Fine-Mapping and Comparative Genomic Analysis Reveal the Gene Composition at the S and Z Self-incompatibility Loci in Grasses.

Self-incompatibility (SI) is a genetic mechanism of hermaphroditic plants to prevent inbreeding after self-pollination. Allogamous Poaceae species exhibit a unique gametophytic SI system controlled by two multi-allelic and independent loci, S and Z. Despite intense research efforts in the last decades, the genes that determine the initial recognition mechanism are yet to be identified. Here, we report the fine-mapping of the Z-locus in perennial ryegrass (Lolium perenne L.) and provide evidence that the pollen and stigma components are determined by two genes encoding DUF247 domain proteins (ZDUF247-I and ZDUF247-II) and the gene sZ, respectively. The pollen and stigma determinants are located side-by-side and were genetically linked in 10,245 individuals of two independent mapping populations segregating for Z. Moreover, they exhibited high allelic diversity as well as tissue-specific gene expression, matching the expected characteristics of SI determinants known from other systems. Revisiting the S-locus using the latest high-quality whole-genome assemblies revealed a similar gene composition and structure as found for Z, supporting the hypothesis of a duplicated origin of the two-locus SI system of grasses. Ultimately, comparative genomic analyses across a wide range of self-compatible and self-incompatible Poaceae species revealed that the absence of a functional copy of at least one of the six putative SI determinants is accompanied by a self-compatible phenotype. Our study provides new insights into the origin and evolution of the unique gametophytic SI system in one of the largest and economically most important plant families.

Direct evidence for crossover and chromatid interference in meiosis of two plant hybrids (Lolium multiflorum×Festuca pratensis and Allium cepa×A. roylei).

Crossing over, in addition to its strictly genetic role, also performs a critical mechanical function, by bonding homologues in meiosis. Hence, it is responsible for an orderly reduction of the chromosome number. As such, it is strictly controlled in frequency and distribution. The well-known crossover control is positive crossover interference which reduces the probability of a crossover in the vicinity of an already formed crossover. A poorly studied aspect of the control is chromatid interference. Such analyses are possible in very few organisms as they require observation of all four products of a single meiosis. Here, we provide direct evidence of chromatid interference. Using in situ probing in two interspecific plant hybrids (Lolium multiflorum×Festuca pratensis and Allium cepa×A. roylei) during anaphase I, we demonstrate that the involvement of four chromatids in double crossovers is significantly more frequent than expected (64% versus 25%). We also provide a physical measure of the crossover interference distance, covering ~30-40% of the relative chromosome arm length, and show that the centromere acts as a barrier for crossover interference. The two arms of a chromosome appear to act as independent units in the process of crossing over. Chromatid interference has to be seriously addressed in genetic mapping approaches and further studies.

B chromosomes are associated with redistribution of genetic recombination towards lower recombination chromosomal regions in perennial ryegrass.

Supernumerary 'B' chromosomes are non-essential components of the genome present in a range of plant and animal species-including many grasses. Within diploid and polyploid ryegrass and fescue species, including the forage grass perennial ryegrass (Lolium perenne L.), the presence of B chromosomes has been reported as influencing both chromosome pairing and chiasma frequencies. In this study, the effects of the presence/absence of B chromosomes on genetic recombination has been investigated through generating DArT (Diversity Arrays Technology) marker genetic maps for six perennial ryegrass diploid populations, the pollen parents of which contained either two B or zero B chromosomes. Through genetic and cytological analyses of these progeny and their parents, we have identified that, while overall cytological estimates of chiasma frequencies were significantly lower in pollen mother cells with two B chromosomes as compared with zero B chromosomes, the recombination frequencies within some marker intervals were actually increased, particularly for marker intervals in lower recombination regions of chromosomes, namely pericentromeric regions. Thus, in perennial ryegrass, the presence of two B chromosomes redistributed patterns of meiotic recombination in pollen mother cells in ways which could increase the range of allelic variation available to plant breeders.

Genetic Loci Governing Androgenic Capacity in Perennial Ryegrass (Lolium perenne L.).

Immature pollen can be induced to switch developmental pathways from gametogenesis to embryogenesis and subsequently regenerate into homozygous, diploid plants. Such androgenic production of doubled haploids is particularly useful for species where inbreeding is hampered by effective self-incompatibility systems. Therefore, increasing the generally low androgenic capacity of perennial ryegrass (Lolium perenne L.) germplasm would enable the efficient production of homozygous plant material, so that a more effective exploitation of heterosis through hybrid breeding schemes can be realized. Here, we present the results of a genome-wide association study in a heterozygous, multiparental population of perennial ryegrass (n = 391) segregating for androgenic capacity. Genotyping-by-sequencing was used to interrogate gene- dense genomic regions and revealed over 1,100 polymorphic sites. Between one and 10 quantitative trait loci (QTL) were identified for anther response, embryo and total plant production, green and albino plant production and regeneration. Most traits were under polygenic control, although a major QTL on linkage group 5 was associated with green plant regeneration. Distinct genetic factors seem to affect green and albino plant recovery. Two intriguing candidate genes, encoding chromatin binding domains of the developmental phase transition regulator, Polycomb Repressive Complex 2, were identified. Our results shed the first light on the molecular mechanisms behind perennial ryegrass microspore embryogenesis and enable marker-assisted introgression of androgenic capacity into recalcitrant germplasm of this forage crop of global significance.

Fine mapping a self-fertility locus in perennial ryegrass.

KEY MESSAGE: A self-fertility locus was fine mapped to a 1.6 cM region on linkage group 5 in a perennial ryegrass population. This locus was the main determinant of pollen self-compatibility. In grasses, self-incompatibility (SI) is characterized by a two-loci gametophytic (S and Z) mechanism acting together in the recognition and inhibition of self-pollen. Mutations affecting the expression of SI have been reported in a few grass species. In perennial ryegrass (Lolium perenne L.), a mutation independent from S and Z, and mapping on linkage group 5 (LG 5), was previously reported to produce self-fertile plants. Here, we describe fine mapping of the self-fertility (SF) gene in a perennial ryegrass population and determine whether there is any effect of other genomic regions on the pollen compatibility. The phenotypic segregation of SF showed a bimodal distribution with one mean at 49% pollen compatibility and the other at 91%. Marker-trait association analysis showed that only markers on LG 5 were significantly associated with the trait. A single gene model explained 82% of the observed variability and no effects of the other regions were detected. Using segregation and linkage analysis, the SF locus was located to a 1.6 cM region on LG 5. The flanking marker sequences were aligned to rice and Brachypodium distachyon reference genomes to estimate the physical distance. We provide markers tightly linked to SF that can be used for introgression of this trait into advanced breeding germplasm. Moreover, our results represent a further step towards the identification of the SF gene in LG 5.

Comparative and Evolutionary Analysis of Grass Pollen Allergens Using Brachypodium distachyon as a Model System.

Comparative genomics have facilitated the mining of biological information from a genome sequence, through the detection of similarities and differences with genomes of closely or more distantly related species. By using such comparative approaches, knowledge can be transferred from the model to non-model organisms and insights can be gained in the structural and evolutionary patterns of specific genes. In the absence of sequenced genomes for allergenic grasses, this study was aimed at understanding the structure, organisation and expression profiles of grass pollen allergens using the genomic data from Brachypodium distachyon as it is phylogenetically related to the allergenic grasses. Combining genomic data with the anther RNA-Seq dataset revealed 24 pollen allergen genes belonging to eight allergen groups mapping on the five chromosomes in B. distachyon. High levels of anther-specific expression profiles were observed for the 24 identified putative allergen-encoding genes in Brachypodium. The genomic evidence suggests that gene encoding the group 5 allergen, the most potent trigger of hay fever and allergic asthma originated as a pollen specific orphan gene in a common grass ancestor of Brachypodium and Triticiae clades. Gene structure analysis showed that the putative allergen-encoding genes in Brachypodium either lack or contain reduced number of introns. Promoter analysis of the identified Brachypodium genes revealed the presence of specific cis-regulatory sequences likely responsible for high anther/pollen-specific expression. With the identification of putative allergen-encoding genes in Brachypodium, this study has also described some important plant gene families (e.g. expansin superfamily, EF-Hand family, profilins etc) for the first time in the model plant Brachypodium. Altogether, the present study provides new insights into structural characterization and evolution of pollen allergens and will further serve as a base for their functional characterization in related grass species.

Pollen-Mediated Movement of Herbicide Resistance Genes in Lolium rigidum.

The transfer of herbicide resistance genes by pollen is a major concern in cross-pollinated species such as annual ryegrass (Lolium rigidum). A two-year study was conducted in the greenhouse, under favorable conditions for pollination, to generate information on potential maximum cross-pollination. This maximum cross-pollination rate was 56.1%. A three-year field trial was also conducted to study the cross-pollination rates in terms of distance and orientation to an herbicide-resistant pollen source. Under field conditions, cross-pollination rates varied from 5.5% to 11.6% in plants adjacent to the pollen source and decreased with increasing distances (1.5 to 8.9% at 15 m distance and up to 4.1% at 25 m in the downwind direction). Environmental conditions influenced the cross-pollination both under greenhouse and field conditions. Data were fit to an exponential decay model to predict gene flow at increasing distances. This model predicted an average gene flow of 7.1% when the pollen donor and recipient plants were at 0 m distance from each other. Pollen-mediated gene flow declined by 50% at 16.7 m from the pollen source, yet under downwind conditions gene flow of 5.2% was predicted at 25 m, the farthest distance studied. Knowledge of cross-pollination rates will be useful for assessing the spread of herbicide resistance genes in L. rigidum and in developing appropriate strategies for its mitigation.

Variation in Alkaloid Production from Genetically Diverse Lolium Accessions Infected with Epichloë Species.

Widespread infection of Epichloë occultans in annual ryegrass in Australia suggests that infection provides its weedy host, Lolium rigidum, some ecological advantage. Initial studies determined the distribution and profiles of known Epichloë alkaloids (epoxy-janthitrems, ergovaline, lolines, lolitrem B, and peramine) in plant extracts using a combination of GC-FID and HPLC techniques utilizing a single accession of Australian L. rigidum. However, the lolines N-acetylnorloline (NANL) and N-formylloline (NFL) were the only alkaloids detected and were highly concentrated in the immature inflorescences of mature plants. Additional glasshouse studies subjected a wide range of Australian L. rigidum haplotypes and international annual Lolium accessions to a suite of analyses to determine alkaloid levels and profiles. Again, NFL and NANL were the key lolines produced, with NFL consistently predominating. Considerable variation in alkaloid production was found both within and between biotypes and accessions evaluated under identical conditions, at the same maturation stage and on the same tissue type. The pyrrolopyrazine alkaloid peramine was also present in 8 out of 17 Australian biotypes of L. rigidum and 7 out of 33 international accessions infected with Epichloë spp.; the highest peramine concentrations were observed in seed extracts from L. rigidum collected from Australia. This study represents the first report of alkaloids from a geographically diverse collection of annual ryegrass germplasm infected with Epichloë spp. when grown under identical controlled conditions.

A synteny-based draft genome sequence of the forage grass Lolium perenne.

Here we report the draft genome sequence of perennial ryegrass (Lolium perenne), an economically important forage and turf grass species that is widely cultivated in temperate regions worldwide. It is classified along with wheat, barley, oats and Brachypodium distachyon in the Pooideae sub-family of the grass family (Poaceae). Transcriptome data was used to identify 28,455 gene models, and we utilized macro-co-linearity between perennial ryegrass and barley, and synteny within the grass family, to establish a synteny-based linear gene order. The gametophytic self-incompatibility mechanism enables the pistil of a plant to reject self-pollen and therefore promote out-crossing. We have used the sequence assembly to characterize transcriptional changes in the stigma during pollination with both compatible and incompatible pollen. Characterization of the pollen transcriptome identified homologs to pollen allergens from a range of species, many of which were expressed to very high levels in mature pollen grains, and are potentially involved in the self-incompatibility mechanism. The genome sequence provides a valuable resource for future breeding efforts based on genomic prediction, and will accelerate the development of new varieties for more productive grasslands.

Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses.

Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species.

Early response mechanisms of perennial ryegrass (Lolium perenne) to phosphorus deficiency.

BACKGROUND AND AIMS: Improving phosphorus (P) nutrient efficiency in Lolium perenne (perennial ryegrass) is likely to result in considerable economic and ecological benefits. To date, research into the molecular and biochemical response of perennial ryegrass to P deficiency has been limited, particularly in relation to the early response mechanisms. This study aimed to identify molecular mechanisms activated in response to the initial stages of P deficiency. METHODS: A barley microarray was successfully used to study gene expression in perennial ryegrass and this was complemented with gas chromatography-mass spectrometry metabolic profiling to obtain an overview of the plant response to early stages of P deficiency. KEY RESULTS: After 24 h of P deficiency, internal phosphate concentrations were reduced and significant alterations were detected in the metabolome and transcriptome of two perennial ryegrass genotypes. Results indicated a replacement of phospholipids with sulfolipids and the utilization of glycolytic bypasses in response to P deficiency in perennial ryegrass. CONCLUSIONS: The transcriptome and metabolome of perennial ryegrass undergo changes in response to reductions in P supply after 24 h.

Identification of ABC transporters from Lolium perenne L. that are regulated by toxic levels of selenium.

Selenium is an essential micronutrient for animals and humans, but can be toxic at higher levels. Manipulation of Se metabolism in plants may enable plants to be tailored to enhance Se content for human and animal consumption and to decontaminate Se polluted soils. Here, we generated subtracted cDNA libraries from perennial ryegrass roots and leaves, enriched for genes which expression is enhanced under toxic levels of selenium. The libraries were sequenced using next generation sequencing technologies to characterize the pool of enriched genes. Within these subtracted libraries, there were a large number of genes involved in the calcium-calmodulin signaling network. Furthermore, in the leaf subtracted cDNA library, we identified 28 ABC transporters. Subsequent expression analysis by quantitative RT-PCR demonstrated the significant accumulation of these transcripts in the leaf tissue of perennial ryegrass under toxic levels of Se. These results suggest a role for ABC transporters in selenium movement and accumulation in perennial ryegrass.

Evolution of glyphosate resistance in a Lolium rigidum population by glyphosate selection at sublethal doses.

The majority of the documented cases of field-evolved herbicide-resistant weed biotypes established that single major genes confer glyphosate resistance. However, the contribution of minor genes endowing substantial plant survival at sublethal herbicide doses may be a potential complementary path to herbicide resistance evolution in weed populations under selection. Here, we subjected a number of susceptible individuals of Lolium rigidum to recurrent glyphosate selection to test the potential for sublethal glyphosate doses to additively select for glyphosate resistance. After 3-4 cycles of glyphosate selection in two distinct environments, the progenies of the initially susceptible population were shifted toward glyphosate resistance. The results indicate progressive enrichment of minor gene trait(s) contributing toward plant survival in the glyphosate-selected progenies. After three generations of selection, the estimated LD(50) values were doubled compared with the original population and up to 33% plant survival was obtained in the glyphosate-selected progeny at the recommended glyphosate label rate. This level of resistance probably was the maximum shift achievable with sublethal glyphosate dose selection in this small population. Cross-pollination was a crucial factor enabling the rapid rate of accumulation of minor glyphosate resistance gene trait(s) that are likely to be present at a relatively high frequency in a small susceptible population. The mechanistic basis of the moderate glyphosate resistance level selected by sublethal glyphosate doses remains unknown and warrants future research. Studying the main factors influencing the evolution of resistant weed populations is crucial for understanding, predicting and managing herbicide resistance.

Long distance pollen-mediated flow of herbicide resistance genes in Lolium rigidum.

Gene flow promotes genetic exchange among plant populations mediating evolutionary dynamics; yet, the importance of gene flow at distance via pollen movement is poorly understood. A field experiment at the landscape level was conducted with Lolium rigidum herbicide-susceptible individuals (population VLR1) placed into an otherwise Lolium-free bushland environment at increasing distances from adjacent large commercial crop fields infested with herbicide-resistant L. rigidum. Herbicide resistance was used as a marker to quantify the distance and the rate of pollen-mediated gene flow. About 21,245 seeds were produced on the isolated, susceptible mother plants of which 3,303 seedlings were tested for herbicide resistance and 664 seedlings were found to be resistant. Pollen-mediated gene flow occurred at 3,000 m (maximum tested distance). Both Mendelian and molecular analyses (sequencing and CAPS markers) confirmed the introgression of herbicide resistance genes. This is the first documented case of long-distance gene flow in L. rigidum. The results are important for future modeling simulations of herbicide resistance evolution and subsequent mobility. The adoption of integrated agronomic strategies, the control of potential receptor plants on fields' margins and conservative use of herbicides can be realistic options to minimize herbicide resistance spread.

Improved fructan accumulation in perennial ryegrass transformed with the onion fructosyltransferase genes 1-SST and 6G-FFT.

Carbohydrate limitation has been identified as a main cause of inefficient nitrogen use in ruminant animals, which feed mainly on fresh forage, hay and silage. This inefficiency results in suboptimal meat and milk productivity. One important molecular breeding strategy is to improve the nutritional value of ryegrass (Lolium perenne) by increasing the fructan content through expression of heterologous fructan biosynthetic genes. We developed perennial ryegrass lines expressing sucrose:sucrose 1-fructosyltransferase and fructan:fructan 6G-fructosyltransferase genes from onion (Allium cepa) which exhibited up to a 3-fold increased fructan content. Further, the high fructan content was stable during the growth period, whereas the fructan content in an elite variety, marketed as a high sugar variety, dropped rapidly after reaching its maximum and subsequently remained low.

Effects of interaction between pollen coat eluates and pistil at the molecular level in self-compatible and self-incompatible plants of Lolium multiflorum Lam.

Two-dimensional electrophoresis (2-DE) of soluble proteins and enzymes was performed and specific activities of 5 enzymes (esterase, pectinesterase, acid phosphatase, protease and diaphorase) were determined in stigmas of Lolium multiflorum (Italian ryegrass) treated with self or foreign pollen coat eluates (pc). Also, a low-molecular-weight fraction of the treated self-compatible (SC) and self-incompatible (SI) stigmas was analyzed by high-pressure liquid chromatography (HPLC). The treatment of stigmas with foreign pollen induced the loss of 42% of the control sample proteins in SC plants but only of 5.5% in SI plants. In contrast, the treatment of stigmas with foreign pollen induced the loss of 15% proteins in SC plants and of 29% in SI plants. Specific activities of esterase, pectinesterase and diaphorase were higher in SC than in SI stigmas. The 2-DE enzyme patterns indicated qualitative relationships between the presence of some isoforms of acid phosphatase or protease and the treatment with self or foreign pc in SC and SI stigmas. No changes were observed in HPLC profiles of the low-molecular-weight fraction from SC and SI stigmas treated or not with pc. The presented results revealed different reactions of SC and SI stigmas to the treatment with self or foreign pc. Further investigations may explain if any of the observed reactions represent specific reorientations in the style, facilitating cross- or self-pollination.

Tracing field hybridization in Ryegrass species using microsatellite and morphological markers.

We have assessed the utility of morphological and microsatellite markers for tracing field hybridization between Lolium multiflorum and Lolium perenne in cereal-enclosed gene flow plots. The presence of awns on the inflorescence of F(1) hybrids was found to be a reliable, but underscoring, indicator of L. multiflorum paternity in L. perenne derived seed as determined by inheritance of species-specific alleles at the microsatellite locus 'H01 H06' in these progeny. A positive correlation was evident in the experimental treatment between the number of pollen donor plants in a given plot and the frequency of hybrid F(1) seed harvested from pollen receptor plants in that plot. These experiments have established the utility of naturally occurring heritable markers for the measurement of gene flow rates in field Ryegrass populations, with particular significance for risk assessment modeling of potential gene flow from transgenic grass cultivars.

Identification and mode of action of self-compatibility loci in Lolium perenne L.

The two-locus gametophytic incompatibility system in perennial ryegrass (Lolium perenne L.) is not always fully effective: obligate selfing of plants sieves self-compatible pollen mutants, and self-fertility becomes fixed in subsequent generations. Self-compatibility (SC) was investigated in an F2 family. In vitro self-pollinations were analysed and recorded and plants were classified as being either partially or fully compatible. Distorted segregation ratios of markers on linkage group (LG) 5 were found, which indicate the possible presence of a gametophytic SC locus. Interval linkage analysis of pollen compatibility after selfing confirmed that this distortion was due to a locus (T) analogous to the S5 locus of rye. However, even though markers in this region were, on average, less than 1 cM apart, the minimum number of plants possessing the unfavoured allele was never less than 6% for any marker locus. We proved that this was because of the presence of another SC locus, exhibiting gametophytic selection, segregating in this population and identified by interval mapping analysis of compatibility classes of in vitro self-pollinations. This locus was located on LG1, and probably corresponds to the S locus. We show that the T locus, a relic of a multilocus system, functions through interaction with the S locus: F2 segregation of incompatibility phenotypes and linked markers demonstrated that the S/t pollen genotype combination, expected to be compatible on selfing, was sometimes incompatible. Further evidence is presented to show that this interaction must be dependent on yet another locus located on LG2. A prime candidate would be the Z incompatibility locus.

Photoperiodic regulation of flowering in perennial ryegrass involving a CONSTANS -like homolog.

Photoperiod and vernalization are the two key environmental factors of the floral induction of perennial ryegrass (Lolium perenne L.). Transition from vegetative to reproductive growth will only occur after an extended vernalization period, followed by an increase in day length and temperature. Here we report on the isolation and characterization of a L. perenne gene (LpCO ) that is homologous to CONSTANS , and which is tightly coupled to the floral inductive long day signal. Like other monocot CO-like proteins, the LpCO contains a zinc finger domain with a non-conserved B-Box2. Although the B-Box2 has been demonstrated to be essential for the function of the Arabidopsis CO (AtCO), LpCO is able to complement the Arabidopsis co-2 mutant, and ectopic expression in Arabidopsis wild type leads to early flowering. The LpCO transcript exhibits diurnal oscillations and is expressed at higher levels during long days.

Self-incompatibility in ryegrass 12. Genotyping and mapping the S and Z loci of Lolium perenne L.

Perennial ryegrass (Lolium perenne L.) is an outcrossing, wind-pollinated species exhibiting a gametophytic two-locus system of self-incompatibility (S and Z). The two incompatibility loci were genotyped in a cross between a doubled-haploid plant crossed as the female parent with a normal heterozygous plant. The S and Z loci were found to segregate in the expected 1:1 ratio and also segregated independently. The two loci were mapped to linkage groups one and two respectively, in accordance with the Triticeae consensus map. In addition, there were notable associations between the segregation of particular alleles mapping to the S locus region of linkage group 1 and those mapping to the WG889/CDO920 loci region of linkage group 3 which resulted in significant segregation distortions. No such associations were found between the Z locus and this region or any other region of the genome. The L. perenne S and Z loci showed conserved synteny with the equivalent loci in rye (Secale cereale L.).