GENETIC DIVERSITY OF MINERAL CONTENTS, NUTRITIONAL TRAITS AND HIGH MOLECULAR GLUTENIN SUBUNITS IN BREAD WHEAT (TRITICUM AESTIVUM)

Abstract

Plant genetic diversity is a key element in any agriculture. Wheat is an annual plant that belongs to the grass family Poaceae Wheat contains carbohydrates, essential amino acids, vitamins, protein and minerals. Institute of Agri-Biotechnology and Genetic Resources (IABGR), Islamabad is a good source of wheat germplasm collected from all over the country. Rust caused by Puccinia spp. cause considerable worldwide damage to wheat production. There are three types of wheat rust viz, stripe rust, stem rust and leaf rust .For the assessment of genetic variability in germplasm collections biochemical markers, such as storage proteins, have received more attention in recent years. High molecular weight glutenin subunits, encoded by Glu- A 1 , Glu-B 1 , and Glu-D 1 loci located on long arms of the homologous group 1 chromosomes of wheat, play a vital role in determining the bread making quality of wheat. Phenotypic identification based on morphological characteristics has been successfully used for genetic diversity analysis. However, morphological traits have a number of limitations, including low polymorphism, low heritability, late expression and may be controlled by epistatic and pleiotropic gene effects) while protein markers, like seed storage proteins, reflect with more accuracy the genotypes, independently from the environmental effects. Single seed was ground to fine powder with the help of mortar and pestle. Protein extraction buffer (400μl) was added to 0.01g of seed flour in eppendorf tube and mixed. The samples were mixed thoroughly by vortexing and centrifuged at 13,000 rpm for 10 min. Electrophoresis was carried out at 100 mA until a blue line of Bromophenol blue reached the bottom of the gel (approximately three and half hour). Then staining and destaining was carried out. 139 accessions of wheat germplasm were evaluated for nutritional characteristics. The experiment was carried out at Grain Quality Testing Laboratory, National Agricultural Research Centre, Islamabad. Fibre, oil, moisture, ash and protein were studied following the standard methods of AOAC (2005). Determination of Minerals Contents was carried out by dry ashing (Boron), wet digestion(Zinc, Copper, Manganese, Iron, Sodium, Potassium and Phosphorus) and Kjeldahl method (Nitrogen). Seed characteristics studied included seed length (By vernier caliper), seed width (By vernier caliper), 100 seed weight, seed colour, seed size and degree of seed shriveling. For the screening of stem rust, plants were inoculated with 09077. Inoculums in the form of uredial suspension in soltor-170 (eight weightnon-phototoxic mineral oil) was sprayed uniformly with a sprayer having five nozzle. The seedlings were left in open air for 1-2 hours to evaporate mineral oil and shifted afterwards to a humidity chamber for 24 hours, after which they were transferred to green house at 18-22 o C. After ten days infection types were recorded. Summary statistic showed that fibre ranged from 0.64 to 1.87 %, oil from 1.18 to 2.49 %, moisture from 6.00 to 8.50 %, ash from 0.77 to 6.86 %, protein from 7.12 to 16.92 %, Nitrogen from 1.25 to 2.97 %, Phosphorus from 0.10 to 0.44 %, Potassium from 0.30 to 0.88 %, Boron from 0.48 to 3.78 ppm, Zinc from 13.50 to 54. 00 ppm, Copper from 1.00 to 9.00 ppm, Manganese from 7.80 to 41.60 ppm, Iron from 8.20 to 300.0 ppm, Sodium from 0.02 to 0.08 %, seed length from 3.36 to 7.43 mm, seed width from 1.67 to 3.15 and 100 seed weight from 2.20 to 5.36 g. Regarding nutritional traits, PC 1 contributed 23.7% and PC 2 contributed 23.4% to the genetic variance of wheat germplasm constituting 139 accessions belonging to Punjab and Baluchistan. Moisture (0.736) and ash (0.505) contributed more positively to PC 1 while oil (0.717) and protein (0.679) imparted maximum genetic variance to PC 2 . First four principal components contributed 62.3% of the total variation as far as mineral contents are concerned. PC 1 contributed 21.1%, PC 2 15.4%, PC 3 13.8% and PC 4 contributed 11.8% to the total variation shown by the wheat germplasm. Nitrogen (0.571), Phosphorus (0.581), Zinc (0.729) and Copper (0.616) imparted maximum genetic variance to PC1, Potassium (0.718) and Iron (0.643) to PC 2 , Boron (0.532) to PC 3 and Manganese (0.768) and Sodium (0.675) contributed more positively to PC 4 . The seed characteristics that contributed more positively to PC 1 included seed length (0.745) and sized width (0.741). To PC 2 seed size (0.514) contributed more positively while seed width (0.597) and seed color (0.659) imparted maximum genetic variance to PC 3 .Regarding combined traits of Punjab and Baluchistan the characteristics which imparted maximum genetic variance to PC 1 included protein (0.828), Nitrogen (0.831) and Zinc (0.687). Moisture (0.638), Phosphorus (0.611) and Boron (0.656) contributed were positively to PC 3 , Iron (0.533) to PC 4 , Sodium (0.539) to PC 5 , and ash (0.589) contributed more positively to PC 7 .Oil was found to be positively correlated with Zinc whereas moisture showed positive association with Phosphorus and Boron. Protein exhibited positively association with Nitrogen and Zinc. P exhibited positive correlation with Boron and Manganese. Zinc showed positive association with Manganese and Iron. Seed lengthwas observed to be positively associated with seed width, and seed width showed positive correlation with 100 seed weight. Wheat germplasm was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to predict the genetic variability on the basis of high molecular weight glutenin sub-units. In Punjab accessions three allelic variants (Null, 1 and 2*) were found at Glu-A 1 locus. Glu-B 1 locus was observed to be highly polymorphic. 19 sub-unit or sub-unit pairs were found at Glu-B1 as 16,(14*+9), (9, 17+18), 17+18, 7**+8, 7**, 7**+8*, 7, 7+8, 7(7**), (6, 7), 7*+9, 7*+8, (8, 13+160, 13+16,9, 7+9, 6+9 AND (7*, 7**+8). Glu-D 1 locus consisted of four allelic sub-units or subunit pairs i.e. 12, 2+12, 4, 5+10.At the Glu-A 1 locus, four allelic variants (Null, 1, 2* and 2’) were observed in 122 wheat accessions belonging to Baluchistan region. Glu-B 1 locus was found to be highly polymorphic. 30 sub-unit pairs or sub-units were found at this locus as 7*+8, 7*+8(8**), 7+8, 7+9, 7(7*)+9, 8*, 7+8*, 7+8**, 7**, 7**+9, 7**+8, 7(7**)+9, 13, 7**+8**, 7(7**), 17+18, 8**(17+18), 14+15, (6, 14+15), (7, 14+15), 20, 9, 7*+9, 7(7*)+8, 13+16, (8*, 7+9), 8*(7*+9), (6, 17+18) and 17. Glu-D 1 locus was comprised of nine allelic subunits or sub-unit pairs i.e. 2+12, 3+12, 2+12*, 10, 12*, 12, 5+10, 5+12*, 5+12. In commercial varieties three allelic variants (Null, 2 and 2*) were observed at the Glu-A 1 locus. The Glu-B 1 locus was found to be highly polymorphic. Out of fourteen allelic variants detected, ten sub-unit pairs or subunits were found at this locus as 7+9, 7*+9, 7**+9, 17+18, 13+16, 7+8, &*+8, 7+8(8*), 14 and 7* (13+16). Glu-D 1 locus was comprised of two allelic sub- unit pairs i.e. 5+10 and 2+12. Total of 192 accessions/commercial varieties were screened against stem rust and stripe rust including eighty seven accessions of Baluchistan, 37 accessions of Punjab and 68 commercial varieties. For stem rust resistance was recorded as resistant, moderately resistant and susceptible. Regarding stem rust, 153 accessions/commercial varieties were recorded to be resistant. While 16 accessions/commercial varieties were found to be susceptible. The data regarding resistance against stripe rust was recorded as resistant, moderately resistant, moderately susceptible and susceptible. Nine accessions of Punjab, 21 Baluchistan accessions and 34 commercial varieties were identified to be resistant. None of the accessions or commercial varieties was found to be susceptible.