Characterization of Nepalese Barley Gene Pool for Leaf Rust Resistance

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.


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.

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 95 o C followed by 30 cycles of 95 o C for 30 sec, annealing as per primer for 1 min, extension at 72 o C for 2 min and final extension at 72 o C for 7 min followed by holding at 4 o C 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 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.

Table 2 ) for Table 1 :
Nepalese barley accessions showing variation on leaf rust resistance characteristics.

Leaf Rust Genotype Leaf Rust Genotype Leaf Rust Genotype Leaf Rust Genotype Leaf Rust 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 Note: *=product size determined based on field data for disease resistance ©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 ©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