INTROGRESSION OF GENES FOR HIGH GRAIN Fe AND Zn OF GROUP 2 CHROMOSOME OF Aegilops INTO WHEAT

Abstract

In the developing world, more than 3 billion peoples are affected by iron and zinc deficiency. Deficiency of Fe leads to impaired physical growth, anaemia, mental retardation, weak learning capacity and ability to do physical work. While Zn pays important role in normal growth and development, maintenance of body tissues, cognitive ability, vision, immune system and cofactor for more than 300 enzymes. Micronutrient deficiency can be alleviated by diet diversification, supplementation, fortification and biofortification. However biofortification is the most sustainable and effective approach for improving nutritional quality of crop plants. There are several approaches for biofortification of crops including agronomic fortification conventional and molecular breeding and genetic engineering. Wheat is the primary staple food for majority of global population and accounts for major proportion for daily dietary calorie intake. Acid digestion of seeds of parents and derivatives were done for mineral micronutrient analysis through atomic absorption spectrophotometer (AAS) and inductively coupled plasma mass (ICPMS). Most of the wheat cultivars are low in mineral micronutrient content. Several Aegilops species had 2-3 folds higher Fe and Zn content in comparison to elite wheat cultivars and can be utilized for biofortification of wheat. Several addition and substitution lines of 2U or 2S chromosome of Ae. kotschyi with high grain Fe and Zn content have been developed. The linkage drag with low yield and harvest index is the major bottleneck for their exploitation. Precise transfer of useful variability from wheat- Ae. kotschyi addition lines into elite wheat cultivars can easily be achieved by induced homoeologous pairing by seed and pollen irradiation, ph1b deletion and 5B deficiency. A total of 189 anchored wheat SSR microsatellite molecular markers specific for group 2 chromosomes were used for analysis of their transferability and polymorphism between genomic DNA of three bread wheat cultivars and six accessions of Aegilops species. A total of 143 markers (75.66%) mapped on group 2 chromosomes of wheat were transferable among all the selected Aegilops species. Polymorphism varied from 37-77% for 2A, 2B and 2D chromosome markers. Among the polymorphic transferable markers, there were 27 markers for chromosome 2A, 22 markers for chromosome 2B and 40 markers for chromosome 2D were polymorphic in all the selected Aegilops species. The high transferability of D genome specific marker indicates greater similarity of D genomes with S and U genome than those to the A and B genomes. A tentative consensus map of A, B, D,U and S genomes was prepared by BioMercator V3.0 These polymorphic markers were highly informative, reliable and useful for molecular characterization of the introgressed derivatives. vi All the five selected wheat-Aegilops derivatives with introgression of group 2 chromosomes from Aegilops species showed consistently high grain Fe and Zn content. Most of the derivatives (except 77-50-8-1) had non-waxy leaf sheaths, a character controlled by group 2 chromosome indicating the introgression of group 2 chromosome of Ae. kotschyi. Wheat-Aegilops derivatives 49-1-73 had also shown introgression of 1U/1S, a confirmed through HMW- glutenin subunit profiling of seeds. 46-1-15-15 with 22 bivalents had brittle rachis and red seed colour indicating disomic addition of chromosome 3k in addition to 2Sk Seeds of wheat-Aegilops addition/substitution lines for group 2 chromosomes were irradiated at 40 krad of gamma radiation. In the introgressed derivatives through seed irradiation (SRH. All the derivatives showed ~70-90% higher grain Fe and Zn indicating that the introgressed chromosomes were responsible for high grain Fe and Zn. These derivatives were further used for induced homoeologous pairing through ph1b mutants, mono 5B approach or irradiation induced transfers 3, WL711/77-36-2), the maximum concentration of grain iron was found in SRH3-62-1 (43.34±1.02 mg/kg) and zinc content 60 ±1.08 mg/kg in SRH3-115-1. GISH analysis of A-108 and A-115 showed several translocations of 2Sk in wheat genome. A-108 was waxy plant indicating translocation of long arm of 2Sk For pollen irradiation, spikes of wheat-Aegilops addition/substitution lines with introgression of group 2 chromosomes were irradiated at 2 krad of gamma radiation and crossed with elite wheat cultivar PBW343. Among the pollen irradiated (PRH. 2, PBW343/49-1-73) plants the maximum concentration of grain iron (64.47±3.17 mg/kg) and zinc (88.42±3.04 mg/kg ) was found in B-45-1.HMW glutenin subunit profiling of all the pollen irradiated hybrids were showed introgression of 1U/1S except PRH1-14, PRH1-48, PRH1-52, PRH1-97 and PRH1-1 progenies. GISH analysis of B-52 and B-56 showed several translocations of U and S chromosome. However absence of chromosome 1 introgression confirms the presence of only 2S and 2U in B-52. Waxy leaf sheaths of B-52 plants also indicates presence of long arm of 2Sk As an alternative strategy for gene transfer, 2U/S wheat-Aegilops derivatives were crossed with ph1bph1b mutant and the F, as the gene for waxiness trait has been mapped on the short arm of group 2 chromosomes. 1 plants were further backcrossed with ph1b mutant. The BC1F1 plants homozygous for ph1bph1b were screened through Ph1 locus specific markers psr574 and psr2120. The shrivelled seeds and leaf yellowing seemed to be associated with the presence of ph1b mutant. Group 2 chromosome specific SSR markers gwm265, barc349, barc11, gwm539 and gwm71 were used for monitoring introgression of group 2 chromosome vii into the BC1F1 plants homozygous for ph1b mutant. The BC1F2 plants were screened for introgression of fragment(s) of group 2 chromosome and grain iron and zinc content. The BC1F3 The mono 5B plants of T. aestivum cv. Pavon were cytologically identified and crossed with Aegilops species as the male parent and the F seeds were mostly shrivelled and plants had yellow leaves. Only a few plants (PH-34, PH-110, PH-199, PH-208, PH-301 and PH-305) having harvest index equivalent to that of PBW343 and 40-65% increase in grain iron and zinc content were selected for further propagation and analysis. There was no translocation was detected through GISH analysis, indicating transfer of small fragment beyond the GISH resolution. 1 plants were analyzed by molecular markers, psr574 and psr128, for the absence of chromosome 5B. These plants were further confirmed for homeologous pairing by cytological analysis. The F1 plants without 5B having 34 chromosomes showed high chromosome pairing up to 2V+4III+2II+1I, while the plants with 5B having 35 chromosomes, had highly reduced homeologous pairing, 6II+23I. The plants with 34 chromosomes without 5B were selected and backcrossed extensively with wheat cultivar PBW343 for the transfer of useful variability of Aegilops for micronutrient biofortification.. Fe and Zn content of mono5B BC1F2 and BC2F1 plants ranged from 43-84 mg/kg and 53-96 mg/kg, respectively. The chromosome number of BC1F2 and BC2F1 All the selected wheat-Aegilops derivatives with translocation of 2S/2U had better system for uptake, transport and translocation. However the overall nutrient content of these derivatives were less than the donor Aegilops species. The biofortification of wheat for grain Fe and Zn content could be achieved up to 40-60%, without any linkage drag. Tagging, localization and pyramiding of the introgressed genes/ QTLs for grain micronutrient content in wheat could be achieved through biofortification of wheat for grain Fe and Zn content.