MAPPING AND CHARACTERIZATION OF DWARF, SOFT GLUME AND BRITTLE CULM MUTANTS IN DIPLOID WHEAT

Abstract

Hexaploid wheat (Triticum aestivum L.) being the second most important crop of the world and the single largest traded crop with a global annual production exceeding 650 million tons from an area of 215.26 million hectare. A total of 78.57 million tons of wheat was produced in India in 2009 from an area of 27.6 million hectare. The diploid wheat Triticum monococcum L. (einkorn), one of the first crop domesticated by humans in the Fertile Crescent 10,000 - 12,000 years ago which is being still cultivated for its edible seeds in the mountainous area of Germany, Switzerland and Italy, is an ideal material for induced variability which could be characterized and transferred to cultivated wheat. The diploid nature of T. monococcum with smaller genome size of 5,700 Mb compared to 17,300 Mb of bread wheat, the existence of a very high level of polymorphism for DNA based markers, conservation of colinearity and synteny with other cereal crops, availability of a large BAC library and resistance against various wheat disease makes this species an attractive diploid model for gene discovery in wheat. The EMS induced dwarf (Rht22), soft glume (sog3) and brittle (brcl, brc2 and brc3) mutants used in the present study were isolated from diploid wheat line T. monococcum ace. pau14087. Rht22 was found to be insensitive to exogenous gibberellic acid (GA3) application. In histological studies, the cell size of the Rht22 was found to be reduced due to loss-of-function of the gene in the mutant. The spike of soft glume (sog3) mutant was more compact with lax glumes. The seeds in the mature spike could be easily threshed manually. On removal of individual spikelets from the spike, the rachis remained intact whereas in T. monococcum the spikelets separate with a wedge of rachis fragments without leaving any intact rachis. Out of three brittle mutants studied morphologically one (brcl) had early flowering and early brittleness (at 40-45 days seedling stage) while the other two (brc2 and brc3) had late flowering and late brittleness (at 65-70 days seedling stage), indicating that brittleness was development stage specific. All the brittle mutants were sensitive to lodging and had brittleness in all parts of plants including leaves, leaf sheaths, spikes, culms and roots. Biochemical studies of brittle culm mutants indicated that all the brittle mutants had less a-cellulose on secondary cell wall than T. monococcum indicating that all of them did not have proper deposition of cellulose microfibrils. Staining by Phloroglucinol-HCL, a lignin specific stain indicated that all the brittle mutants had slight increase in lignin content than the wild type and had very little cellulose deposition on secondary cell wall of sclerenchymatous cells. After treatment with sodium chlorite which removes all hemicellulose and lignin it was found that in brittle mutants all cells became deformed due to their less cellulose in cell wall whereas in wild type cell shape remained intact. Atomic Force Microscopy studies of surface of brittle and wild type indicated that the surfaces of all brittle mutants were more rough than the wild type In order to map the dwarfing, soft glume and brittle culm mutants, mapping populations were developed by crossing the dwarf (Rht22), soft glume (sog3) and brittle (brcl, brc2 and brc3) mutants with Triticum boeoticum acc.pau 5088, a non-brittle, tall and hard threshing wild diploid progenitor of T. monococcum. T. boeoticum ace. 5088 was chosen as a parent for development of mapping populations on the basis of its high level of polymorphism with T. monococcum. Based on the data on recombination frequency, the flanking markers xbarc37 and xbarcll3 mapped at distance of 1.9 cM and 10.3 cM, respectively from the brcl on chromosome 6AS, flanking markers xcfd62 and xcfd2170 were mapped at distance of 0.8 cM and 2.9 cM, respectively from the brc-2 on chromosome 3AL, whereas flanking markers xgwml35 and xwmc470 mapped at distance of 2.1cM and 3.9 cM, respectively from the brc3 on chromosome 1AL. Similarly flanking markers xwmc89 and xbarcl07 mapped at distance of 0.9 cM and 0.6 cM, respectively from the Rht22 on chromosome 4AL and markers xgwm473 and xbarc69 maped at the distance of 1.8 cM and 28.3 cM, respectively from sog3 on chromosome 7AL. For cloning candidate gene of Rht22, gene specific primers were synthesized covering the DELLA domain of gibberellic acid insensitive dwarf mutant gene Rht-Dl of hexaploid wheat. Amplification was observed in all the dwarf mutants along with T moncoccum and T boeoticum. After sequencing Rht22 amplicon two bp deletion was found in DELLA domain. PCR primers based on deletion in DELLA motif amplified Rht22 only which confirmed the two bp deletion in DELLA motif which was also confirmed by DOT MATRIX view. Sequence alignment of Rht22 shows high similarity with other DELLA domain defected dwarfing gene of hexaploid wheat, barley, mays and rice. Translated protein of Rht22 shows frame shift in amino acids. 3D of Rht22 shows high similarity with 3D of Rht-Dl. Soft glume (sog3) mutant mapped on chromosome 7A which is syntenic to 7H chromosome of barley where a naked seed mutant nud has been mapped suggesting that sog3 may be orthologous to nud of barley which shows one major ortholog on rice chromosome 6 and one minor ortholog on rice 2 chromosome. Three rice chromosomes, 2, 1 and 5 syntenic to T. monococcum 6,3 and 1 respectively with brittle mutants brcl, brc2 and brc3 Ifech had two CesA and CSL orthologs. BLASTN analysis of the six rice CesA and CSL genes identified about five EST contigs with high similarity (bit score >200) but none of them was bin mapped.