BIOCHEMICAL MARKER SECALIN FOR THE SELECTION OF RUST RESISTANCE IN WHEAT BREEDING

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1 , February ; Volume 3(1) ISSN No BIOCHEMICAL MARKER SECALIN FOR THE SELECTION OF RUST RESISTANCE IN WHEAT BREEDING Manikandan N Department of Biotechnology, Nandha Arts & Science College, Bharathiar University, Tamilnadu, India. Received January 28, 2015; Revision February 05, 2015; Accepted February 23, 2015 Available Online February 23, 2015 KEYWORDS Secalin SDS-PAGE Biochemical marker Rust resistance 1B/1R translocations ABSTRACT Five Indian wheat cultivars and their hybrid with MACS 2496 were selected for the present study. They were screened for seed storage proteins Secalin by the SDS-PAGE under non-reducing conditions for the presences of sec-1 which is a characteristic feature of 1BL/1RS translocation. Total soluble proteins were also estimated in all the varieties by the Lowry s method. Results revealed that all the hybrids possess the sec-1 band denoting the 1BL/1RS translocation and conferring the transfer of gene complex Sr31 + Lr26 + Yr9. This study reveals that Secalin can be used as a biochemical marker for the confirmations of 1B/1R translocations. * Corresponding author srimani84@gmail.com (Manikandan N) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher ( All rights reserved.

2 68 Manikandan 1 Introduction Wheat (Triticum spp.) is the most important staple food of the world. In India it is one of the main food crops, next to rice. Wheat occurs in three ploidy level such as diploid (T. monococcum - AA); tetraploid (T. durum - AABB) and hexaploid (T. aestivum -AABBDD) (Bai et al., 1995). Wheat crop is highly susceptible for many diseases like rust, kernel bunt, powdery mildew and loose smut. Wheat rusts are different types and affecting various parts of the plant like black (stem) rust caused by Puccinia graminis pers.f.sp tritici Erick s and Henn, brown (leaf) rust caused by P. recondita Rob ex Desm.f.sp. tritici and yellow (stripe) rust caused by P. striiformis West which are considered as the most destructive often causing % yield loss around the world. All these three rust diseases are caused by fungi which are obligate parasite in nature (Bai et al., 1995). Loses due to rust can be reduced by cultural practices, removal of an alternative host, chemical control and genetic control of the pathogens (McIntosh & Brown 1997). At the genetic level the rust protection is achieved by crossing with wild types of wheat. The wild relatives of cultivated plants have co-evolved with the crop pathogens and so are extremely useful sources of protective genes. Although many of the wild relatives of wheat are Triticum species many are most distant for example the protective genes Lr24 and Sr24 came from tall wheat Thinopurum poniticum and the genes Lr26, Sr31 and Yr9 come from Rye (Secale cereale).the most common method for transferring one or a few genes into a cultivar from wild relative is by back crossing. In breeding for rust resistance the resistance gene complex (Sr31+Lr26+Yr9) is transferred from rye to wheat by 1BL/1RS translocation (Mago et al., 2005) In the biochemical marker study the proteins which can be used for the identification of rust resistance genes. The rye chromosome 1 is having rust resistance gene complex Sr31+Lr26+Yr9. That rye chromosome also codes block of Secalin storage proteins which can be detected by polyacrylamide gel electrophoresis can be used as a biochemical marker for the confirmations of 1B/1R translocations. Secalin are major storage protein of rye. Amounts of these proteins are closely related to 1B/1R translocation (Afshari 2006).The objective of present work is to identify the rust resistance genes in wheat by using secalin as a biochemical marker. 2 Materials and Methods 2.1 Seed Sample collection Five Indian wheat cultivars HP 1102, HD 2285, HD 2324, J 24, Kalyansona and their hybrid obtained from the cross between MACS 2496 were selected for the present study. The seed samples were collected from IARI, Regional station, Wellington, The Nilgiris. All the wheat parental cultivars were susceptible to all the three rusts, whereas all the hybrids were resistant all the three rusts. 2.2 Extraction of Secalin The seeds were powered and the fine flour was used for the extraction of secalin. 50 mg of the flour was mixed with 1.4 ml of dimethyl sulfoxide and vortexed at 20,000 rpm for 10 minutes. The suspension was then centrifuged at 15,900 rpm for 5 minutes. The pellet obtained was resuspended in 1.4 ml of 70% ethanol. After vortexing for 10 minutes it was again centrifuged at15,900 rpm for minutes. The separated pellet was mixed with 160 µl of 70% ethanol by stirring with syringe needle and incubated for 5 minutes at o C in water bath. Then it was centrifuged at 10,000 rpm for 2 minutes and the supernatant was collected (Afshari, 2006). 2.3 Estimation of protein by Lowry s method Proteins react with folin s reagent to give blue colored complex compounds having 2 or more peptide bonds take a characteristic purple colour when treated with dil CuSO 4 in alkaline solution. The colour is apparently caused by coordination complex of the copper atom with 4 nitrogen atoms. About 0.1 ml of sample was diluted to 0.2 ml and taken in test tubes. To the entire samples 1 ml alkaline copper reagent was added. The mixture was incubated at room temperature for 10 minutes. The 300 µl of folin s phenol reagent was added and shaken well. After 45 minutes blue colour was read at 660 nm in a spectrometer. From the standard calibration curve the slope was calculated and the amount of protein present in the sample was estimated (Lowry et al., 1951). 2.4 Molecular weight determination by SDS-PAGE SDS-PAGE was carried outusing the buffer of Singh et al., (1991). Theseparating gel contained 12% acrylamidecrosslinked with 0.213% N N,-Methylene-bis-acrylamide (75:1 weight ratioof acrylamide: bisacrylamide), 0.1% SDS in 375mM Tris.Cl buffer (ph 8.8). Thestacking gel contained 4% acrylamidecross linked with 0.11%N N,-Methylene-bisacrylamide, 0.1% SDSand 125mM Tris.Cl buffer (ph 6.8). The gel sandwich was fixed to the electrophoresis chamber and the chamber was filled with 1X SDS electrophoresis buffer, no leakage was confirmed. The chamber assembly was connected to a 100mA power pack having position to set constant current. The protein samples, 40µl per well along with molecular weight markers are loaded. The current was set initially at 10mA and the gel was allowed to run. When the dye front touches the separating gel mark the current is increased to 30mA. It was stopped when dye reached the bottom of the gel. Total run took around 4 hours. The gel was removed from glass plates and was placed in 100ml fixing solution for 30 minutes.

3 Biochemical marker secalin for the selection of rust resistance in wheat breeding. 69 S. no Varieties Secalin OD at 660 nm Protein content (µg) 1 HP 1102 (Sr 31) HP HD 2285 (Sr 31) HD Kalyansona(Sr31) Kalyansona J 24 (Sr 31) J HD 2329 (Sr 31) HD MACS WH542 (Sr 31) Table 1 Total Protein content and sec-1 of hexaploid wheat parents and their hybrids. (+) - Presence of secalin(-) -Absence of secalin After fixing the gel in fixing solution it was placed in a solution containing 0.05% coommassie blue, 50% methanol and10% acetic acid for overnight. The gel was then rinsed with distilled water and destained in 7% acetic acid and 5% methanol for 2 hours. 3 Results and Discussion The protein content of hybrid varieties found to be increased th an their parent(table-1). The protein content of hybrids HP 1102 (Sr31), HD 2285 (Sr31), Kalyansona (Sr31), J 24 (Sr31) and HD 2329 (Sr31) were slightly increased than their recurrent parents HP 1102, HD 2285, Kalyansona, J 24 and HD 2329 respectively, this was observed from the chart (Graph-1). The screening of seed storage proteins secalin of hybrids from the cross between rust susceptible wheat cultivar and MACS 2496 (Sr31+Lr19+Yr9) together with their recurrent parents was determined by SDS-PAGE for the presence of Sec-1 band of secalin protein. The samples were loaded on the well and run for about 3 and half hours revealed that all the hybrids HP 1102 (Sr31), HD 2285 (Sr31), Kalyansona (Sr31), J 24 (Sr31), HD 2329 (Sr31)and the MACS 2496 having sec-1 band which is a characteristic feature of 1BL/1RS translocations. But all the parental varieties HP 1102, HD 2285, Kalyansona, J 24 and HD 2329 does not have the sec-1 band (Figure-1 and Figure- 2). Javornik et al. (1991) compared four methods such as N banding of metaphase chromosome, APAGE of gliadins, SDS- PAGE of unreduced proteins and isoelectric focusing of subtilisin inhibitor for detecting 1BL/1RS translocations in 59 Yugoslav wheat varieties and recommended isoelectric focusing of subtilisin inhibitor. Gupta & Shepherd (1992) screened wheat cultivars from as many as 31 countries recommended the use of SDS-PAGE of unreduced proteins for identify 1BL/1RS translocations in wheat. Wheat - rye translocation 1BL/1RS is associated with the loss of LMW glutinin and gliadins in wheat and the introduction of secalin. The increase in protein content because of the 1BL/1RS translocation on quality (Kumlay et al., 2003).The translocation of 1Rs also increases the disease resistance, high yield and environmental stability (Gobba et al., 2007). Graph 1 Total Protein comparison of hybrids and their hybrids.

4 70 Manikandan Figure1 SDS-PAGE separation of secalin (Sec-1) as a marker for 1BL/1RS translocations. Lane 1 - MACS 2496 (Donor marker); Lane 2 - HP 1102; Lane 3 - HP 1102 (Sr31); Lane 4 - HD 2285 (Sr31); Lane 5 - HD 2285; Lane 6 - Kalyansona (Sr31); Lane 7 - Kalyansona Figure 2 SDS-PAGE separation of secalin (sec-1) as a marker for 1BL/1RS translocations. Lane 1 - WH 547 (Sr31); Lane 2 - J 24 (Sr31); Lane 3 - J 24; Lane 4 - HD 2329 (Sr31); Lane 5 - HD The sec-1 electrophoretic pattern of MACS 2496a standard of 1BL/1RS translocation similar with the hybrids, whereas the parents do not have band for rye segment. This indicates that the hybrids having the gene complex Sr31 + Lr26 + Yr9 and it was absent in parental varieties. The rye carries genes for resistance to three wheat rust diseases, namely Lr26 for resistance to leaf rust, Yr9 for resistance to stripe rust and Sr31 for resistance to stem rust. Since Sec-1 is tightly linked with the three rust resistance genes electrophoresis it is a useful method to identify and confirm the presence of three rust resistance genes in current wheat populations. SDS-PAGE was used to examine wheat cultivars for resistance to three rusts. The SDS-PAGE results showed that hybrids have Sec-1 bands and are therefore likely to carry the 1BL.1RS translocation and the linked genes Yr9, Lr26 and Sr3. The present study concluded that the hybrid varieties had more protein content than their respective parents, Because of the presence of the seed storage protein secalin. The presence of secalin in wheat seeds confirms the presence of resistance gene complex (Sr31+Lr26+Yr9). References Afshari F (2006) Protein marker assisted identification of Yr9, Lr26, Sr31 genes in a group of Iranian wheat cultivars. Journal of Agricultural Science and Technology 8:

5 Biochemical marker secalin for the selection of rust resistance in wheat breeding. 71 Bai D, Scoles GJ, Knott DR (1995) Rust resistance in Triticum cylindricum Ces. (4x, CCDD) and its transfer into duram and hexaploid wheats. Genome 38:8-16. Gobaa S, Bancel E, Kleijer G, Stamp P, Branlard G (2007) Effect ofthe 1BL.1RS translocation on the wheat endosperm, as revealed by proteomic analysis. Proteomics 7: Gupta RB, Shepherd KW(1992) Identification of Rye Chromosome Translocations and Substitutions in Hexaploid Wheats Using Storage Proteins as Genetic Markers.Plant Breeding109: Javornik B, Sikovié T, Vapa L, Koebner RMD, Rogers WJ (1991) A comparison of methods for identifying and surveying the presence of 1BL.1RS translocations in bread wheat. Euphytica 54: Kumlay AM, Baenziger PS, Gill KS, Shelton DR, Graybosch RA, Lukaszewski AJ, Wesenberg DM (2003) Understanding the Effect of Rye Chromatin in Bread Wheat. Crop science 43: Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin Phenol reagent. Journal of Biological Chemistry 193: Mago R, Miah H, Lawrence CJ, Wellings CR, Spielmeyer W, Bariana HS, McIntosh RA, Pryor AJ, Ellis JG (2005) Highresolution mapping and mutation analysis separate the rust resistance genes Sr31, Lr26 and Yr9 on the short arm of rye chromosome 1.Theoretical and Applied Genetics112: McIntoshRA, Brown GN (1997) Anticipatory breeding for resistance to rust diseases in wheat. Annual Review of Phytopathology35: Singh NK, Shepherd KW, Cornish GB(1991) A simplified SDS-PAGE procedure for separating LMW subunits of glutenin. Journal of Cereal Science 14: