TaCKX gene family, at large, is associated with thousand-grain weight and plant height in common wheat.

KEY MESSAGE: We used SMRT sequencing and explored the haplotypes of TaCKX genes, linked with thousand-grain weight and plant height, and developed the functionally validated markers, which can be used in the marker-assisted breeding program. Cytokinin oxidase/dehydrogenase (CKX) enzymes catalyze the permanent degradation of cytokinins. Identification of the TaCKX alleles associated with yield traits and the development of functional markers is the first step in using these alleles in marker-assisted breeding program. To identify the alleles, we sequenced the genome fragments, containing TaCKX genes from 48 wheat genotypes, by PacBio® sequencing. Six out of 22 TaCKX genes were found polymorphic, forming 14 distinct haplotypes. Functional markers were developed and validated for all the polymorphic TaCKX genes. Four specific haplotypes, i.e., TaCKX2A_2, TaCKX4A_2, TaCKX5A_3, and TaCKX9A_2, were found significantly associated with high thousand-grain weight (TGW) and short plant height (PH) in Chinese wheat micro-core collection (MCC) and GWAS open population (GWAS-OP), whereas TaCKX1B_2 in GWAS-OP and TaCKX11A_3 in MCC were significantly associated with high TGW and short PH. The mean values of TGW and PH for cumulative favorable haplotypes from chromosome 3A, i.e., TaCKX2A_2, TaCKX4A_2, and TaCKX5A_3, were significantly higher as compared to the cumulative unfavored haplotypes, and the change was additive in manner. Frequency distribution analysis revealed that since the 1960s, the frequency of the favorable haplotypes and TGW has gradually increased in Chinese wheat cultivars. Expression profiling in the seed tissue excised at 2, 4, 6, and 8 days after anthesis depicted that the favorable haplotypes are significantly less expressive as compared to the unfavored haplotypes. We conclude that the functional markers developed in this study can be used to select the favorable haplotypes of TaCKX genes in wheat marker-assisted breeding programs.

Genome-wide identification and expression analysis of new cytokinin metabolic genes in bread wheat (Triticum aestivum L.).

Cytokinins (CKs) are involved in determining the final grain yield in wheat. Multiple gene families are responsible for the controlled production of CKs in plants, including isopentenyl transferases for de novo synthesis, zeatin O-glucosyltransferases for reversible inactivation, ß-glucosidases for reactivation, and CK oxidases/dehydrogenases for permanent degradation. Identifying and characterizing the genes of these families is an important step in furthering our understanding of CK metabolism. Using bioinformatics tools, we identified four new TaIPT, four new TaZOG, and 25 new TaGLU genes in common wheat. All of the genes harbored the characteristic conserved domains of their respective gene families. We renamed TaCKX genes on the basis of their true orthologs in rice and maize to remove inconsistencies in the nomenclature. Phylogenetic analysis revealed the early divergence of monocots from dicots, and the gene duplication event after speciation was obvious. Abscisic acid-, auxin-, salicylic acid-, sulfur-, drought- and light-responsive cis-regulatory elements were common to most of the genes under investigation. Expression profiling of CK metabolic gene families was carried out at the seedlings stage in AA genome donor of common wheat. Exogenous application of phytohormones (6-benzylaminopurine, salicylic acid, indole-3-acetic acid, gibberellic acid, and abscisic acid) for 3 h significantly upregulated the transcript levels of all four gene families, suggesting that plants tend to maintain CK stability. A 6-benzylaminopurine-specific maximum fold-change was observed for TuCKX1 and TuCKX3 in root and shoot tissues, respectively; however, the highest expression level was observed in the TuGLU gene family, indicating that the reactivation of the dormant CK isoform is the quickest way to counter external stress. The identification of new CK metabolic genes provides the foundation for their in-depth functional characterization and for elucidating their association with grain yield.

A wheat dominant dwarfing line with Rht12, which reduces stem cell length and affects gibberellic acid synthesis, is a 5AL terminal deletion line.

Dwarfing and semi-dwarfing are important agronomic traits that have great potential for the improvement of wheat yields. Rht12, a dominant gibberellic acid (GA)-responsive dwarfing gene from the gamma-ray-induced wheat mutant Karcagi 522M7K, is located in the long arm of chromosome 5A, which is closely linked with the locus Xwmc410. Rht12 is likely an ideal gene for GA biosynthesis and deactivation research in common wheat. However, information on the Rht12 locus and sequence is lacking. In this study, Rht12 significantly shortened stem cell length and decreased GA biosynthetic components. Using bulked segregant RNA-Seq, wheat 660k single nucleotide polymorphism chip detection, and newly developed simple sequence repeat markers, Rht12 was mapped to a 11.21-Mb region at the terminal end of chromosome 5AL, and was found to be closely linked with the Xw5ac207SSR marker with a 10.73-Mb fragment deletion in all of the homologous dwarfing plants. Transcriptome analyses of the remaining 483-kb region showed significantly higher expression of the TraesCS5A01G543100 gene encoding the GA metabolic enzyme GA 2-ß-dioxygenase in dwarfing plants than in high stalk plants, suggesting that Rht12 reduces plant height by activating TaGA2ox-A14. Taken together, our findings will promote cloning and functional studies of Rht12 in common wheat.