Nepal Journal of Biotechnology. D e c . 2 0 1 6 Vol. 4, No. 1: 13-18 ISSN 2091-1130(Print)/ISSN 2467-9319 (online) ORIGINAL RESEARCH ARTICLE ©NJB, Biotechnology Society of Nepal 13 Nepjol.info/index.php/njb Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance Resham Babu Amgai1*, Sumitra Pantha2, Madan Raj Bhatta3 1Biotechnology Division, Nepal Agriculture Research Council, Khumaltar, Lalitpur, Nepal. 2Agriculture Botany Division, Nepal Agriculture Research Council, Khumaltar, Lalitpur, Nepal. 3National Plant Genetic Resource Centre, Nepal Agriculture Research Council, Khumaltar, Lalitpur, Nepal. Abstract Barley (Hordeum vulagare L) is the major crop for the people living in the high hills and mountainous region of Nepal. Leaf rust (caused by Puccinia hordei) is one of the major production threats for barley cultivation. A lot of variation can be observed on Nepalese barley accessions with respect to leaf rust resistance characteristics. Two hundred and forty one barley accessions were screened for leaf rust resistance characteristics on heading stage at Khumaltar, Lalitpur, Nepal. Among them, one hundred and nine Nepalese barley accessions showing promising for disease resistance were screened using six SSR markers linked to leaf rust resistance genes. Bonus and Local Jau was used as the resistant and susceptible check respectively. Leaf rust resistance genes Rph1, Rph2, Rph3, Rph7, QBLR-P and QTL on chromosome 5HS were detected on Nepalese barley accessions using respective SSR markers. Eight Nepalese barley accessions showed presence of three and more leaf rust resistant genes. The poor relationship between the field disease resistance and molecular markers linked with specific leaf rust resistance gene proved that Nepalese barley gene pool contains other leaf resistance genes. Keywords: leaf rust, Puccinia hordei, resistant gene, Nepalese barley, Simple Sequence Repeats (SSR) *Corresponding Author Email: reshamamgain@yahoo.com Introduction Barley (Hordeum vulgare L.) occupies total area of 29598 hectare and total production 33782 metric ton with average productivity of 1.141 metric ton/ha in Nepal [1]. The maximum area of the crop lies in the mid-western development region. Out of the total barley area, more than 50 % is in the hill region, while 40% is in the mountain region [2]. However, its production and productivity is declining due to diseases and unpredictable climatic condition of the mountain area [3]. Leaf rust (caused by Puccinia hordei) is one of the major problematic diseases for barley production in Nepal [4]. Therefore, rust resistant barley varieties are another need of mountain farmers. Nepal harbours hundreds of the barley landraces. High level of genetic variation can be observed in Nepalese barley [5]. Similarly, a lot of variation was observed among the Jumla collection of Nepalese barley for many yield attributing characters [6]. Many of these landraces possess one or more characteristics for abiotic and biotic stress tolerance [2,3]. Variation on rust resistance characteristics is very important for rust resistance breeding program. Therefore, use of these germplasm for rust resistance gene pyramiding is highly beneficial to Nepalese farmers. Identification of particular rust resistance gene and its incorporation is the only option for the development of barley varieties for leaf rust rust resistance. Use of the molecular marker for this purpose is highly sought for this. Material and Methods Germplasm Collection One hundred and fifty five Nepalese barley accessions (NPGR No.s) collected from different parts of Nepal were obtained from National Plant Genetic Resources Centre (NPGRC); and forty seven Jumla collection (JC# series), two local collection (Acc# series) and thirty six barley breeding lines (NB, B, GR and Xveola series) were collected from Hill Crops Research Program (HCRP), Dolakha of Nepal Agricultural Research Council (NARC) (Table 1). Similarly, one hundred and nine Nepalese barley accessions were selected based on their disease resistance data (Table 2) for Nepal Journal of Biotechnology. D e c . 2 0 1 6 Vol. 4, No. 1:13-18 Amgai et al. ©NJB, Biotechnology Society of Nepal 14 Nepjol.info/index.php/njb Table 1: Nepalese barley accessions showing variation on leaf rust resistance characteristics. Genotype Leaf Rust Genotype Leaf Rust Genotype Leaf Rust Genotype Leaf Rust Genotype Leaf Rust 1522 2060 2496 7436 JC#14 1535 2062 2505 7441 JC#15 1537 2064 2506 8252 JC#16 1538 2065 2507 9436 JC#17 1540 2066 2508 9963 JC#18 1543 2069 2511 12069 JC#19 80s 1544 2072 2512 12538 JC#21 40s 1545 2073 2513 40s 20774 JC#22 1546 2074 2514 22463 JC#23 1547 2075 2515 112-14 JC#24 1550 2078 2518 Acc#1545 JC#25 1574 2079 2520 Acc#1574 JC#26 60s 1575 2080 2523 Arupos/oy- B-oy JC#27 1576 2081 2525 B86019-1-0 JC#28 1589 2082 2527 B86019-1K2 JC#29 1664 2083 2530 B86023-1K ts JC#31 1999 2084 2532 B86065-1-4 JC#32 80s 2000 2086 2533 B86099-1K JC#33 80s 2001 2087 2534 tms B86099-2-1 JC#34 20s 2002 2088 2539 B86099-2K JC#35 2003 2089 2542 B86115 JC#36 60s 2004 2090 5177 B86122-1-5 JC#37 90s 2005 2181 5617 B86122-1-5- 0K3 JC#38 ts 2008 2244 6035 B86122-1K JC#39 2009 2447 6036 B86152-2 JC#41 2010 2453 6038 B86152-2-2-0- 0K JC#42 60s 2011 2454 6041 B86157-1-1-5- 0-0K3 JC#43 20s 2013 2456 6044 B86615-1-4 JC#44 40ms 2014 2457 6045 B90K-007-1 JC#45 40s 2016 2458 6055 B90K-01-2K JC#48 40s 2018 2459 6063 80s B90K-014-1 ts JC#49 2023 2461 6235 B90K-024-1 JC#50 2024 2464 6289 B90K-038 JC#51 60s 2027 2465 6292 B90K-090 Jumla coll 2029 2467 6293 Bonus LG- 51/Xve 2033 2468 6304 COQ/KI/Pes cii Local Jau 2035 2469 6309 GR-25-85 Maticos 2037 2470 6310 JC#01 NB-1003- 37 2040 2471 6311 JC#02 NB-1003- 37/1034 2042 2472 6315 JC#03 NB-1003- 37/1038 2043 90ms 2473 6316 JC#04 NB-1003- 37/1214 2046 2482 6319 JC#05 NB- 1207/CI 2048 2483 6320 JC#06 20s Xveola-12 2049 2485 6326 JC#07 60s XVeola-13 2050 2486 6327 JC#08 10s XVeola-28 2051 2487 6334 JC#09 30s Xveola-38 2056 2488 6342 JC#11 20s Xveola-43 2057 2491 6447 JC#12 2058 2494 40s 6557 JC#13 Note: r-resistance, mr-moderately resistance, tms-trace moderately susceptible, tmr-trace moderately resistance, ts-trace susceptible, s- susceptible; blank=no disease i.e. 0 score Nepal Journal of Biotechnology. D e c . 2 0 1 6 Vol. 4, No. 1:13-18 Amgai et al. ©NJB, Biotechnology Society of Nepal 15 Nepjol.info/index.php/njb Table 2: Nepalese barley accessions used for screening leaf rust resistance using simple sequence repeats marker 1535 2002 2046 2081 2458 2483 6036 6327 8242 JC#18 1537 2003 2051 2082 2459 2489 6293 6340 8244 JC#46 1538 2004 2062 2084 2461 2494 6304 6342 8245 JC#49 1540 2008 2064 2086 2464 2496 6309 6343 8246 Local Jau 1544 2009 2069 2087 2467 2505 6310 6344 8252 Maticos 1545 2010 2072 2088 2468 2506 6314 6350 9436 NB-1003-37 1572 2014 2073 2089 2470 2508 6315 6557 9963 NB-1003-37/1038 1574 2023 2074 2090 2471 2515 6316 7436 Arupos/oy- B-oy Solu Uwa 1575 2026 2075 2441 2478 2537 6319 7441 B86122-1-5- 0K3 Xveola-12 1594 2033 2078 2456 2480 2541 6320 8239 Bonus XVeola-13 1601 2043 2080 2457 2482 2542 6326 8240 GR-25-85 Xveola-43 2000 Note: Name as numbers without any alphabet denote the Nepalese Plant Genetic Resource (NPGR) number. Table 3: SSR markers used to identify presence of leaf rust resistance gene in Nepalese barley gene pool Marker Name Sequence-F [5'… … 3'] Sequence-R [5'… … 3'] Annealing Temperature Stripe rust resistance gene PCR product size Chromosome No. Ref AY642926- CA11 CCAAAAAC AATTGAGAA AACCCTA CCTCCC TGAGAG ACCTCCTAT T 58 Rph7 183 3H [12] Bmac096 GCTATGGCG TACTATGTA TGGTTG TCACGATGA GGTATGATCA AAGA 58 Rph2 173 5HS [13] Bmag0225 AACACA CA AAATATTAC AT CA CGAGTAGTTC CC ATG TGA C 58 Rph7 162 3H [14] Bmag337 ACAAAGAG GGAGTAGTA CGC GACCCATGAT ATATGAAGA TCA 55 QTL 129-150 5HS [13] EBmac075 5 AGCCTTGTG TATCAGGAC A CTGCTGGTGT TCTCTAAAAG T 55 Rhp3 Rhp19 144-155 7HL [15] Ris44 ACGGATCTA CTTTAGCTA GCA AAACAACCC CACACAATC 58 QBLR-P 105-110* 7HL [16] Note: *=product size determined based on field data for disease resistance Nepal Journal of Biotechnology. D e c . 2 0 1 6 Vol. 4, No. 1:13-18 Amgai et al. ©NJB, Biotechnology Society of Nepal 16 Nepjol.info/index.php/njb Table 4: Nepalese barley germplasm with different leaf rust resistant gene identified using different molecular markers Rph7 (AY642926- CA11) Rph2 (Bmac096) Rph3 & Rph1 (EBmac0755) Rph7 (Bmag0225) QTL on 5HS (Bmag0337) QBLR-P (Ris44) 2480 GR-25-85 1538 GR-25-85 1540 GR-25-85 2062 2470 2008 1545 1601 1544 2483 2471 2033 1574 2010 1545 2541 2515 2062 2010 2023 1574 Solu Uwa 6036 2478 2080 2033 1575 1537 2480 2505 2043 1601 2074 2483 2508 2051 2002 2082 2489 6304 2478 2003 2506 2515 6310 2480 2004 NB-1003-37 2541 6315 2482 2072 NB-1003- 37/1038 6342 6343 2489 2073 Xveola-43 8252 6350 2494 2075 9963 2000 2505 2078 1594 2026 2515 2080 2506 6309 2537 2081 2537 Arupos/oy-B-oy 2541 2084 6557 Bonus 6036 2086 JC#46 JC#49 6340 2088 JC#49 Local Jau 6342 2089 NB-1003-37/1038 Maticos 6344 2090 Solu Uwa NB-1003-37 8246 2456 Xveola-43 XVeola-13 8252 2457 9963 2459 1537 2461 2000 2464 2014 2467 2046 2468 2074 2470 2082 6304 2506 6310 2542 6314 6557 6315 7436 6316 8239 6319 8245 6326 B86122-1-5-0K3 6327 JC#49 1535 Local Jau 2009 NB-1003-37 2064 NB-1003-37/1038 2069 Solu Uwa 2458 Xveola-43 2471 6293 6309 6320 Bonus NB-1003-37 Note: combination on parenthesis is respective SSR markers used to detect particular gene. Nepal Journal of Biotechnology. D e c . 2 0 1 6 Vol. 4, No. 1:13-18 Amgai et al. ©NJB, Biotechnology Society of Nepal 17 Nepjol.info/index.php/njb Table 5: Nepalese barley germplasm having three and more leaf rust resistance gene detected by molecular markers Genotype Rph3 & Rph1 QTL on 5HS Rph2 QBLR-P Rph7 GR-25-85 0 0 1 1 1 NB-1003-37 0 1 1 1 1 NPGR No. 2506 1 1 1 0 0 Xveola-43 1 1 1 0 0 NB-1003-37/1038 1 1 1 0 0 NPGR No. 2515 1 1 1 0 0 JC#49 1 1 0 0 1 Solu Uwa 1 1 0 0 1 Note: 1=present, 0=absent molecular marker screening. Bonus and Local Jau were used as resistant and susceptible check respectively. Leaf Rust Evaluation at Field Barley lines were screened for leaf rust at heading stage at Khumaltar, Lalitpur, Nepal during normal barley growing season. Bonus (origin Sweden) and Local Jau (Nepalese landrace) was used as resistance and susceptible check respectively. Two rows (spacing 20cm) of 1.5m long per accession were sown. The resistance and susceptible checks were repeated after every 15 test lines. Two spreader rows of Local Jau were sown around the disease screening plots. Disease scoring was conducted according to the modified Cobb scale [7]. Molecular Marker Six SSR markers were selected for screening leaf rust resistance gene (Table 3). Molecular markers are selected based on their linkage with particular leaf rust resistance gene. DNA extraction, PCR reaction and data analysis Genomic DNA of barley accessions was prepared using modified CTAB method as described by Sul and Korban [8]. Each PCR reaction was conducted with100ng of genomic DNA, 1 µM of each primer and 7.5 µl of 2x GoTaqGreen PCR Master Mix (Promega Corporation, Madison, WI, USA). PCR mixture was amplified in MJ Research PTC-100TM Programmable Thermal Controller (MJ Research, Inc, Watertown, MA, USA) with the following temperature regimes: initial denaturation for 2 min at 95oC followed by 30 cycles of 95oC for 30 sec, annealing as per primer for 1 min, extension at 72oC for 2 min and final extension at 72oC for 7 min followed by holding at 4oC as described on Table 3 and Scottish Crop Research Institute [9]. Amplified PCR products were separated in 2% analytical grade agarose gel at 100V for 1H. Gels were stained with 0.1 µg/ml ethidium bromide (Promega Corporation, Madison, WI, USA) and then visualized under UV trans illuminator gel documentation system (Wilber Lourmat, Marne- La-Valleen, France) using 1 µg guide size DNA ladder (Genetix, Biotech Asia Pvt. Ltd.). The presence and absence of particular band size was scored for screening disease resistance genes. Results and Discussion A lot of variation was observed in Nepalese barley germplasm for leaf rust resistance characteristics (Table 1). Leaf rust resistance gene Rph1, Rph2, Rph3, Rph7, QBLR-P and QTL on 5HS was detected on Nepalese barley accessions using respective SSR markers. Twelve landraces showed presence of Rph2, 22 accessions showed presence of Rph1 and Rph3, 27 accessions showed presence of Rph7, forty two accessions showed presence of QTL on chromosome 5HS and 47 accessions possessed leaf rust resistant QTL QBLR-P (Table 4). Similarly, eight Nepalese barley accessions showed presence of three and more leaf rust resistant gene (Table 5). Nepalese barley germplasm showed good resistance with leaf rust which may be due to the presence of leaf rust resistance major genes and quantitative trait loci (QTLs) as detected by different SSR markers. Tyryshkin [10] and Henderson [11] also concluded that Nepalese barley has good resistance against the leaf rust. The released hulless barley variety “Solu Uwa” show good resistance with leaf rust and have Nepal Journal of Biotechnology. D e c . 2 0 1 6 Vol. 4, No. 1:13-18 Amgai et al. ©NJB, Biotechnology Society of Nepal 18 Nepjol.info/index.php/njb QTLs (Table 4) and major genes including Rph7 (Figure 1). Similarly, adult plant resistance for leaf rust was also observed by Tyryshkin [10] for Nepalese barley NB-3002 while screening world collection of barley for leaf rust and proved to have one dominant gene. This gene may be Rph7. Similarly, the poor relationship between the field disease resistance and molecular markers linked with specific leaf rust resistance gene proved that Nepalese barley gene pool contains new leaf resistance genes that cannot be defined by the tested molecular markers. Conclusion Many Nepalese barley landraces showed field resistance with leaf rust, however, some promising lines still lacks any major resistant genes as defined by molecular markers and need to be incorporated from other lines to address future unwanted leaf rust spread. Barley genotypes with more than three resistant genes could be the choice of donor parents for leaf rust resistant breeding through molecular marker assisted selection in Nepal. Acknowledgement This work was conducted under Global Biodiversity Trust Grant No. GS10027. References 1. GoN: Statistical information on Nepalese agriculture 2012/13. Ministry of Agriculture Development. Agri-Business Promotion and Statistics Division, Kathmandu, Nepal, 2013. 2. Upreti RP: Status of food barley in Nepal. In-S. Grando and H. Gormez Macpherson (eds.), Food Barley: Importance, Uses and Local Knowledge. Proceedings of the International Workshop on Food Barley Improvement, 14-17 January 2002, Hammamet, Tunisia. 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