Frost survival and gene expression in timothy (Phleum pratense L.) cultivars as affected by age and selection in diverse field environments.

The sustainable production of perennial grasses in Northern Norway is at risk due to the ongoing climate change. The predicted increase in temperatures and variable weather patterns are further expected to create challenges for winter survival of timothy (Phleum pratense L.). Knowledge about the molecular mechanisms underlying freezing tolerance is crucial for developing robust cultivars. The current study is aimed at identifying genes involved in freezing stress response of timothy and studying gene expression differentiation due to field selection in contrasting environments using RNAseq. Four timothy cultivars were field tested for three years in Tromsø and Vesterålen, in Northern Norway. The surviving material from the field tests, along with plants raised from the original seed lots, were subjected to freezing tests. LT50 values varied across cultivars and materials. Many genes coding for transcription factors and proteins known to play an important role in freezing tolerance, like dehydrins, c-repeat binding factors, and late embryogenesis abundant proteins were upregulated with decreasing temperatures. Moreover, genes associated with glycolysis/gluconeogenesis, TCA cycle, glutathione metabolism, proteasome pathways and genes encoding autophagy-related proteins, plasma membrane-associated proteins, sugar and amino acid transporters had elevated expression in field survivors compared to plants raised from the original material. The lower freezing stress tolerance of field survivors despite the elevated expression of several stress-responsive genes might be due to a combination of selection in the field and the age effect. Furthermore, differences in freezing stress response between northern and southern adapted cultivars and surviving material from two field trial locations are discussed.

Limited genetic diversity found among genotypes of the Entada landrace (Ensete ventricosum, (Welw.) Chessman) from Ethiopia.

The Entada landrace of enset (Ensete ventricosum (Welw.) Chessman) is probably the most unique indigenous crop in Ethiopia, being maintained and utilized by the Ari people in the South of Ethiopia. Here we describe genetic diversity, selection signatures and relationship of Entada with cultivated and wild enset using 117 Entada genotypes collected from three Entada growing regions in Ethiopia (Sidama, South and North Ari). A total number of 1,617 high-quality SNP markers, obtained from ddRAD-sequences, were used for the diversity studies. Phylogenetic analysis detected a clear distinction between cultivated enset, Entada and wild enset with Entada forming a completely separated clade. However, extremely short branch lengths among the Entada genotypes indicate very little molecular evolution in the Entada lineages. Observed and expected heterozygosities were high, 0.73 and 0.50, respectively. Overall, our results strongly indicate that the Entada genotypes we have studied originated from one or a few clonal lineages that have been propagated and spread among farmers as clones. Prolonged clonal propagation of heterozygous genotypes from a single or few founding lineages has led to populations with very little or no diversity between genotypes, and high heterozygosity within genotypes. Signatures of directional selection were identified at eight loci based on an FST outlier analysis. Four candidate genes detected are involved in axillary shoot growth and might be involved in controlling natural sucker formation in Entada.

Improving abiotic stress tolerance of forage grasses - prospects of using genome editing.

Due to an increase in the consumption of food, feed, and fuel and to meet global food security needs for the rapidly growing human population, there is a necessity to obtain high-yielding crops that can adapt to future climate changes. Currently, the main feed source used for ruminant livestock production is forage grasses. In temperate climate zones, perennial grasses grown for feed are widely distributed and tend to suffer under unfavorable environmental conditions. Genome editing has been shown to be an effective tool for the development of abiotic stress-resistant plants. The highly versatile CRISPR-Cas system enables increasingly complex modifications in genomes while maintaining precision and low off-target frequency mutations. In this review, we provide an overview of forage grass species that have been subjected to genome editing. We offer a perspective view on the generation of plants resilient to abiotic stresses. Due to the broad factors contributing to these stresses the review focuses on drought, salt, heat, and cold stresses. The application of new genomic techniques (e.g., CRISPR-Cas) allows addressing several challenges caused by climate change and abiotic stresses for developing forage grass cultivars with improved adaptation to the future climatic conditions. Genome editing will contribute towards developing safe and sustainable food systems.

Candidacy of a chitin-inducible gibberellin-responsive gene for a major locus affecting plant height in rice that is closely linked to Green Revolution gene sd1.

Appropriate plant height is crucial for lodging resistance to improve the rice crop yield. The application of semi-dwarf 1 led to the green revolution in the 1960s, by predominantly increasing the rice yield. However, the frequent use of single sd1 gene sources may cause genetic vulnerability to pests and diseases. Identifying useful novel semi-dwarf genes is important for the genetic manipulation of plant architecture in practical rice breeding. In this study, introgression lines derived from two parents contrasting in plant height, Zhenshan 97 and Pokkali were employed to locate a gene with a large effect on plant height by the bulk segregant analysis method. A major gene, ph1, was mapped to a region closely linked to sd1 on chromosome 1; the additive effects of ph1 were more than 50 cm on the plant height and 2 days on the heading date in a BC(4)F(2) population and its progeny. ph1 was then fine mapped to BAC AP003227. Gene annotation indicated that LOC_OS01g65990 encoding a chitin-inducible gibberellin-responsive protein (CIGR), which belongs to the GRAS family, might be the right candidate gene of ph1. Co-segregation analysis of the candidate gene-derived marker finally confirmed its identity as the candidate gene. A higher expression level of the CIGR was detected in all the tested tissues in tall plants compared to those of short plants, especially in the young leaf sheath containing elongating tissues, which indicated its importance role in regulating plant height. ph1 showed a tremendous genetic effect on plant height, which is distinct from sd1 and could be a new resource for breeding semi-dwarf varieties.

Mapping and validation of quantitative trait loci for spikelets per panicle and 1,000-grain weight in rice (Oryza sativa L.).

This study identified four and five quantitative trait loci (QTLs) for 1,000-grain weight (TGW) and spikelets per panicle (SPP), respectively, using rice recombinant inbred lines. QTLs for the two traits (SPP3a and TGW3a, TGW3b and SPP3b) were simultaneously identified in the two intervals between RM3400 and RM3646 and RM3436 and RM5995 on chromosome 3. To validate QTLs in the interval between RM3436 and RM5995, a BC(3)F(2) population was obtained, in which TGW3b and SPP3b were simultaneously mapped to a 2.6-cM interval between RM15885 and W3D16. TGW3b explained 50.4% of the phenotypic variance with an additive effect of 1.81 g. SPP3b explained 29.1% of the phenotypic variance with an additive effect of 11.89 spikelets. The interval had no effect on grain yield because it increased SPP but decreased TGW and vice versa. Grain shape was strongly associated with TGW and was used for QTL analysis in the BC(3)F(2) population. Grain length, grain width, and grain thickness were also largely controlled by TGW3b. At present, it is not clear whether one pleiotropic QTL or two linked QTLs were located in the interval. However, the conclusion could be made ultimately by isolation of TGW3b. The strategy for TGW3b isolation is discussed.

Genetic dissection of rice grain shape using a recombinant inbred line population derived from two contrasting parents and fine mapping a pleiotropic quantitative trait locus qGL7.

BACKGROUND: The three-dimensional shape of grain, measured as grain length, width, and thickness (GL, GW, and GT), is one of the most important components of grain appearance in rice. Determining the genetic basis of variations in grain shape could facilitate efficient improvements in grain appearance. In this study, an F7:8 recombinant inbred line population (RIL) derived from a cross between indica and japonica cultivars (Nanyangzhan and Chuan7) contrasting in grain size was used for quantitative trait locus (QTL) mapping. A genetic linkage map was constructed with 164 simple sequence repeat (SSR) markers. The major aim of this study was to detect a QTL for grain shape and to fine map a minor QTL, qGL7. RESULTS: Four QTLs for GL were detected on chromosomes 3 and 7, and 10 QTLs for GW and 9 QTLs for GT were identified on chromosomes 2, 3, 5, 7, 9 and 10, respectively. A total of 28 QTLs were identified, of which several are reported for the first time; four major QTLs and six minor QTLs for grain shape were also commonly detected in both years. The minor QTL, qGL7, exhibited pleiotropic effects on GL, GW, GT, 1000-grain weight (TGW), and spikelets per panicle (SPP) and was further validated in a near isogenic F2 population (NIL-F2). Finally, qGL7 was narrowed down to an interval between InDel marker RID711 and SSR marker RM6389, covering a 258-kb region in the Nipponbare genome, and cosegregated with InDel markers RID710 and RID76. CONCLUSION: Materials with very different phenotypes were used to develop mapping populations to detect QTLs because of their complex genetic background. Progeny tests proved that the minor QTL, qGL7, could display a single mendelian characteristic. Therefore, we suggested that minor QTLs for traits with high heritability could be isolated using a map-based cloning strategy in a large NIL-F2 population. In addition, combinations of different QTLs produced diverse grain shapes, which provide the ability to breed more varieties of rice to satisfy consumer preferences.

ForageGrassBase: molecular resource for the forage grass meadow fescue (Festuca pratensis Huds.).

Meadow fescue (Festuca pratensis Huds.) is one of the most important forage grasses in temperate regions. It is a diploid (2n = 14) outbreeding species that belongs to the genus Festuca. Together with Lolium perenne, they are the most important genera of forage grasses. Meadow fescue has very high quality of yield with good winter survival and persistency. However, extensive genomic resources for meadow fescue have not become available so far. To address this lack of comprehensive publicly available datasets, we have developed functionally annotated draft genome sequences of two meadow fescue genotypes, 'HF7/2' and 'B14/16', and constructed the platform ForageGrassBase, available at http://foragegrass.org/, for data visualization, download and querying. This is the first open-access platform that provides extensive genomic resources related to this forage grass species. The current database provides the most up-to-date draft genome sequence along with structural and functional annotations for genes that can be accessed using Genome Browser (GBrowse), along with comparative genomic alignments to Arabidopsis, L. perenne, barley, rice, Brachypodium and maize genomes. We have integrated homologous search tool BLAST also for the users to analyze their data. Combined, GBrowse, BLAST and downloadable data gives a user-friendly access to meadow fescue genomic resources. To our knowledge, ForageGrassBase is the first genome database dedicated to forage grasses. The current forage grass database provides valuable resources for a range of research fields related to meadow fescue and other forage crop species, as well as for plant research communities in general. The genome database can be accessed at http://foragegrass.org.

De novo and reference transcriptome assembly of transcripts expressed during flowering provide insight into seed setting in tetraploid red clover.

Red clover (Trifolium pratense L.) is one of the most important legume forage species in temperate livestock agriculture. Tetraploid red clover cultivars are generally producing less seed than diploid cultivars. Improving the seed setting potential of tetraploid cultivars is necessary to utilize the high forage quality and environmentally sustainable nitrogen fixation ability of red clover. In the current study, our aim was to identify candidate genes involved in seed setting. Two genotypes, 'Tripo' with weak seed setting and 'Lasang' with strong seed setting were selected for transcriptome analysis. De novo and reference based analyses of transcriptome assemblies were conducted to study the global transcriptome changes from early to late developmental stages of flower development of the two contrasting red clover genotypes. Transcript profiles, gene ontology enrichment and KEGG pathway analysis indicate that genes related to flower development, pollen pistil interactions, photosynthesis and embryo development are differentially expressed between these two genotypes. A significant number of genes related to pollination were overrepresented in 'Lasang', which might be a reason for its good seed setting ability. The candidate genes detected in this study might be used to develop molecular tools for breeding tetraploid red clover varieties with improved seed yield potentials.

Freezing tolerance revisited-effects of variable temperatures on gene regulation in temperate grasses and legumes.

Climate change creates new patterns of seasonal climate variation with higher temperatures, longer growth seasons and more variable winter climates. This is challenging the winter survival of perennial herbaceous plants. In this review, we focus on the effects of variable temperatures during autumn/winter/spring, and its interactions with light, on the development and maintenance of freezing tolerance. Cold temperatures induce changes at several organizational levels in the plant (cold acclimation), leading to the development of freezing tolerance, which can be reduced/lost during warm spells (deacclimation) in winters, and attained again during cold spells (reacclimation). We summarize how temperature interacts with components of the light regime (photoperiod, PSII excitation pressure, irradiance, and light quality) in determining changes in the transcriptome, proteome and metabolome.

Global transcriptome changes in perennial ryegrass during early infection by pink snow mould.

Lack of resistance to pink snow mould (Microdochium nivale) is a major constraint for adaptation of perennial ryegrass (Lolium perenne L.) to continental regions with long-lasting snow cover at higher latitudes. Almost all investigations of genetic variation in resistance have been performed using cold acclimated plants. However, there may be variation in resistance mechanisms that are functioning independently of cold acclimation. In this study our aim was to identify candidate genes involved in such resistance mechanisms. We first characterized variation in resistance to M. nivale among non-acclimated genotypes from the Norwegian cultivar 'Fagerlin' based on relative regrowth and fungal quantification by real-time qPCR. One resistant and one susceptible genotype were selected for transcriptome analysis using paired-end sequencing by Illumina Hiseq 2000. Transcriptome profiles, GO enrichment and KEGG pathway analysis indicate that defense response related genes are differentially expressed between the resistant and the susceptible genotype. A significant up-regulation of defense related genes, as well as genes involved in cell wall cellulose metabolic processes and aryl-alcohol dehydrogenase (NADP+) activity, was observed in the resistant genotype. The candidate genes identified in this study might be potential molecular marker resources for breeding perennial ryegrass cultivars with improved resistance to pink snow mould.

Population Structure, Genetic Variation, and Linkage Disequilibrium in Perennial Ryegrass Populations Divergently Selected for Freezing Tolerance.

Low temperature is one of the abiotic stresses seriously affecting the growth of perennial ryegrass (Lolium perenne L.), and freezing tolerance is a complex trait of major agronomical importance in northern and central Europe. Understanding the genetic control of freezing tolerance would aid in the development of cultivars of perennial ryegrass with improved adaptation to frost. The plant material investigated in this study was an experimental synthetic population derived from pair-crosses among five European perennial ryegrass genotypes, representing adaptations to a range of climatic conditions across Europe. A total number of 80 individuals (24 of High frost [HF]; 29 of Low frost [LF], and 27 of Unselected [US]) from the second generation of the two divergently selected populations and an unselected (US) control population were genotyped using 278 genome-wide SNPs derived from perennial ryegrass transcriptome sequences. Our studies investigated the genetic diversity among the three experimental populations by analysis of molecular variance and population structure, and determined that the HF and LF populations are very divergent after selection for freezing tolerance, whereas the HF and US populations are more similar. Linkage disequilibrium (LD) decay varied across the seven chromosomes and the conspicuous pattern of LD between the HF and LF population confirmed their divergence in freezing tolerance. Furthermore, two F st outlier methods; finite island model (fdist) by LOSITAN and hierarchical structure model using ARLEQUIN, both detected six loci under directional selection. These outlier loci are most probably linked to genes involved in freezing tolerance, cold adaptation, and abiotic stress. These six candidate loci under directional selection for freezing tolerance might be potential marker resources for breeding perennial ryegrass cultivars with improved freezing tolerance.

Expression patterns of photoperiod and temperature regulated heading date genes in Oryza sativa.

In plants, flowering is a major biological phenomenon, which is regulated by an array of interactions occurring between biotic and abiotic factors. In our study, we have compared the expression profiles of flowering genes involved in the flowering pathway, which are influenced by conditions like photoperiod and temperature from seedling to heading developmental stages in two Oryza sativa indica varieties, viz., Zhenshan 97 and Minghui 63 using a expression network approach. Using the network expression approach, we found 17 co-expressed genes having the same expression profile pattern as three key photoperiod flowering genes Hd1, Ehd1 and Hd3a. We also demonstrated that these three co-expressed genes have a similar simulation pattern as temperature flowering genes. Based on our observations, we hypothesize that photoperiod and temperature regulate flowering pathways independently. The present study provides a basis for understanding the network of co-expressed genes involved in flowering pathway and presents a way to demonstrate the behavior of specific gene sets in specific cultivars.

Quantitative trait loci for rice yield-related traits using recombinant inbred lines derived from two diverse cultivars.

The thousand-grain weight and spikelets per panicle directly contribute to rice yield. Heading date and plant height also greatly influence the yield. Dissection of genetic bases of yield-related traits would provide tools for yield improvement. In this study, quantitative trait loci (QTL) mapping for spikelets per panicle, thousand-grain weight, heading date and plant height was performed using recombinant inbred lines derived from a cross between two diverse cultivars, Nanyangzhan and Chuan7. In total, 20 QTLs were identified for four traits. They were located to 11 chromosomes except on chromosome 4. Seven and five QTLs were detected for thousand-grain weight and spikelets per panicle, respectively. Four QTLs were identified for both heading date and plant height. About half the QTLs were commonly detected in both years, 2006 and 2007. Six QTLs are being reported for the first time. Two QTL clusters were identified in regions flanked by RM22065 and RM5720 on chromosome 7 and by RM502 and RM264 on chromosome 8, respectively. The parent, Nanyangzhan with heavy thousand-grain weight, carried alleles with increased effects on all seven thousand-grain weight QTL, which explained why there was no transgressive segregation for thousand-grain weight in the population. In contrast, Chuan7 with more spikelets per panicle carried positive alleles at all five spikelets per panicle QTL except qspp5. Further work on distinction between pleiotropic QTL and linked QTL is needed in two yield-related QTL clusters.