THE EGYPTIAN SOCIETY OF GENETICS Volume 43 July 2014 No . 2 MAPPING OF QTLs CONTROLLING RESISTANCE TO LEAF STRIPE ( Pyrenophora graminea ) IN EGYPTIAN BARLEY

Leaf stripe in barley, caused by Pyrenophora graminea, is an important seed-borne disease in organically grown as well as in conventionally grown Nordic and Mediterranean barley districts. In the present work, a QTL map for F2 barley and 7 LSLs) distributed on 8 linkage groups and spanning 2099.4 cM of the barley genome. The size of linkage groups varied from 117.8 cM for LG2 (2H) to 482.9 cM for LG6 (6H) with an average length of 262.4 cM. Based on the used anchor SSR markers, only four linkage groups were assigned to chromosomes, where LG2, LG5, LG6 and LG7 were assigned to chromosomes 2H, 5H, 6H and 7H, respectively. Single point analysis was used to identify genomic regions controlling the leaf stripe severity and plant height traits. A total of 40 QTL were identified for the disease severity (DS) and plant height (PH) traits (20 QTL for disease severity and 20 QTL for plant height). This work represents the first genetic linkage map for barley population derived from an intraspecific cross between At20 and At29 showing chromosomal regions associated with disease severity and plant height traits in barley.

cold climate during the sowing season.In susceptible varieties, the disease causes brown stripes on the leaves, stunted growth and severe yield reductions (Tekauz, 1983;Porta-Puglia et al., 1986).The fungus is unable to cause secondary infection through leaf-to-leaf transmission and therefore behaves as a true, seedborne disease.
Detecting of sources for resistance to leaf stripe and an understanding of their genetic background are very crucial to develop new resistant varieties for such disease.Resistance to leaf stripe is controlled by several genes and dependents on the source of resistance.
Genome research promises to promote continued and enhanced plant genetic improvement.As a world's leading crop and a model system for studies of many biological processes, genomics research of barley has advanced rapidly in the past few years.Genetic linkage maps are useful tools for studying genome structure, organization and evolution and to estimate gene effects of important agronomic traits, identifying introgression and for tagging genes of interest to facilitate marker aided selection (MAS) and map based cloning.Genetic linkage maps saturated with polymorphic molecular markers have already been generated for many species of economic and scientific interest including: barley (Rostoks et al., 2005;Stein et al., 2007;Adawy et al., 2013), durum wheat (Diab et al., 2013;Patil et al., 2013;Kumar et al., 2013), cotton (Adawy et al., 2008 and2013), maize (Stange et al., 2013), rice (Li et al., 2011;YueHui et al., 2013), tomato (Gonzalo and van der Knaap, 2008;Shirasawa et al., 2010;Sim et al., 2012), and hexaploid wheat (Röder et al., 2004;Quarrie et al., 2005;Paux et al., 2008;Bennett et al., 2012).
The Simple Sequence Repeats (SSRs) are PCR-based marker and have proved to be useful in barley research as they offer reproducibility, multi-allelic nature, co-dominant inheritance, genome specificity, relative abundance, and good genome coverage (Varshney et al., 2005;Ganal and Roder, 2007).They have been used in the localization of genes to chromosomes (Roder et al., 2004), identification of quantitative trait loci (QTL) for yield and quality traits (Ganal and Roder, 2007).Meanwhile, the Amplified Fragment Length Polymorphism (AFLPs), developed by Vos et al. (1995).AFLP fragments are inherited in a Mendelian fashion as dominant or codominant markers, making the technique amenable to tracking inheritance of genetic loci in a segregating population.A new frontier type of genetic marker is start codon targeted (SCoT) polymorphism, which is a novel, simple and reliable gene targeted marker technique based on designed primers targeting conserved region surrounding the translation initiation codon ATG (Collard and Mackill, 2009).This type was successfully employed in development of QTL mapping in both durum wheat and barley (Adawy et al., 2013;Diab et al., 2013).

Mapping population
The mapping population used in this work was a segregating F 2 population of 60 individuals derived from the intraspecific cross between At20 and At29.The female parent was cv "At29," which is characterized by leaf stripe resistance and the male parent was cv "At20" which is characterized by leaf stripe sensitivity.The F 2 trials were grown in controlled conditions using one of the greenhouses in the Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza, Egypt.

DNA isolation
DNA was isolated from the two parents and the 60 F 2 plants using a DNAeasy Plant Mini Kit (Qiagen, Santa Clarita, CA).

Molecular markers
Four different types of molecular markers (13 LSL, 15 SSR, 18 SCoT and 10 AFLP primer pairs or primer combinations) were used to screen polymorphism between the two parental accessions (At20 and At29).Markers found to be polymorphic were then used to survey the segregation among the 60 individuals of the F 2 mapping population.

Simple sequence repeats (SSR) analysis
Forty-eight anchor microsatellite markers located on barley chromosomes were chosen from Grain Genes database and different published papers for analysis.SSR analysis was conducted as described by Hussein et al. (2006) and Adawy (2007).Analysis of segregation among the 60 F 2 individuals was performed using 15 anchor SSR primer pairs that showed a polymorphic pattern between the parents.

Start codon targeted (SCoT) analysis
SCoT analysis was performed as described by Collard and Mackill (2009).
Initially, Forty-eight 18-mer SCoT primers were screened against the two parental varieties.Eighteen out of these 48 primers showed polymorphic pattern.
Therefore, they were used to survey the segregation among the 60 F 2 individuals.

Amplified fragment length polymorphism (AFLP) analysis
The AFLP analysis was performed using the AFLP® Analysis System II (Invitrogen, USA) as described by Vos et al. (1995).Initially, 22 AFLP primer combinations (PCs) were tested on the two parental varieties.Among these, the best ten PCs were selected on the basis of the number of bands, clarity of pattern, and distribution on the gel.These were tested again on the parental varieties and the 60 F 2 individuals.

Leaf stripe linked (LSL) analysis
Thirteen markers linked to leaf stripe resistance were chosen from previously published papers for analysis (Tacconi et al., 2001;Bulgarelli et al., 2004;Biselli et al., 2010) on barley chromosomes.Some of these LSL markers were used as CAPS markers through cutting with restriction enzymes.
Initially, all primers were screened against the two parental varieties.Six out of these 13 primers showed polymorphic pattern.Therefore, they were used to survey the segregation among the 60 F 2 individuals.

Phenotypes and traits measurements
The parents and F 2 mapping populations were evaluated for two morphological traits (disease severity and plant height).

Genetic linkage map construction and QTL detection
The 104 markers that showed polymorphism between the parental varieties were used to construct the genetic linkage map.Linkage analysis and map construction were performed using Map Manager QTX V.1.4(Manly and Cudmore, 1997).The graphic representation of the linkage groups was created using Map Chart 2.1 (Voorrips, 2002).The association between phenotype and genotype was investigated using single point analysis (SPA), using Windows QTL Cartographer V. 2.5 (Wang et al., 2007).

RESULTS AND DISCUSSION
An intraspecific F 2 population of 60 plants derived from the cross between the resistant variety "At29" and the susceptible variety "At20" to leaf stripe pathogen (Pyrenophora graminea) and was helpful for identifying loci controlling leaf stripe resistance and plant height.
From the survey of the two parents and F 2 population using 15 SSR, 10 AFLP primers combinations, 18 SCoT and 13 LSL, a total of 1098 major bands were observed (30 SSR, 821 AFLP, 218 SCoT and 29 LSL).Of these major bands, only 239 (21.7%) were polymorphic between the two parental varieties and among population giving 15 for SSR, 161 for AFLP, 50 SCoT and 13 LSL (Tables 1, 2, 3 and 4).

Molecular markers analysis
Different molecular markers including SSR, SCoT, LSL and AFLP were employed to identify markers that reveal differences between parents and among the F 2 individuals.

Simple sequence repeat analysis (SSR)
Forty eight microsatellite primer pairs previously mapped the barely chromosomes (Grain Genes database) were used in this study.These primers were screened against parental varieties "At20" and "At29", in an attempt to detect polymorphic markers.Only 15 SSR primers (31.25%) showed polymorphism between the two parents.The analysis of segregation among the 60 F 2 individuals was performed using these 15 SSR polymorphic primers.These primers exhibited 15 SSR alleles (Table 1 and Fig. 1) with an allele size ranged from 120 bp to 700 bp.

Amplified fragment length polymorphism analysis (AFLP)
Among the twenty two AFLP primer combinations used to screen the two parents, only ten AFLP primer combinations (45.5%) showed polymorphism.The selected primer combinations, the total number of amplicons, polymorphic amplicons and polymorphism percentage are presented in Table (2).A total of 821 major AFLP bands were observed, 161 of these (19.6%) were polymorphic between the two parents.The number of amplicons/ primer combination ranged from 68 (E-AGC/M-CAG) to 98 (E-AGG/M-CTT).While, the number of polymorphic amplicons varied from 8 in primer combination (E-ACG/M-CTC) to 25 in primer combinations (E-AGG/M-CTT).
The average number of polymorphic fragments per combination was 16.1.

Analysis of leaf stripe linked markers (LSL)
Thirteen leaf stripe linked primers initially screened against parental varieties.These primers reported as linked markers to leaf stripe resistance genes (Tacconi et al., 2001;Bulgarelli et al., 2004;Biselli et al., 2010).Some of these primers were developed as Cleaved Amplified Polymorphic Sequence (CAPS) markers through cutting with restriction enzymes.Six LSL of the 13 primers showed polymorphism between the two parents and consequently used in the analysis of segregation among the 60 F 2 individuals.These primers amplified a total of 28 amplicons including 15 monomorphic DNA fragments.While, 13 fragments were polymorphic (46.4%) (Table 3 and Fig. 3).

Start codon targeted analysis (SCoT)
Among 48 SCoT primers initially screened against parental genotypes, only 18 SCoT primers (37.5%) showed polymorphism between the two parents and consequently used in the analysis of segregation among the 60 F 2 individuals.These primers amplified a total of 218 amplicons including 168 monomorphic DNA fragments.While, 50 fragments were polymorphic, corresponding to a level of polymorphism of 22.9% (Table 4 and Fig. 4).

Linkage analysis and map construction
Two hundred thirty nine markers were used to construct the genetic map.Only 104 markers including (6 SSRs, 63 AFLPs, 7 LSL and 28 SCoT) were mapped in 8 linkage groups spanning 2099.4 cM of the barely genome.The resulting linkage groups were numbered as LG1 to LG8.The size of linkage groups varied from 117.8 cM for LG2 to 482.9 cM for LG6 with an average length of 262.4 cM.The number of markers located on each linkage group also varied from 6 to 28 markers.The distribution of markers, linkage group assignment and map coverage across the 8 barely linkage groups are summarized in Table (5).The main marker type contributing to this linkage map was AFLPs (60.5% AFLPs, 5.7% SSRs, 6.7% LSL and 26.9% SCoT).As shown in Table (5) and Fig. (5) some linkage groups were composed entirely of AFLPs (LG 1 and 3), whereas, others consisted of AFLPs and SCoTs (LG4), or AFLPs, SCoTs and SSRs (LG2 and 5).Additional markers are needed to provide bridges for joining unlinked markers.

Assignment of linkage groups to the chromosomes
Linkage groups were assigned to barley chromosomes according to published map locations of the SSR markers as reference point.Only 15 SSRs primers revealed polymorphic patterns between the two parents.Based on the presence of these SSR markers, only four linkage groups (LG) were assigned to chromosomes, where, LG2, LG5, LG6 and LG7 were assigned to chromosomes 2H, 5H, 6H and 7H, respectively.Five anchored SSR markers were used to assign the genetic linkage groups to barley chromosomes as for 2H (SSR-3), 5H (SSR-10 and SSR5), 6H (SSR-9) and 7H (SSR1).Nine SSR assigned markers were unlinked; representing 53% of the total SSR assigned markers used to construct the genetic map.
Development of genetic linkage map represents the first step towards tracing out the valuable alleles in a segregating population.The manipulation of large number of genes will enables dissection of the complex traits to provide new ideas for efficient plant improvements, by natural selection or breeders; this depends on creating, evaluating, and selecting the right combination of alleles (Flavell, 1995).
Several barley maps were reported by using different molecular marker techniques.Sequence-related amplified polymorphism (SRAP) markers along with SSR markers were used to construct a barley linkage map with 175 loci covering 1,475 cM with a mean density of 8.7 cM per locus (Guo et al., 2013).Diversity arrays technology (DArT) were also used to cover 1,503 cM of barley genome with an average density of 2.33 cM (Rodríguez-Suárez et al., 2012).Different molecular marker system (AFLP, SSR, CAPS, SCoT, STS and others) were used to develop 1644.8 cM.Barley map targeting net blotch resistance genes, caused by Pyrenophora teres f. teres, in an F 2 across between two barley varieties "AT4" (net blotch resistant) and "Femina" (net blotch susceptible) were used to develop a segregating F 2 population (Adawy et al., 2013).
There are useful resistances loci to leaf stripe have been reported in the last few years.The first attempt was reported by Giese et al. (1993), they localized the 'Vada resistance' gene on barley chromosome 2 (2H).After a few years, Thomsen et al. (1997) mapped this qualitative resistance gene in variety 'Alf' on the long arm of chromosome 2 (2H) and proposed Rdg1 as its designation and several sources of complete and quantitatively resistance found in European and non-European germplasm (Skou et al., 1994;Pecchioni et al., 1999).(resistant) (Biselli et al., 2010).
In this investigation a genetic linkage map, for the first time, integrates SCoT marker technology in barley mapping for leaf stripe resistance.The early maps were based on restriction fragment length polymorphism (RFLP) markers (Tacconi et al., 2001), while later the polymerase chain reaction (PCR)based markers became dominant for genetic map construction, e.g.Amplified fragment length polymorphisms (AFLPs) (Biselli et al., 2010).SCoT marker system used with other three user-friendly PCRbased marker systems (SSR, AFLP and LSL) to generate a genetic linkage map in cross between At20 and At29.Also, the constructed linkage map contains four marker types that were not mapped collectively in any other barley maps.A genetic map constructed in 8 linkage groups consisted of 104 loci and spanning a total of 2099.4 cM with an average distance between loci of 262.4 cM.

Quantitative trait loci linked to leaf stripe
Twenty QTLs were defined by marker interval that included the LOD score peaks as responsible for leaf stripe severity (DS) this result is supported by the high portion of phenotypic variance.
Many linked loci (AFLP-11, AFLP-123, SCoT-34, AFLP-84 and SCoT-17) were located on barley chromosome 7H.In this content, our results agree with Tacooni et al. (2001).They developed a NIL population from the susceptible barley variety "Mirco" and the resistant barley variety "Thibaut" using RAPD, STS, AFLP and CAPS markers and reported a resistance QTL in chromosome 7H.
Another high number of Loci (AFLP-42, SSR-3, AFLP-46 and SCoT-1) were located on chromosome 2H , this is supported by a previous study on the "Vada" variety resistance to leaf stripe isolate Dg2 in which a major QTL was detected on chromosome 2H (Arru et al., 2002).Also, Pecchioni et al. (1996) identified a qualitatively acting factor conferring resistance to barley leaf stripe mapped on chromosome 2H.By using Restriction Fragment Length Polymorphism (RFLP) markers and based on Doubled Haploid (DH) barley lines de-rived from the F 1 cross between the variety "Proctor" (resistant) and the variety "Nudinka" (susceptible).Also, Arru et al. (2003) reported similar results by using a medium-density, molecular marker map derived from a 'Steptoe' (partially resistant) × 'Morex' (susceptible).There map demonstrates one QTL is common to the resistance for the two isolates, on the long arm of chromosome 2H, two QTLs are linked on chromosome 3H, a further two QTLs are isolate-specific for one isolate on chromosome 2H and for the other isolate on chromosome 5H.This is in agreement with (Biselli et al., 2010), they mapped to chromosome 2H a major gene with its positive allele contributed by two segregating populations of L94 (susceptible) X Vada (resistant) and Arta (susceptible) X H. spontaneum 41-1 (resistant) detected for two different pathogen isolates.
Start Codon Targeted marker system was generally reproducible and revealed a high number of linked loci compared with other markers ,this was also supported in many plant species (Luo et al., 2011;Bhattacharya et al., 2013;Mulpuri et al., 2013), this encourage using SCoT to expand the quantitative trait loci identification on conserved region in plant genes surrounding the ATG translation start (or initiation) codon that has been well characterized in previous studies (Joshi et al., 1997;Sawant et al., 1999).
By using BLAST tool provided by "The International Barley Genome Sequencing Consortium" database, SCoT-21, SCoT34 and SCoT-35 were highly matched to atpase plasma membrane-typelike gene which responsible for pumping protons out of cells, and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi (Justesen et al., 2013).SCoT-17 and SCoT-3 gave high similarity ratio with phosphatidylserine synthase 2-like gene which play crucial role as a structural component and involved in cell signaling (Sturbois-Balcerzak et al., 2001).
SCoT-1 gave a high similarity score with "E3 ubiquitin-protein ligase like", the importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting (Ardley et al., 2005), SCoT-47 gave also high similarity ratio with udp-glucuronic acid decarboxylase, this enzyme participates in starch and sucrose metabolism and nucleotide sugars metabolism (Rubenstein et.al., 1974).
On the other hand, SCoT-13 gave a high similarity ratio with "respiratory burst oxidase homolog protein" which involved in the generation of reactive oxygen species (ROS) during incompatible interactions with pathogens and in UV-B and abscisic acid ROS-dependent signaling and might be required for ROS signal amplification during light stress (Kwak et al., 2003).
Seven Leaf Stripe Linked (LSL) markers out of 13 were mapped and only two were linked to quantitative pathogen resistance loci, although LSL-1 (NBS-1) was reported to be located on chromosome 7H (Biselli et al., 2010).In this study, it has been separated in independent linkage group (LG8), this could be maintained by increasing mapped markers to fill gaps between the two linkage groups in future studies.On the other hand, LSL-5 (P-9) was successfully linked to chromosome 7H as confirmed previously by Bulgarelli et al. (2004).Most of LSL markers were concentrated on chromosome 7H (LSL-5, LSL-6, LSL-4 and LSL-2) which in high consistency with previous published results reported by (Bulgarelli et al., 2004;Biselli et al., 2010), also it highlights the link between chromosome 7H and leaf stripe pathogen resistance.
Using of AFLP in this study was helpful to detect a high number of QTLs (AFLP-154, AFLP-42, AFLP-45, AFLP-46, AFLP-11, AFLP-123, AFLP-84, AFLP-125, AFLP-52 and AFLP-81) ,this support the idea of using this technique to identify more loci linked to pathogen resistance for leaf stripe and other plant pathogens.Using SSR marker system for tracing pathogen resistance genes were reported in barley net blotch (Adawy et al., 2013), soil-borne viruses (Werner et al., 2006) and genotyping (Macaulay et al., 2001).In our study, the using of SSR was useful to detect two QTLs (SSR-3 and SSR-9).

QTL associated with plant height
Controlling of plant height is a highly important trait can be used for reducing yield loss caused by lodging and to increase harvest index (Bezant et al., 1996).Plant height show continuous variation and the inheritance of this trait is complex and usually supposed to include numerous genetic factors that interact with environmental conditions (Kjaer et al., 1995).

Correlation between traits
In this study, plant height and disease resistance are possible to have similar locations.Shared genomic loci for agronomic traits have also been reported in barley in previous studies between yield and yield components; Biomass, tiller number in fertile and infertile, harvest index, plant height and spike length (Babaiy et al., 2011), rain filling, malt protein, malt fragility, wort viscosity, diastatic power and ferment-ability (Bichoński and Śmiałowski, 2004) and heading date and plant height (Shahinnia et al., 2006).
QTL results obtained from this work as some genomic regions were occupied by overlapped QTLs on linkage groups LG4 (SCoT-34 and SCoT-17) and LG6 (SSR-9) for both disease resistance and plant height.
According to Yang et al. (2007), our results could be caused by: (1) two strongly linked genes affecting different traits, (2) one single gene that produces a series of effects in related traits, (3) one gene affecting two or more independent traits, (4) two linked genes with effects in the same traits.

SUMMARY
Leaf stripe in barley, caused by Pyrenophora graminea, is an important seed-borne disease in organically grown as well as in conventionally grown Nordic and Mediterranean barley districts.In the present work, a QTL map for F 2 barley mapping population derived from an intraspecific cross between At20 x At29 was constructed using 104 DNA markers (6 SSRs, 28 SCoTs, 63 AFLPs and 7 LSLs) distributed on 8 linkage groups and spanning 2099.4 cM of the barley genome.The size of linkage groups varied from 117.8 cM for LG2 (2H) to 482.9 cM for LG6 (6H) with an average length of 262.4 cM.Based on the used anchor SSR markers, only four linkage groups were assigned to chromosomes, where LG2, LG5, LG6 and LG7 were assigned to chromosomes 2H, 5H, 6H and 7H, respectively.Single point analysis was used to identify genomic regions controlling the leaf stripe severity and plant height traits.A total of 40 QTL were identified for the disease severity (DS) and plant height (PH) traits (20 QTL for disease severity and 20 QTL for plant height).This work represents the first genetic linkage map for barley population derived from an intraspecific cross between At20 and At29 showing chromosomal regions associated with disease severity and plant height traits in barley.Fig.

M P
The objectives of this study were to construct a QTL map with chromosomal assignment and to determine the most significant QTL controlling leaf stripe severity in a barley mapping population derived from the intraspecific cross between At29 (leaf stripe resistant) and At20 (leaf stripe susceptible) aiming to facilitate the breeding of barley through marker-assisted selection of varieties characterized by leaf stripe resistant trait, which is controlled by multiple genes and difficult to handle through conventional breeding programs.
By using STS and CAPS markers developed from RFLPs a qualitative resistance gene to P. graminea, named Rdg2 and carried by a highly resistant sixrowed winter barley variety, has been mapped on the short arm of chromosome 7H(Tacconi et al., 2001).Gene confers resistance to P. graminea isolate Dg5 and has been mapped to the long arm of chromosome 2H, using a

Fig
Fig. (4): SCoT patterns of the two parents and F 2 individuals derived from the cross At20and At29 as revealed by primer SCoT-15.M is the standard DNA marker 100 bp ladder, P 1 (cv.At20) and P 2 (cv.At29).

Table ( 2
): Selective nucleotides of AFLP primer combinations, number of total bands, polymorphic bands and percentage of polymorphism.

Table ( 4
): Primer name, primer sequence, number of total bands, polymorphic bands and percentage of polymorphism as detected by SCoT.

Table ( 6
): The most significant QTL detected by the single point analysis.This analysis fits the data to the simple linear regression model y = b 0 + b 1 x + e.The results below give the estimates for b 0 , b 1 and the F statistic for each marker.