program of C. ciliaris in the search of new genotypes tolerant to salinity.

Delineation by fluorescence in situ hybridization of a single hemizygous chromosomal region associated with aposporous embryo sac formation in Pennisetum squamulatum and Cenchrus ciliaris.

Apomixis is a means of asexual reproduction by which plants produce embryos without meiosis and fertilization; thus the embryo is of clonal, maternal origin. We previously reported molecular markers showing no recombination with the trait for aposporous embryo sac development in Pennisetum squamulatum and Cenchrus ciliaris, and the collective single-dose alleles defined an apospory-specific genomic region (ASGR). Fluorescence in situ hybridization (FISH) was used to confirm that the ASGR is a hemizygous genomic region and to determine its chromosomal position with respect to rDNA loci and centromere repeats. We also documented chromosome transmission from P. squamulatum in several backcrosses (BCs) with P. glaucum using genomic in situ hybridization (GISH). One to three complete P. squamulatum chromosomes were detected in BC(6), but only one of the three hybridized with the ASGR-linked markers. In P. squamulatum and in all BCs examined, the apospory-linked markers were located in the distal region of the short arm of a single chromosome. All alien chromosomes behaved as univalents during meiosis and segregated randomly in BC(3) and later BC generations, but presence of the ASGR-carrier chromosome alone was sufficient to confer apospory. FISH results support our hypotheses that hemizygosity, proximity to centromeric sequences, and chromosome structure may all play a role in low recombination in the ASGR.

Agrobacterium-mediated transient GUS gene expression in buffel grass (Cenchrus ciliaris L.).

The study was conducted to standardize a protocol for Agrobacterium-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.). Embryogenic calli, produced from one-year-old mature seeds of buffel grass, were used as target cells for Agrobacterium-mediated transformation. A. tumefaciens strain LBA4404, harbouring pCAMBIA-1301 or pCAMBIA-2301, was used for co-cultivation with embryogenic calli from three genotypes (IG-3108, IG-9757 and IG-97101). Co-culturing of calli with Agrobacterium for 30 minutes, followed by co-cultivation with 0.1 mM acetosyringone for 3 days was found to be optimum for maximum transformation efficiency. Presence of acetosyringone during co-cultivation was found to be necessary for transformation. Transient GUS (beta-glucuronidase) gene expression was used to monitor T-DNA delivery into the target cells. Significant genotypic variations in response to transformation were observed among the tested genotypes. A very high frequency (63.3%) of GUS gene expression was obtained following Agrobacterium-mediated gene transfer into embryogenic calli. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of buffel grass with genes of agronomic importance.

Selection and validation of reference genes for gene expression analysis in apomictic and sexual Cenchrus ciliaris.

BACKGROUND: Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris. FINDINGS: Among tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction. CONCLUSION: Our gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation.