Development of EST Derived Microsatellite Markers in Buffel Grass and Their Cross Transferability to Other Forage Grasses

Buffel grass (Cenchrus ciliaris L.) is an important perennial grass grown widely in arid and semiarid regions of the world. It is one of the prominent species of the Dichanthium-Cenchrus-Lasiurus grass cover spread over the Peninsular India (Dabadghao and Shankarnarayan, 1973). C. ciliaris is a warm season, C4 grass well adapted to survive harsh conditions, elevated CO2 levels and wide range of climates and soil regimes. It is considered an excellent pasture grass as it provides highly nutritious and palatable forage during drought periods. Despite its excellent forage characteristics genetic improvement through conventional breeding methods has been difficult due to its apomictic mode of reproduction, and is mostly confined to the selection of elite lines from natural variants. (Bhat et al., 2001). Knowledge of genetic diversity and phenetic relationships among accessions is prerequisite for breeding programmes. The study of morphological variability is the only approach for assessing genetic diversity especially in minor crops. An assessment based only on agro-morphological traits might be biased because distinct morphotypes can result from spontaneous mutations. However, with molecular markers, genetic resources can accurately be assessed and characterized (Capo-chichi et al., 2004). Buffel grass has been extensively studied to understand the genetic regulation of apomixis targeting the genomic regions shared and conserved across grass family, while limited studies have been done so far to assess the genetic variation and utilization in breeding for agronomically important traits and absence of adequate genomic resources impeded further improvement. Cenchrus cultivars and accessions have previously been characterized using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeat (ISSR) and Sequence tagged site (STS) markers. While microsatellites are the most preferred markers due to their locus specificity, codominant nature, high polymorphism and reproducibility, their development and application is highly time consuming and expensive and has been limited to a few agriculturally important crops (Powell et al., 1996). An alternative way is to search the abundant sequence data already available in public databases to identify SSR’s. Nevertheless, markers developed in this way present a valuable resource for subsequent comparison between the model species and the related species. Therefore, the objectives of the present study were to develop EST based SSR markers and test their transferability potential in other related forage grasses

Enhancing Fodder Oat (\u3cem\u3eAvena sativa\u3c/em\u3e) Production in Problem Soils Using Phosphate Solubilizing Fungi Isolated from Acid and Salt Affected Soils of India

Tropical and subtropical soils are predominantly acidic, and often extremely phosphorus-deficient with high phosphorus sorption (fixation) capacities. Phosphorus (P) is one of the major essential macronutrients for plants, present at levels of 400–1200 mg/kg of soil. One of the drawbacks of fertilization is that only a fraction of the P added is eventually assimilated by plants, due to high reactivity of soluble phosphate with other elements the rest becomes unavailable to plants by forming complexes with either, Al, Fe, Ca or Mn depending on soil type (Rodríguez and Fraga, 1999). Even though some soils may have high levels of total P, they can still be P-deficient due to low levels of soluble phosphate available to plants. Available P concentrations for maximum pasture production are estimated to be between 20 and 50 µg/g. Because of the spiraling cost of phosphatic fertilizers coupled with low recovery (10- 30%) of phosphorous applied in the field, the developing tropical countries are attempting to utilize their indigenous reactive ground phosphate rock as a cheap alternative (Sabannavar and Lakshman, 2009). Many soil bacteria, Pseudomonas, Bacillus, Burkholderia, Arthrobacter, Alcaligenes, Serratia, Enterobacter, Acinetobacter and Flavobacterium and fungi especially Aspergillus, Penicillium, Trichoderma have the ability to solubilize elemental phosphate (Pi) and make it available to plants. They are used as biofertilizers for supplying the P requirement of the plants. However, scanty information is available on the occurrence of PSMicroorganisms (PSMs) in acid and salt affected soils. The present investigation was aimed to isolate PSMs from acid and salt affected soils that could survive and solubilize insoluble phosphate efficiently in the presence of higher salt concentration and acidic medium so as to obtain efficient isolates for application as a potential biofertilizer in acid and saline or problematic soils

Establishment of High-Efficiency Agrobacterium-Mediated Transformation of Callus Derived from \u3cem\u3eSehima nervosum\u3c/em\u3e, an Important Range Grass Species

Sehima nervosum is one of the important rangeland grass in India, It is commonly known as Saen grass in India, white grass in Australia, and has also been reported from the Central East Africa and Sudan. It is a good forage grass and maybe utilized for grazing as well as for hay preparation. It is a perennial grass, prefers hot and dry climate and survive even in limited rainfalls. As this natural grass is found inherently rich in precursors for several industrially important biomolecules, fractionation of these precursors seems to be a promising endeavour. Production of nutraceuticals (prebiotic xylo-oligosaccharides) from the lignocellulosic biomass of this grass is promising, as this grass does not compete with food crops, and is comparatively less expensive than conventional agricultural food-stocks. However, germplasm of this grass has narrow genetic variability. Being largely apomictic in reproduction, generation of variability through hybridization approaches have been limited. Utilization of biotechnological tools is one of the potential ways for introducing variability and transfer of desirable traits. The development of an efficient genetic transformation procedure for Sehima could facilitate physiological and molecular biology studies as well as the production of transgenic cultivars for higher productivity and quality. To the best of our knowledge there are no reports on in vitro callus induction, regeneration and transformation in Sehima. Herein, for the first time, efficient in vitro callus induction from mature seed explant and transformation efficiency in Sehima is reported. Here we standardized a reproducible, rapid and efficient Agrobacterium mediated transformation using Agrobacterium strain EHA105 harbouring binary vector pCAMBIA 1305

Transcriptome Analysis of Differentially Expressed Genes at Pre-Meiotic Developmental Stage in Pennisetum Hybrids with Contrasting Modes of Reproduction

Apomixis is an asexual reproduction through seeds where embryo develops without meiosis and fertilization. It is widely distributed throughout plant kingdom, but is more prevalent in families like Asteraceae, Rosaceae and Poaceae (Carman, 1997). This trait is highly desirable for fixing heterosis in F1 hybrids with significant implications for crop improvement (Dwivedi et al., 2007). Therefore it is necessary to unravel the molecular and genetic basis of apomixis to tap its potential. Pennisetum is an important genus of the Poaceae family which contains a wide range of species exhibiting wide variability in morphological, molecular, and reproductive traits (Jauhar, 1998). It includes many apomictic wild relatives of cultivated pearl millet (Pennisetum glaucum), some of them used extensively for introgression and molecular studies on apomixis, such as P. squamulatum, P. ciliare, and P. orientale (Ozias-Akins and Van Dijk, 2007; Kaushal et al., 2010). In order to identify putative genes involved in expression of apomixis, the genes showing differential expression across sexual and apomictic genotypes may be identified and characterized. A variety of methods are available for such molecular differential screening. These include differential display, fingerprinting techniques like cDNA AFLP, Subtractive hybridization, Micro array and Gene Chip technologies. These methods are employed for different purposes based on their convenience, sensitivity, automation and throughput. Texa with contrasting modes of reproduction are resources to identify genes involved in apomixis phenomenon. Broadly, the differentiation in reproduction pathway between apomictic and sexual lines is at three steps viz. pre-meiotic (including genes involved in preparing of ovule to enter into apomeiotic pathway), meiotic (genes involved in apomeiosis and embryo-sac development) and post-meiotic (genes involved in embryo-sac maturation and preparing for parthenogenesis). The present study was aimed to carry out a comprehensive transcriptome survey to identify differentially expressed transcripts in ovules of aposporous Pennisetum hybrid during the pre-meiotic stage of apomictic reproduction

Recent Trends in Breeding of Tropical Grass and Forage Species

Germplasm enrichment in major tropical grasses and their characterization for emerging environmental challenges have been major focussed area in the recent past. Breeding efforts in tropical grasses are still limited to few selected species viz. Panicum spp, Cenchrus spp, Pennisetum spp and Bracharia spp and all other grasses use of land races for varietal development through selection have been major source of improvement. The pace of breeding efforts in the tropical grasses have been slowed because of many inherent characteristics viz. apomixis, poor seed set, high photo and thermo sensitivity often creating problem in designing and implementing an effective breeding programme. Identification of sexual lines using the modern tools of biotechnology have given new ways for the improvement in these group of crops. This paper provides overview of the recent development that has taken place in the germplasm collection, utilization and significant achievement made through genomic and biotechnological research

Ploidy Dependent Expression of Apomixis Components in Guinea Grass (\u3cem\u3ePanicum maximum\u3c/em\u3e Jacq.)

Apomixis is an asexual method of reproduction through seeds. The potential of apomixis has been envisaged as “asexual revolution” by virtue of its capacity to fix hybrid vigour, a much desirable feature in breeding of agricultural crops. The genetic mechanism of apomixis regulation is complex and is believed to be largely affected by polyploidy (Nogler 1984). Expression of apomixis essentially contains three components, viz. apomeiosis (formation of unreduced egg cell), parthenogenesis (fertilization independent embryo development) and functional endosperm development (autonomous or psuedogamous). In contrast to previous reports, the evidence has now gathered that these three components can be functionally uncoupled and recombination is possible between these components (Kaushal, et al., 2008). Such recombinations lead to diversity in seed development pathways and also provide a mechanism to modify the ploidy levels. Uncoupling of apomeiosis from parthenogenesis may yield high frequency of triploids and haploids. Utilizing this partitioning principle we have generated a ploidy series following a Hybridization–supplemented Apomixis-components Partitioning Approach (HAPA) in guinea grass, a model crop for polyploidy and apomixis research, (Kaushal et al., 2009). From a single 4x (2n=32) progenitor, a ploidy series has been developed represented by 3x, 4x, 5x, 6x, 7x, 8x, 9x and 11x cytotypes. This ploidy series offers advantage of studying ploidy regulated gene expression. There have been sporadic reports on effect of polyploidy in expression of apomixis per se; however information on effect of polyploidy on individual apomixis components is not available. The guinea grass ploidy series with sequentially added monoploid genome doses has been used in present study to understand the effect of ploidy levels on phenotypic expression of partitioned apomixis components

Partitioning Apomixis Components to Understand and Utilize Gametophytic Apomixis

Apomixis is a method of reproduction to generate clonal seeds and offers tremendous potential to fix heterozygosity and hybrid vigor. The process of apomictic seed development is complex and comprises three distinct components, viz., apomeiosis (leading to formation of unreduced egg cell), parthenogenesis (development of embryo without fertilization) and functional endosperm development. Recently, in many crops, these three components are reported to be uncoupled leading to their partitioning. This review provides insight into the recent status of our understanding surrounding partitioning apomixis components in gametophytic apomictic plants and research avenues that it offers to help understand the biology of apomixis. Possible consequences leading to diversity in seed developmental pathways, resources to understand apomixis, inheritance and identification of candidate gene(s) for partitioned components, as well as contribution towards creation of variability are all discussed. The potential of Panicum maximum, an aposporous crop, is also discussed as a model crop to study partitioning principle and effects. Modifications in cytogenetic status, as well as endosperm imprinting effects arising due to partitioning effects, opens up new opportunities to understand and utilize apomixis components, especially towards synthesizing apomixis in crops

Transcriptomic data of pre-meiotic stage of floret development in apomictic and sexual types of guinea grass (Panicum maximum Jacq.)

Guinea grass (Panicum maximum Jacq), an important fodder crop of humid and sub-humid tropical regions, reproduces through apomixis, a method of clonal propagation through seeds. Lack of knowledge of the genetic and molecular control of this phenomena has hindered the genetic improvement of this crop. The dataset provided here represents the first RNA-Seq based assembly and analysis of florets at pre-meiotic stage from the apomictic and sexual genotypes of guinea grass. The raw sequence files in FASTQ format were deposited in the NCBI SRA database with accession number SRP115883. A total of 24.8 Gb raw sequence data, corresponding to 17,96,65,827 raw reads was obtained by paired end sequencing. We used Trinity for de-novo assembly and identified 57,647 transcripts in sexual and 49,093 transcripts in apomictic type. This transcriptome data will be useful for identification and comparative analysis of genes regulating the mode of reproduction in grasses. Keywords: Florets, Pre-meiosis, Apomixis, RNA-Seq, Panicum maximu