INTROGRESSION AND MOLECULAR MAPPING OF HIGH Fe AND Zn CONTENT FROM AEGILOPS INTO WHEAT

Abstract

Biofortification through genetic manipulations is the best approach for improving micronutrient content of the staple food crops for alleviating the micronutrient deficiency of Fe and Zn affecting more than 2 billion people worldwide. Identification of sources with high grain Fe and Zn content in wheat germplasm and understanding its genetics are the pre-requisites for their manipulation. Triticum and seven Aegilops species with nonprogenitor S, U and M genomes along with 14 semi-dwarf bread and durum wheat cultivars, grown over two years at IIT Roorkee, were analyzed for iron and zinc content. The wheat and durum cultivars had very low content and limited variability for iron and zinc content. The Aegilops species showed up to 2-3 fold higher grain iron and zinc content than the cultivars. An Interspecific hybridization was made between a wheat line and an Aegilops kotschyi accession 3790 selected for high grain iron and zinc content. Wheat xAe. kotschyi F, hybrid with low chromosome pairing was highly male and female sterile. This was extensively back crossed with wheat cultivars to get some seed set. Flag leaf analysis of sterile F, hybrid showed intermediate content of iron and zinc between their parents; suggesting superiority of Aegilops kotschyi for better uptake and translocation for the micronutrients. The BC, F, and BC2F, plants were allowed to self and plants with high grain iron and zinc content were selected among subsequent generations. Grain iron and zinc content ofthe selected derivatives showed 60% to 140% increased Fe and Zn content as compared to the recipient wheat cultivars. On the basis of morphological and cytological analysis 13 plants with high grain iron and zinc content were selected. Selected plants were finally subjected to cytological as well as molecular characterization. Application of anchored wheat SSR markers indicated alien introgression of group 2and group 7chromosomes of Ae. kotschyi in the high grain iron and zinc containing derivatives suggesting that the genes controlling high grain micronutrient content in the Ae. kotschyi accession are on the group 2 and group 7 chromosomes. Four different interspecific hybrids involving three accessions of Aegilops longissima Schweinf. &Muschl. with high grain iron and zinc content and three Triticum turgidum L. subsp. durum (Desf.) Husn. cultivars with low micronutrient content were made for durum wheat biofortification and were investigated for chromosome pairing, fertility, putative amphiploidy and micronutrient content. The chromosome pairing in the 21 chromosome F, hybrids (ABS1) varied from 0-6 rod bivalents and occasionally one trivalent. All the Fi hybrids however, unexpectedly showed partial but variable fertility. The detailed meiotic investigation indicated the simultaneous occurrence of two types of aberrant meiotic divisions viz., first division restitution (FDR) and single division meiosis (SDM) leading to dyads and unreduced gamete formation and fertility. The F2 seeds being putative amphiploids (AABBS'S1) had nearly the doubled chromosome number (42) of the F| hybrids, regular meiosis and fertility. The F, hybrids and putative amphiploids were intermediate between the two parents for different morphological traits. The putative amphiploids with bold seed size had higher grain ash and ash iron and ash zinc content as compared to durum wheat cultivars, suggesting that Ae. longissima also possesses better genetic system(s) for uptake and seed sequestration of iron and zinc which could be transferred to elite durum and bread wheat cultivars and exploited. The amphiploids can be used to transfer useful variability and development of alien addition and substitution lines in wheat background. Wild Triticum and Aegilops species including Triticum boeoticum (AmAm) have higher grain Fe and Zn content compared to the bread and durum wheat cultivars. A Triticum boeoticum accession pau5088 had relatively higher grain Fe and Zn content. A recombinant inbred line (RIL) population involving the accession with T. monococcum (pau14087) was evaluated for grain Fe and Zn content at two locations over two years. The grain Fe and Zn concentrations in the RIL population ranged from 13.1 to 70.9mg/kg and 16.6 to 69.0mg/kg, respectively. A linkage map with 179 molecular markers available for the RIL population was used for mapping QTL for accumulation of grain Fe and Zn. The QTL mapping led to the identification of two QTL for accumulation of grain Fe on chromosomes 2A and 7A and one QTL for accumulation of grain Zn on chromosome 7A. The QTL for accumulation of grain Fe were designated as QFe.pau-2A and QFe.pau-7A and these mapped in marker interval Xwmc382-Xbarcl24 and Xqwm473-Xbarc29, respectively, each explaining 12.6%> and 11.7% of the total phenotypic variation. The QTL for accumulation of grain Zn, which mapped in the marker interval Xcfd31-Xcfa2049, designated as QZn.pau-7A and accounted 18.8% of the total phenotypic variation. The chromosomal locations of the QTLs have been validated from the introgression of group 2 and group 7 chromosomes of Ae. kotschyi in the high grain iron and zinc derivatives. Seed samples of 63 landraces of wheat were collected from farmers' fields of hilly areas of Himalaya in Uttarakhand were analyzed for morphological trait, grain iron and zinc content, hardness index, HMW subunit and diversity for SSR markers . Genetic diversity in among 78 genotypes (cultivars and landraces of wheat) was studied using morphological traits, micro-satellite markers, micronutrient content, grain texture and SDS-PAGE of HMW-GS. The dendrograms based on molecular markers and morphological data clearly separated landraces of wheat from cultivars with few exceptions. The landraces had higher diversity for HMW-glutenin subunits coded by Glu-Bl, with distinct subunit combinations 6+ 8, 7+9, 13 + 16, than within the wheat cultivars analyzed. Most ofthe landraces are clearly distinct from the indigenous and modern wheat cultivars released in India in the 20th century. Useful variation was found for high grain iron content and grain hardness. The landraces with resistance to yellow rust and powdery mildew, and higher grain iron content and distinct HMW-GS subunits can be exploited as such or used in appropriate breeding programs. Some of the landraces with very soft grains can be used for biscuit making. It will be desirable to conserve and protect the landraces as geographical indications of Uttarakhand. Development of addition, substitution and translocation lines ofAe. kotschyi and Ae. longissima in wheat background will be of great help in wheat biofortification. The precise transfer of Ae. kotschyi and Ae. longissima grain Fe and Zn content and their marker assisted pyramiding in elite wheat cultivars can easily double the micronutrient content over the existing levels in wheat cultivars. Fine mapping and cloning of the putative QTL for high Fe and Zn content will lead to thorough understanding of the pathways for their uptake, translocation and sequestration in grains.