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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 maximum

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.

In angiosperms, meiotic failure coupled with the formation of genetically unreduced gametophytes in ovules (apomeiosis) constitute major components of gametophytic apomixis. These aberrant developmental events are generally thought to be caused by mutation. However, efforts to locate the responsible mutations have failed. Herein, we tested a fundamentally different hypothesis: apomeiosis is a polyphenism of meiosis, with meiosis and apomeiosis being maintained by different states of metabolic homeostasis. Microarray analyses of ovules and pistils were used to differentiate meiotic from apomeiotic processes in Boechera (Brassicaceae). Genes associated with translation, cell division, epigenetic silencing, flowering, and meiosis characterized sexual Boechera (meiotic). In contrast, genes associated with stress responses, abscisic acid signaling, reactive oxygen species production, and stress attenuation mechanisms characterized apomictic Boechera (apomeiotic). We next tested whether these metabolic differences regulate reproductive mode. Apomeiosis switched to meiosis when premeiotic ovules of apomicts were cultured on media that increased oxidative stress. These treatments included drought, starvation, and H2O2 applications. In contrast, meiosis switched to apomeiosis when premeiotic pistils of sexual plants were cultured on media that relieved oxidative stress. These treatments included antioxidants, glucose, abscisic acid, fluridone, and 5-azacytidine. High-frequency apomeiosis was initiated in all sexual species tested: Brassicaceae, Boechera stricta, Boechera exilis, and Arabidopsis thaliana, Fabaceae, Vigna unguiculata, Asteraceae, Antennaria dioica. Unreduced gametophytes formed from ameiotic female and male sporocytes, first division restitution dyads, and nucellar cells. These results are consistent with modes of reproduction and types of apomixis, in natural apomicts, being regulated metabolically.

Panicum maximum (guinea grass) is a model crop for apomixis and polyploidy studies. It is predominantly tetraploid (2n = 32) and is characterized by gametophytic apomixis, Panicum-type apospory and pseudogamous endosperm development. The three components of apomixis, viz. apomeiosis, parthenogenesis and functional endosperm development, can be uncoupled in this crop. An exhaustive single progenitor-derived ploidy series comprising of 32 accessions representing 3x, 4x, 5x, 6x, 7x, 8x, 9x and 11x cytotypes was utilized in present study to understand ploidy effects on expression of apospory as well on uncoupled components in two phases of progeny formation i.e. in matured ovules (using embryo-sac analysis) and in matured self-pollinated seeds (using Flow Cytometric Seed Screen method). Rise in ploidy enhanced the formation of sexual embryo-sacs (ES) thereby increasing the frequency of facultative accessions at higher ploidy level. Our results suggested that the eventual phenotype depends on relative doses of apospory and sexual factors in the genome. Ploidy level was also found affecting the penetrance and expressivity of uncoupled apomixis components. Formation of BIII hybrids (3n) appeared to be more stabilised and less affected by the ploidy change, however, formation of M1 (1n) progenies increased with the rise in ploidy. Ploidy effects on traits such as occurrence of multiple ES, autonomous endosperm development, and twin embryos were also studied. Flexibility of guinea grass to tolerate excessive genome burden and successful formation of seeds overcoming endosperm balance number and endosperm imprinting constraints is also discussed.

†BackgroundandAims During seed fill in cereals, nutrients are symplasmically unloaded to vascular parenchyma in ovules, but thereafter nutrient transport is less certain. In Zea mays, two mechanisms of nutrient passage through the chalaza and nucellus have been hypothesized, apoplasmic and symplasmic. In a recent study, nutrients first passed non-selectively to the chalazal apoplasm and were then selectively absorbed by the nucellus before being released to the endosperm apoplasm. This study reports that the promoter of OUTER CELL LAYER3 (PSbOCL3 )f romSorghum bicolor (sorghum) directs gene expression to chalazal cells where the apoplasmic barrier is thought to form. The aims were to elucidate PSbOCL3 expression patterns in sorghum and relate them to processes of nutrient pathway development in kernels and to recognized functions of the homeodomain-leucine zipper (HD-Zip) IV transcription factor family to which the promoter belongs. †Methods PSbOCL3 was cloned and transformed into sorghum as a promoter‐GUS (b-glucuronidase) construct. Plant tissues from control and transformed plants were then stained for GUS, and kernels were cleared and characterized using differential interference contrast microscopy. †KeyResults A symplasmic disconnect between the chalaza and nucellus during seed fill is inferred by the combination of two phenomena: differentiation of a distinct nucellar epidermis adjacent to the chalaza, and lysis of GUSstained chalazal cells immediately proximal to the nucellar epidermis. Compression of the GUS-stained chalazal cells during kernel maturation produced the kernel abscission zone (closing layer). †Conclusions The results suggest that the HD-Zip IV transcription factor SbOCL3 regulates kernel nutrition and abscission. The latter is consistent with evidence that members of this transcription factor group regulate silique abscission and dehiscence in Arabidopsis thaliana. Collectively, the findings suggest that processes of floral organ abscission are conserved among angiosperms and may in some respects differ from processes of leaf abscission.

Apomixis is a mode of asexual reproduction through seeds. The apomictic process bypasses both meiosis and egg cell fertilization, producing offspring that are exact genetic replicas of the mother plant. Cenchrus ciliaris (buffelgrass) is a valuable forage grass, which reproduces through aposporous apomeiosis. We identified one sexual plant in this obligate apomictic population. The objective of this work was to identify molecular markers linked to apospory trait through bulked segregant analysis. Facultative apomictic F1 plants obtained from open pollinated sexual plant were selfed to derive F2 population. Individual F2 plants were classified through embryo sac analysis as sexual, facultative apomictic or obligate apomictic. Two bulks namely, obligate apomictic and sexual were prepared by pooling DNA of individual identified plants and used in PCR analysis. Out of 208 primers tested, 20 were found to be polymorphic. A 600 bp RAPD amplicon specific to apomictic bulk, obtained with OPF-08 primer was cloned and sequenced. Based on the sequence information, a pair of SCAR (sequence characterized amplified region) primers was designed which generated a 600 bp amplicon (named OPF08-600) only in the apomictic plants. Southern hybridization of genomic DNA of apomictic and sexual plants using the OPF08-600 fragment as probe indicated the presence of two copies of the SCAR sequence in only the apomictic plants. Gel elution of restricted DNA fragments and PCR analysis confirmed the presence of duplicated copies of the SCAR sequence in the apomictic plants.

An alloplasmic cytoplasmic male sterile (CMS) line of Brassica juncea was developed by repeated backcrossing of the sexual hybrid (Diplotaxis erucoides [multiplication] Brassica rapa) with B. juncea cv. 'Pusa Bold'. The CMS line is comparable to the B. juncea nuclear donor line for all morphological traits except male fertility. Southern and cleaved amplified polymorphic sequence (CAPS) analyses showed that the CMS line carries organellar genomes from the wild species that differ from the organellar genomes of B. juncea. Northern analysis with three mitochondrial gene probes revealed altered atp[alpha] transcription pattern associated with male sterility. The restorer of CMS (Moricandia arvensis) B. juncea was found to restore male fertility to the new CMS system. The mode of fertility restoration was found to be monogenic and gametophytic. The study identified CAPS markers for the psbB gene that could be useful in quick identification of the erucoides CMS line. Broad similarities and important differences observed at the molecular level between M. arvensis and D. erucoides CMS systems of B. juncea are discussed.

Though many tissue-specific promoters have been identified, few have been associated specifically with the angiospermous megasporangium (nucellus). In the present study the 2000-bp regulatory region upstream to the watermelon, Citrullus lanatus (Thunb.) Matsum & Nakai, gene WM403 (GenBank accession no. AF008925), which shows nucellus-specific expression, was cloned from watermelon gDNA and fused to the β-glucuronidase reporter gene (GUS). The resulting plasmid, WM403 Prom::GUS(+), which also contained NPTII, was transformed into Arabidopsis thaliana ecotype Co1-0. Seedlings were selected on kanamycin-containing medium, and transformants were confirmed by PCR. GUS assays of T(3) transformants revealed weak promoter activation in epidermal layers of the placenta and locule septum during premeiotic ovule development but strong activation in the nucellus, embryo sac and early embryo, from early embryo sac formation to early globular embryo formation. Expression in seeds was absent thereafter. These results indicate that the WM403 promoter may be useful in driving nucellus-specific gene expression in plants including candidate genes for important nucellus-specific traits such as apospory or adventitious embryony.

A cytoplasmic male sterile (CMS) line of Brassica juncea was derived by repeated backcrossing of the somatic hybrid (Diplotaxis catholica + B. juncea) to B. juncea. The new CMS line is comparable to euplasmic lines for almost all characters, except for flowers which bear slender, needle-like anthers with aborted pollen. Detailed Southern analysis revealed two copies of coxI gene in the CMS line. One copy, coxI-1 is similar to the coxI gene of B. juncea, whereas the second copy, coxI-2 is present in a novel rearranged region. Northern analysis with eight mitochondrial gene probes showed altered transcript pattern only for the coxI gene. Two transcripts of 2.0 and 2.4 kb, respectively, were detected in the CMS line. The novel 2.4 kb transcript was present in floral bud tissue but absent in the leaf tissue. In plants where male sterility broke down under high temperature during the later part of the growing season, the 2.4 kb coxI transcript was absent, which suggested its association with the CMS. The two coxI genes from the CMS line showed two amino acid changes in the coding region. The novel coxI gene showed unique repeats in the 5′ region suggesting recombination of mitochondrial genomes of the two species. The possible role of the duplicated coxI gene in causing male sterility is discussed.

Cenchrus ciliaris (buffel grass) is a valuable forage grass, reproducing through aposporous apomixis. Inter Simple Sequence Repeat (ISSR) markers converted to SCAR marker linked to apospory in this crop is reported. Out of twenty-five ISSR primers tested in DNA bulks from apomictic and sexual progenies, five primers detected polymorphism. The 1.1 kb amplicon derived from the primer ISSR-856 showed co-segregation with apomictic mode of reproduction. Accordingly, a pair of SCAR (Sequence Characterized Amplified Region) primers were designed which also generated a 1.1kb amplicon (Apo-856) only in the apomictic plants. Importance of such molecular markers in genetics and breeding of C. ciliaris is discussed.