A point mutation in Phytochromobilin Synthase alters the circadian clock and photoperiodic flowering of Medicago truncatula

Plants use seasonal cues to initiate flowering at an appropriate time of year to ensure optimal reproductive success. The circadian clock integrates these daily and seasonal cues with internal cues to initiate flowering. The molecular pathways that control the sensitivity of flowering to photoperiod (daylength) are well described in the model plant Arabidopsis. However, much less is known in crop species, such as the legume family species. Here we performed a flowering time screen of a TILLING population of Medicago truncatula and found a line with late-flowering and altered light-sensing phenotypes. Using RNA-sequencing, we identified a nonsense mutation in the Phytochromobilin Synthase (MtPΦBS) gene, which encodes an enzyme that carries out the final step in the biosynthesis of the chromophore required for phytochrome (PHY) activity. The analysis of the circadian clock in the MtpΦbs mutant revealed a shorter circadian period, which was shared with the phyA mutant. The MtpΦbs and MtphyA mutants showed downregulation of FT floral regulators MtFTa1, MtFTb1/b2 and a shift in phase for morning and night core clock genes. Our findings show that PHYA is necessary to synchronize the circadian clock and integration of light signaling to promote expression of the MtFT genes to precisely time flowering.

A TILLING by sequencing approach to identify induced mutations in sunflower genes

The Targeting Induced Local Lesions in Genomes (TILLING) technology is a reverse genetic strategy broadly applicable to every kind of genome and represents an attractive tool for functional genomic and agronomic applications. It consists of chemical random mutagenesis followed by high-throughput screening of point mutations in targeted genomic regions. Although multiple methods for mutation discovery in amplicons have been described, next-generation sequencing (NGS) is the tool of choice for mutation detection because it quickly allows for the analysis of a large number of amplicons. The aim of the present work was to screen a previously generated sunflower TILLING population and identify alterations in genes involved in several important and complex physiological processes. Twenty-one candidate sunflower genes were chosen as targets for the screening. The TILLING by sequencing strategy allowed us to identify multiple mutations in selected genes and we subsequently validated 16 mutations in 11 different genes through Sanger sequencing. In addition to addressing challenges posed by outcrossing, our detection and validation of mutations in multiple regulatory loci highlights the importance of this sunflower population as a genetic resource.

Sorghum Brown Midrib19 (Bmr19) Gene Links Lignin Biosynthesis to Folate Metabolism

Genetic analysis of brown midrib sorghum (Sorghum bicolor) mutant lines assembled in our program has previously shown that the mutations fall into four allelic groups, bmr2, bmr6, bmr12 or bmr19. Causal genes for allelic groups bmr2, bmr6 and bmr12, have since been identified. In this report, we provide evidence for the nature of the bmr19 mutation. This was accomplished by introgressing each of the four bmr alleles into nine different genetic backgrounds. Polymorphisms from four resequenced bulks of sorghum introgression lines containing either mutation, relative to those of a resequenced bulk of the nine normal midrib recurrent parent lines, were used to locate their respective causal mutations. The analysis confirmed the previously reported causal mutations for bmr2 and bmr6 but failed in the case of bmr12-bulk due to a mixture of mutant alleles at the locus among members of that mutant bulk. In the bmr19-bulk, a common G → A mutation was found among all members in Sobic.001G535500. This gene encodes a putative folylpolyglutamate synthase with high homology to maize Bm4. The brown midrib phenotype co-segregated with this point mutation in two separate F2 populations. Furthermore, an additional variant allele at this locus obtained from a TILLING population also showed a brown midrib phenotype, confirming this locus as Bmr19.

Characterization of Advanced Tilling and Its Application in Cotton

Among all the fibre crops, cotton is most important economic fibre crop. It is known as white gold because it contributes in the economic, industrial and agricultural sectors. In GDP, it contributes to about 1.6% and have share 7.8% in agricultural products. Pakistan is ranked 5th in world in term of consumption and has 4th in term of total cotton production in 2015-2016. The seed cotton yield and production are stagnant for more than decade in Pakistan, biotic and abiotic stresses are considered as major reasons of this stagnancy. Mutagenesis is an important tool in crop improvement. In breeding programs, mutation is an important tool for creating the variations. Powerful reverse genetic strategies allow the detection of induced point mutation. TILLING (Target Induced Local Lesions in Genomes) is genomic approach which is used for the screening of mutant and germplasm collection for the for the allelic variant in targeted gene. This kind of research explores an advanced TILLING population for various parameters. The main advantage of TILLING is that this technique can be used for any plant species, irrespective of its genome size, ploidy level and method of propagation.

Characterisation of Cellulose Synthase Like F6 (CslF6) Mutants Shows Altered Carbon Metabolism in β-D-(1,3;1,4)-Glucan Deficient Grain in Brachypodium distachyon

Brachypodium distachyon is a small, fast growing grass species in the Pooideae subfamily that has become established as a model for other temperate cereals of agricultural significance, such as barley (Hordeum vulgare) and wheat (Triticum aestivum). The unusually high content in whole grains of β-D-(1,3;1,4)-glucan or mixed linkage glucan (MLG), considered a valuable dietary fibre due to its increased solubility in water compared with cellulose, makes B. distachyon an attractive model for these polysaccharides. The carbohydrate composition of grain in B. distachyon is interesting not only in understanding the synthesis of MLG, but more broadly in the mechanism(s) of carbon partitioning in cereal grains. Several mutants in the major MLG synthase, cellulose synthase like (CSL) F6, were identified in a screen of a TILLING population that show a loss of function in vitro. Surprisingly, loss of cslf6 synthase capacity appears to have a severe impact on survival, growth, and development in B. distachyon in contrast to equivalent mutants in barley and rice. One mutant, A656T, which showed milder growth impacts in heterozygotes shows a 21% (w/w) reduction in average grain MLG and more than doubling of starch compared with wildtype. The endosperm architecture of grains with the A656T mutation is altered, with a reduction in wall thickness and increased deposition of starch in larger granules than typical of wildtype B. distachyon. Together these changes demonstrate an alteration in the carbon storage of cslf6 mutant grains in response to reduced MLG synthase capacity and a possible cross-regulation with starch synthesis which should be a focus in future work in composition of these grains. The consequences of these findings for the use of B. distachyon as a model species for understanding MLG synthesis, and more broadly the implications for improving the nutritional value of cereal grains through alteration of soluble dietary fibre content are discussed.

Fast and Inexpensive Phenotyping and Genotyping Methods for Evaluation of Barley Mutant Population

To further develop barley breeding and genetics, more information on gene functions based on the analysis of the mutants of each gene is needed. However, the mutant resources are not as well developed as the model plants, such as Arabidopsis and rice. Although genome editing techniques have been able to generate mutants, it is not yet an effective method as it can only be used to transform a limited number of cultivars. Here, we developed a mutant population using ‘Mannenboshi’, which produces good quality grains with high yields but is susceptible to disease, to establish a Targeting Induced Local Lesions IN Genomes (TILLING) system that can isolate mutants in a high-throughput manner. To evaluate the availability of the prepared 8043 M3 lines, we investigated the frequency of mutant occurrence using a rapid, visually detectable waxy phenotype as an indicator. Four mutants were isolated and single nucleotide polymorphisms (SNPs) were identified in the Waxy gene as novel alleles. It was confirmed that the mutations could be easily detected using the mismatch endonuclease CELI, revealing that a sufficient number of mutants could be rapidly isolated from our TILLING population.

Development of a PVY Resistant Flue-Cured Tobacco Line via EMS Mutagenesis of eIF4E

Recessive resistance against potyviruses, such as Potato virus Y (PVY), relies on mutations in the eukaryotic translation initiation factor 4E (eIF4E) or one of its isoforms. The eIF4E1-S mutants of burley tobacco (Nicotiana tabacum L.) exhibit recessive resistance against PVY strains. Here, we developed a TILLING population of flue-cured tobacco (N. tabacum cv. Yunyan87) using ethyl methanesulfonate (EMS) to identify eIF4E1-S mutants. M3 plants homozygous for a nonsense mutation in exon 1 of the eIF4E1-S gene demonstrated resistance against PVYMN. These M3 plants were backcrossed to ‘Yunyan87’, and BC4F3 plants were screened using derived cleaved amplified polymorphic sequence (dCAPS) markers. BC4F3 plants showing agronomic traits comparable to the recurrent parent ‘Yunyan87’ and resistance against PVYO, PVYN, and PVYNTN strains were identified. These genotypes would provide useful germplasm for future tobacco improvement and would aid in basic research on PVY resistance in flue-cured tobacco.