DEVELOPMENT AND MOLECULAR ANALYSIS OF HIGH GRAIN IRON AND ZINC WHEAT-^EG/LOPS DERIVATIVES

Abstract

Biofortification of crops is the most feasible and economical approach for overcoming the global challenge of iron and zinc deficiency which currently affects over 3 billion people of the world. Wheat is the staple food crop of about one-third of the world population, but popular wheat cultivars have very low grain micronutrient content indicating the importance of exploration and utilization of its gene pools for transfer of useful variability for biofortification for grain iron and zinc. Eighty accessions of nine species of wild Triticum and Aegilops, namely Ae. peregrina (US), Ae. longissima (S1), Ae. kotschyi (US), Ae. ovata (UM), Ae. cylindrica (CD), Ae. ventricosa (DN), T. dicoccoides (AB), T. araraticum (AB), and T. boeoticum (A) were evaluated for their grain micronutrient contents. Aegilops species showed up to 2-3 folds higher grain iron and zinc contents than the bread and durum wheat cultivars. Interspecific hybridization was done between a wheat cultivar and selected accessions of Ae. peregrina (1155-1-1, 13772, 3519, 3477 and 1155-5-3). The F, hybrids with the expected chromosome number 35, had little chromosomal pairing, high male and fermale sterility and had no seed set. Therefore, extensive backcrossing was done to get some BCi seeds. The BCi plants were further backcrossed as they also did not set seeds. The resulting fertile BC2 plants were screened for high iron and zinc content. Twelve BC2F2 plants with recovered wheat background and high grain iron and zinc content were further characterized using cytology, HMW- glutenin profiles, anchored wheat microsatellite markers and GISH/ FISH. Application of SSR markers showed introgression of chromosomes of group 4 and 7 of Ae. peregrina in the selected derivatives. Further application of GISH/ FISH revealed translocation of 7U in one of the derivatives whereas in others addition of 7U and 4S and 7S was seen. Thus it may be concluded that chromosomes of group 4 and 7 of Ae. peregrina had genes/ QTL for high grain iron and zinc content. Two synthetic amphiploids of Triticum aestivum (AABBDD) landrace Chinese Spring (Ph1) with Ae. peregrina (UUS'S1) accessions 3477 and 1155-5-3 were developed through colchicine treatment of sterile Fi hybrids. The F, hybrids and amphiploid plants were intermediate between the parents for plant morphology and spike characteristics. The amphiploids of advanced generations, however, had variable frequency of univalents at meiotic metaphase-I. HMW glutenin subunits of amphiploids along with the parents showed the additive presence and expression of the parental genomes in the amphiploids. The amphiploids with bolder seeds had higher grain iron and zinc content than the wheat parents and comparable to those of theirAe. peregrina parents suggesting that Ae. peregrina possesses distinctive genetic system for the micronutrient uptake and/or translocation than the wheat cultivars. The variable chromosome number in PMCs in advanced generation of amphiploids suggests somatic chromosome elimination in the amphiploids. The amphiploids can be used for transfer of high grain iron and zinc content to wheat and development of alien addition and substitution lines. A few T. aestivum cultivars and accessions of the Aegilops species were investigated for the release of phytosiderophores in vitro under iron and zinc sufficient and deficient media and root and shoot iron and zinc conent was estimated. All the Aegilops species had significantly higher mean and range with 3-4 times higher release of phytosiderophores than the wheat cultivars under both nutrient sufficient and deficient conditions throughout the investigation. The wheat cultivars as well as Aegilops species had comparable rate of increase of phytosiderophores in iron or zinc deficient media with peak at 11th and 14th days, respectively which li leveled off rapidly among the wheat cultivars and continued to be high among Aegilops species. The absolute amount of iron and zinc expressed on the shoot and root dry weight basis after 18th days on iron and zinc deficient media showed nearly three times higher concentration in both roots and shoots of Aegilops species than that of the wheat cultivars. Aegilops species secrete higher amounts of phytosiderophores than T. aestivum cultivars under micronutrient deficient as well as sufficient media. Further more the deficiency symptoms like chlorosis started to appear later in the Aegilops species than the cultivars. Fertile introgressive derivatives of Ae. peregrina were also found to secrete 2-3 times higher amount of phytosiderophores than the wheat cultivars. The higher grain iron and zinc content in the Aegilops species reported earlier may be attributed to their diverse and efficient mechanism for phytosiderophore mediated micronutrient uptake and translocation system which could be exploited for biofortification ofwheat. Wheat-Aegilops addition lines for chromosomal identification of the genes controlling high grain iron and zinc content and also for the release of phytosiderophores. Addition lines of group 2(U/S), 4Uand 7UofAe. peregrina, IS, 2S and 5S, 6S, 7S of Ae. longissima and 2Uand 5U of Ae. umbellulata in Chinese spring background were found to harbour genes for high grain iron whereas for zinc content, mainly group 7 chromosomes was found responsible. Higher release of phytosiderophores was observed in addition lines of group 2(U/S), 4(U) and 7(S) of Ae. peregrina, 2S1 and 6S1 of Ae. longissima and 2U and 5U of Ae. umbellulata. Addition lines with high grain iron and zinc content also showed higher phytosiderophore release. Significant positive correlation (r2=0.56) between grain iron and zinc content and phytosiderophore release under iron deficient media strongly suggests that higher release of phytosiderophores may be responsible for higher grain m iron and zinc in these addition lines. These addition lines with two to three fold high grain iron and zinc content could be used for precise introgression of genes to the elite wheat cultivars for the improvement in their grain micronutrient content and enhanced efficiency of uptake of these minerals in problematic soils A diploid wheat Recombinant Inbred Line (RIL) population of T. monococcum accession paul4087 x T. boeoticum accession pau5088 was studied for phytosiderophore release under iron deficiency conditions over 3 years. The amounts of phytosiderophore released varied from 3.0 - 22.1 \x,g Fe mobilized per plant. A linkage map with 169 molecular markers available for the RIL population was used for mapping QTL for phytosiderophore release. The QTL mapping led to the identification of two highly linked QTL for phytosiderophore release on chromosome 6A. These two QTL were mapped in the marker intervals Xbarcl 13- Xgwm670 and Xgwm670 - XgwmlOn explaining 15.8 % and 21.6 % contribution of the total phenotypic variation respectively. The chromosomal locations of the QTL on group 6 in the RIL population is also supported by the facts that Ae. longissima addition line with 6S1 chromosome has high phytosiderophore release and in barley also the genes for hydoxy-mugineic acid synthesis are located on chromosome 6H which is syntenous to wheat chromosome 6. The precise transfer of Ae. peregrina 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 phytosiderophore release will lead to thorough understanding of the pathways for their uptake from soil and translocation to grains.