TRANSFER OF GENES FOR HIGH GRAIN Fe AND Zn CONTENT OF GROUP 7 CHROMOSOMES OF Aegilops TO WHEAT

Abstract

More than 2 billion people in the developing world are affected by iron (Fe) and zinc (Zn) deficiency. Fe deficiency often leads to anemia, impaired physical growth, mental retardation, weak learning capacity and ability to do physical labour. Zn acts as necessary component in more than 200 enzyme systems for normal growth and development, maintenance of body tissue, sexual function, brain development, cognitive ability, vision and immune system. Micronutrient deficiency can be alleviated by supplementation, diet diversification, fortification and biofortification. Biofortification is the most sustainable, targeted and cost effective approach for improving nutritional quality of staple crops. There are several approaches to biofortify crops, including agronomic biofortification, conventional or molecular breeding and genetic engineering. Wheat is the second most consumed cereal in Asia after rice. The polyploid nature of wheat provides considerable genetic buffering thus allowing interogression of useful variability from related species. High yielding cultivars of wheat are the poor sources of important micronutrients especially Fe and Zn. Wheat is also rich in anti- nutritional compounds like phytic acid and fibres which reduces the bioavailability of the micronutrients. In 2002, the Consultative Group for International Agricultural Research (CGIAR) and HarvestPlus initiated a program to develop biofortified crops with focus on three critical micronutrients Fe, Zn and vitamin A. The related wild Triticum and Aegilops species with useful variability for high grain Fe and Zn content can be utilized for biofortification of wheat. Several wild progenitor and non-progenitor species of wheat were used for development of alien addition, translocation and substitution lines for transfer of useful variability. The quantitative trait loci (QTL) for grain Fe and Zn were mapped on chromosome 2A and 7A in diploid wheat. Introgression of chromosome 2S and 7U from Aegilops kotschyi to wheat led to high grain Fe and Zn content. Several Aegilops alien addition and substitution lines of group 2 and 7 with high grain Fe and Zn are also available. Transfer of useful variability from non-progenitor species can be easily achieved by induced homoeologous pairing, through ph1b deletion and 5B defciency. The wheat ph1b mutation, which promotes meiotic pairing between homoeologous chromosomes, was employed to induce recombination between Ae. kotschyi 396 chromosome 7S and 7U and their wheat homoeologue. Radiation hybrid is also very useful approach for gene transfer and gene localization. Presence of high density microsatellite maps of wheat and modern cytological techniques like GISH and FISH can be used for precise transfer and tagging of genes responsible for grain micronutrient content. GISH is of potentially wide application in plant ii breeding programmes involving alien translocations. This study was undertaken to reduce linkage drag in substitution lines of 7S and 7U chromosomes of Ae. kotschyi 396 and Ae. kotschyi 3790, respectively through fine transfer of genes for high grain iron and zinc content. Anchored wheat SSR markers of group 7 of wheat were used for transferability and polymorphism between Aegilops donor and recipient wheat cultivars. A total of 173 markers of group 7 chromosomes were screened using PCR. 77.45 % (134 markers) of these 174 markers were found to be transferable. All the markers which were transferable were not polymorphic among wheat and Aegilops species. Polymorphic proportion varied for 41-70 % of 7A, 7B and 7D chromosome markers, from the long and short arms. A total of 51.49 % (69 markers) markers were found to be polymorphic out of 134 transferable markers of group 7 of wheat. Polymorphic markers between wheat and Aegilops species were analysed for transferability to 7S and 7U chromosomes using 7S substitution and 7U addition line as the genes for Fe and Zn are mostly located on these chromosomes supported by micronutrient data of substitution and addition lines of 7S and 7U in wheat and Chinese Spring background. Out of 69 polymorphic markers of group 7 chromosomes of wheat 36 52.29% were transferable to 7S and 7U chromosome, 28.98% specific to 7S, 14.49% for 7U and 8.69% for both the chromosomes. The tentative consensus map of 7S and 7U chromosomes of Ae. kotschyi was prepared by Join Map using markers which were found polymorphic between Triticum aestivum and Ae. kotschyi and transferable to 7S and 7U chromosomes. These 7S and 7U specific markers were used for molecular characterization of introgressed derivatives. In the present study, two kinds of radiation hybrid approaches, seed irradiation and pollen irradiation were used for precise gene transfer. Wheat-Aegilops substitution line CS(PhI)/Ae. kotschyi 396//PBW343-3///PBW373(48)-41-6 of 7S for 7D chromosome was seed irradiated at 40 krad of gamma radiation. These irradiated seeds were grown in the field and the plants were crossed with recurrent parent WL711 to get SRH1 plants. For pollen irradiation, spikes of wheat-Aegilops 7S and 7U substituted lines CS(PhI)/Ae. kotschyi 396//PBW343-3///PBW373(48)-41-6 and CS(PhI)/Ae. kotschyi 3790//UP2338- 2///WL711(63)-2-13, respectively were irradiated at 2 krad of gamma radiation and used for pollination of recipient wheat cultivar PBW343 with Lr24 and GPC1. PRH1 plants were selfed and screened for micronutrients content and transfer of small fragment/genes with high grain Fe and Zn using polymorphic marker of group 7 chromosome and (GISH). Nitric acid digested seed samples of SRH1, SRH3, PRH1, PRH2, BC1F2, (ph1b) BC1F3 (ph1b) and BC2F2 were analysed for micronutrients using AAS and ICP-MS. The SRH2 plants had Fe and Zn concentrations in the range of 46.8 to 127.4 mg/kg and 41.25 to 110.10 mg/kg, respectively, iii The SRH2 plants had Fe and Zn concentrations in the range of 23.18 to 92.34 mg/kg and 27.15 to 72.90 mg/kg, respectively, as compared to 32.20 mg/kg Fe and 40.56 Zn for the wheat cultivar WL711. The plant SRH3-28-2 had 70% increase in grain Fe and 15% increase in grain Zn and plant no. SRH3-14-2- and SRH2-28-6- had 187% and 97% increase in grain Fe and 40% and 47% in grain Zn content, respectively. These plants had short arm and terminal transfers of 7S. In some plants of PRH1 of 48-41-6 20-125% increase in grain Fe content and 40-140% increase of grain Zn content or 40-60 % increase of both the elements was observed over PBW343. The plants of PRH2 of 48-41-6 had Fe and Zn content in the range of had Fe and Zn concentrations in the range of 13.89 to 150.52 mg/kg and 27.11 to 192.48 mg/kg respectively. PRH1-82 and PRH1-124 had 7S chromosome translocations. Grain Fe concentration varied between 18.9 mg/kg to 77.45 mg/kg and grain Zn concentration varied between 23.32 mg/kg to 164.9 mg/kg. for the plants of PRH2 of 48-41-6. The plants of PRH2 of 63-2-13 had Fe and Zn concentrations in the range of 4.04 to 133.16 mg/kg and 22.12 to 124.15 mg/kg respectively. PRH1-312 had short arm translocation. It was found that Fe content of PRH2 plants varied in the range of 19.3 mg/kg to 71.5 mg/kg and Zn content varied in the range of 22.4 mg/kg to 48.03 mg/kg. For an alternative strategy for gene transfer, 7S substitution lines of Aegilops were also crossed with ph1bph1b deletion to obtain F1 plants (ph1bph1//7S/7D) and again backcrossed with ph1b mutant plants. The BC1F1 plants were screened for homozygous ph1bph1b through Ph1 locus specific marker psr574 and 7S monosomic 7S by wheat anchored 7S specific SSR The plant with ph1bph1b and 7S monosomic were selected selfed to get BC1F2. Backcross derivative were further screened for high grain Fe and Zn content. The seeds of BC1F2 were mostly shriveled because of ph1bph1b and leaf yellowing. The shriveled seeds and leaf yellowing seems to be associated with the absence of ph1 locus. Only a few plants obtained had equivalent harvest index to that of the cultivar and 40-60% increase of the Fe or Zn or both the element. Plant BC1F2-471 and BC1F2-487 had multiple translocations and long arm of 7S chromosome, respectively. The BC1F3 had Fe and Zn concentrations in the range of 22.7 to 53.95 mg/kg and 16.58 to 62.12 mg/kg respectively. The derivatives of all three hybrids approaches i.e. seed irradiation, pollen irradiation and ph1b hybrids, which had high grain Fe and Zn content were also found resistant to powdery mildew and had 7S short arm transferred, indicating that the genes for micronutrient uptake and powdery mildew resistance might be linked on short arm of 7S chromosome. Plant PRH2 -124 had translocation of 7S chromosome telomeric region, was resistant to powdery mildew and plant PRH2 -82 had 7S chromosome without telomeric region, was found iv susceptible to the powdery mildew, indicating that powdery mildew resistance gene could be present in sub-telomeric region of the 7S chromosome. Powdery mildew resistance might be linked to SSR markers wmc405 and barc126 as indicated by SSR marker data on seed irradiated hybrids. Genes for micronutrient uptake were also linked to these markers, further proving the linkage of powdery mildew and micronutrient uptake genes. Mono 5B plants of Triticum aestivum cv. Pavon were cytologically identified and crossed with Aegilops 3790 as the male parent. The F1 plants were screened by molecular markers psr574. The absence of these markers indicated the absence of 5B i.e. 34 chromosomes in total. The ABDUS hybrids were also confirmed absence of 5B by cytological analysis at meiosis. The F1 plants with 34 chromosomes (without 5B) showed high chromosome pairing up to, 2V+4III+2II+1I, while the plants with 35 chromosomes (with 5B) had reduced homoeologous pairing, with 6II+23I. Plants with 34 chromosomes (without 5B) were selected and backcrossed extensively with wheat cultivar PBW343 with Lr24 and GPC1 for transfer of useful variability of Aegilops for micronutrients biofortification. Fertile derivative were further screened for high grain Fe and Zn content. Fe and Zn content of mono 5B BC2F2 plants ranged from 43-114 mg/kg and 141-238 mg/kg due to concentration effect. The chromosome number of BC2F1 plants varied 42-48 with 2-7 univalents. The Derivatives of all types of hybrids i.e. SRH, PRH, ph1b induced and 5B deficiency induced, with very high Fe and Zn content had poor tillering, seed set, and low harvest index, indicated that micronutrient content was negatively correlated with yield and harvest index. This negative correlation might be due to distribution of fixed amount of micronutrient per plant among less number of seeds the plants. Plants with shrivelled seeds in the hybrid progenies also had high Fe and Zn content suggesting that the negative correlation between seed size and micronutrient concentration, could be due more aleurone area per unit mass of shrivelled seeds as compared to the bold seeds. All the selected plants with chromosomal translocations had better genetic system for Fe and Zn uptake from the soil and transport within the plants but the overall concentrations of these micronutrients in the seeds was however less than the donor Aegilops species. The biofortification of wheat for Fe and Zn content could be achieved up to 40-50% without any linkage drag. Pyramiding of these introgressed genes/QTLs from different sources through molecular breeding can be done to achieve enhanced biofortification of these micronutrients.