CRISPR/Cas9 editing of wheat Ppd-1 gene homoeologs alters spike architecture and grain morphometric traits.

Mutations in Photoperiod-1 (Ppd-1) gene are known to modify flowering time and yield in wheat. We cloned TaPpd-1 from wheat and found high similarity among the three homoeologs of TaPpd-1. To clarify the characteristics of TaPpd-1 homoeologs in different photoperiod conditions for inflorescence architecture and yield, we used CRISPR/Cas9 system to generate Tappd-1 mutant plants by simultaneous modification of the three homoeologs of wheat Ppd-1. Tappd-1 mutant plants showed no off-target mutations. Four T0-edited lines under short-day length and three lines under long-day length conditions with the mutation frequency of 25% and 21%, respectively. These putative transgenic plants of all the lines were self-fertilized and generated T1 and T2 progenies and were evaluated by phenotypic and expression analysis. Results demonstrated that simultaneously edited TaPpd-1- A1, B1, and D1 homoeologs gene copies in T2_SDL-8-4, T2_SDL-4-5, T2_SDL-3-9, and T2_LDL-10-9 showed similar spike inflorescence, flowering time, and significantly increase in 1000-grain weight, grain area, grain width, grain length, plant height, and spikelets per spike due to mutation in both alleles of Ppd-B1 and Ppd-D1 homoeologs but only spike length was decreased in T2_SDL-8-4, T2_SDL-4-5, and T2_LDL-13-3 mutant lines due to mutation in both alleles of Ppd-A1 homoeolog under both conditions. Our results indicate that all TaPpd1 gene homoeologs influence wheat spike development by affecting both late flowering and earlier flowering but single mutant TaPpd-A1 homoeolog affect lowest as compared to the combination with double mutants of TaPpd-B1 and TaPpd-D1, TaPpd-A1 and TaPpd-B1, and TaPpd-A1 and TaPpd-D1 homoeologs for yield enhancement. Our findings further raised the idea that the relative expression of the various genomic copies of TaPpd-1 homoeologs may have an impact on the spike inflorescence architecture and grain morphometric features in wheat cultivars.

Genome-wide characterization and expression analysis of pseudo-response regulator gene family in wheat.

Pseudo-response regulator (PRR) gene family members play a significant role in plant circadian clocks, flowering time inflorescence architecture development during transition from vegetative growth phase to reproductive phase. In current study, we analyzed the expression profiling, phylogenetic relationship, and molecular characterization of PRR gene family members of common wheat by using IWGSC Ref seq v1.1 wheat genome database with a coverage rate of 90%. By using bioinformatic approach total 20 candidate gene sequences were identified and divided into six groups and four clades. It was found that mostly genes have same number of exons and introns showed similar features because they originated through duplication events during evolution processes. Although all the proteins have conserved PRR domains, but some are distinct in their sequences suggesting functional divergence. By comparative synteny analysis it was revealed that Group 1, 2, 3 and 11-D of group 4 have duplication events while group 5 and TaPRR9-B,10-D showed conservation with previously identified PRR members from rice. While expression variation of six groups from each analysis matches with each other. Five groups highly expressed in leaf, spike, and roots in pattern like leaf > spike > root at all three stages booting, heading and anthesis of spike development. This suggests that TaPRR genes play important roles in different photoperiod signaling pathways in different organs at different stages of spike development and flowering via unknown pathway. These findings will also provide comprehensive knowledge about future investigations on wheat PRR family members involved in complex network of circadian system for plant development.