Analysis of three types of resistance gene analogs in PmU region from Triticum urartu

ZHANG Lei, ZHENG Xing-wei, QIAO Lin-yi, QIAO Ling, ZHAO Jia-jia, WANG Jian-ming, ZHENG Jun

1 College of Agriculture, Shanxi Agricultural University, Taigu 030801, P.R.China

2 Institute of Wheat Research, Shanxi Academy of Agricultural Sciences, Linfen 041000, P.R.China

3 Shanxi Key Laboratory of Crop Genetics and Molecular Improvement, Shanxi Academy of Agricultural Sciences, Taiyuan 030031,P.R.China

Abstract Resistance gene analog (RGA) screening of mapped disease-resistant genes not only helps to clone these genes but also helps to develop efflcient molecular markers for resistance breeding. The present study focused on the PmU region located on chromosome 7AuL of Triticum urartu, and recently, a nucleotide binding site (NBS)-encoding gene, Pm60, was cloned from the same chromosome arm. In this research, NBS, protein kinase (PK), and ATP-binding cassette (ABC), the three disease resistance-related gene families, were analyzed within PmU region by using informatics tools, and an expression experiment was conducted to verify their functions in vivo. Comparative genomic analysis revealed that 126 RGAs were included on chromosome 7AuL, and 30 of the RGAs as well as Pm60 were found in the PmU region. Transcriptome database analysis of T. urartu revealed 14 PmU-RGAs with expression data, and three PmU-NBSs exhibited signiflcant changes in expression after inoculation with Blumeria graminis f. sp. tritici (Bgt); TRIUR3_14879 was up-regulated, while TRIUR3_00450 and TRIUR3_06270 were down-regulated. Cluster analysis showed that these three PmU-NBSs were clustered far from the cloned wheat resistance genes. Then, qRT-PCR was performed to investigate the expression of 14 PmU-RGAs and Pm60 after inoculation with Bgt race E09; the results showed that Pm60 was speciflcally expressed in UR206 which carrying PmU, but not in susceptible UR203; while TRIUR3_14879 was signiflcantly up-regulated and TRIUR3_00450 was downregulated in UR206 after inoculation. These results indicated that PmU is Pm60, and TRIUR3_14879 and TRIUR3_00450 may also be involved in the defense against Bgt.

Keywords: cluster analysis, expression analysis, nucleotide binding site (NBS), powdery mildew, protein kinase (PK)

1. Introduction

Wheat powdery mildew, caused byBlumeria graminisf.sp.tritici(Bgt), has become a major wheat fungal disease worldwide due to increased planting density and fertilizer usage, as well as the simple genetic background of the cultivars. Signiflcant yield loss resulting from severe disease damage has previously been reported (Mwaleet al. 2014).Thus, the extensive mapping of new resistance (R) genes and their further use in breeding resistant cultivars is a costeffective method to reduce the damage (Paillardet al. 2000).

Thus far, more than 70 powdery mildew R genes have been formally designated from 49 loci in wheat (Zhanget al.2016), and seven of these genes, includingPm3b,Pm3c,Pm3f,Pm3g,Pm21,Pm38, andPm60, have been cloned(Yahiaouiet al. 2004; Srichumpaet al. 2005; Tommasiniet al. 2006; Krattingeret al. 2009; Caoet al. 2011; Zouet al.2017). Among the seven cloned genes,Pm3b,Pm3c,Pm3f,Pm3g, andPm60encode nucleotide binding site (NBS)proteins, whilePm21encodes a protein kinase (PK), andPm38encodes an ATP-binding cassette (ABC) transporter.NBS proteins, as intracellular receptors, can speciflcally recognize the effector proteins encoded by pathogens and activate the corresponding immune process, termed effector-triggered immunity (ETI), in the plant defense system (Elmoreet al. 2011). Both PK and ABC proteins are involved in microbe-associated molecular pattern-triggered immunity, which is meditated by surface-localized pattern recognition receptors (Zipfel 2009). With the exception ofPm38, which is located on wheat chromosome 7D, the other six cloned R genes are all located in the wheat A genome.

The wild einkorn wheat,Triticum urartu(2n=14, AuAu) is an A-genome donor of both hexaploid and tetraploid wheat(Dvoraket al. 1993), which possesses excellent qualities(Alvarezet al. 2013). SomeT.urartuaccessions provide good resistance to wheat diseases, such as powdery mildew, leaf rust, and stem rust (Maet al. 1997; Qiuet al. 2005; Rouse and Jin 2011; Zouet al. 2017), which rendersT.urartua promising genetic resource for resistance improvement in wheat.Qiuet al. (2005) reported thatT.urartuUR206 conferred resistance to 15Bgtraces, including E09, from major wheatgrowing areas in China. UR206 was then crossed with UR203,which is susceptible toBgt, to construct the genetic population;subsequently a powdery mildew resistance genePmUfrom UR206 was mapped to the long arm of chromosome 7AuL,which is located in the same arm of the chromosome withPm60(Zouet al. 2017). The publication of the draft genome ofT.urartu(Linget al. 2013) has made it feasible to analyze the region of a single chromosome. In the present study,we isolated the NBS, PK, and ABC sequences from thePmUregion and analyzed their expression patterns with the inoculation ofBgt;this is expected to provide references for further gene cloning and functional marker development.

2. Materials and methods

2.1. ldentification of ABC, PK, and NBS sequences in the PmU region

The whole-genome sequences and annotated protein sequences of theT.urartuG1812 (PI428198) were downloaded from the AGDB database (http://dd.agrinome.org/). Hidden Markov Model (HMM) flles of the NBS family(PF00931), the PK family (PF00069), and the ABC family(PF00005) obtained from the Pfam database (http://pfam.sanger.ac.uk/) were employed to retrieveT.urartuprotein sequences by using HMMER3.0 (Wheeler and Eddy 2013)with an E value≤1e-5. Then, the selected sequences were assessed for conserved domains by the simple modular architecture research tool (SMART) (Letunicet al. 2015).ABC, PK, and NBS sequences were mapped to their corresponding chromosomes inT.urartuaccording to their scaffold location; for sequences without position information,their chromosomal localizations were determined by blasting the wheat A-genome sequences (downloaded from the URGI database, http://wheat-urgi.versailles.inra.fr/). The theoretical product sequences of thePmUlinkage markersXpsp3094,Xwmc346,Xwmc525, andXwmc273(Qiuet al. 2005) (downloaded from the GrainGene database,http://wheat.pw.usda.gov/) were recruited to retrieve the genomic sequences of wheat chromosome 7A to acquire the corresponding physical and genomic location ofPmUin the wheat genome. Then, the Tu-scaffolds containing NBS, PK, and ABC sequences on chromosome 7AuL were selected to screen for homologous scaffolds by aligning with the correspondingPmUregion in wheat, and theTuABCs,TuPKs, andTuNBSsgenes included therein were referred to as resistance gene analog (RGAs) sequences in thePmUregion (abbreviated asPmU-RGAs).

2.2. Transcriptome database analysis

SixT.urartuaccessions were inoculated withBgtrace E09 at the two-leaf stage (Zhanget al. 2016), and the RNASeq data at 0, 4, and 24 h (Appendix A) were downloaded from the sequence read archive (SRA) database of NCBI(https://www.ncbi.nlm.nih.gov/sra/). With reference to the draft genome ofT.urartu, the fragment per kilobase of exon model per million mapped reads (FPKM) of thePmU-RGAs were determinedviaTophat and Cufflinks Software (Trapnellet al. 2009; Pollieret al. 2013). The FPKMΔT=Tn–T0values were then used to analyze the expression data ofPmURGAs after inoculation at 4 and 24 h, and the results were output with MeV (http://www.tm4.org/mev.html).

2.3. Cluster analysis and homology analysis

The encoded protein sequences of cloned R genes in wheat and other species, such asArabidopsis thaliana, rice, and barley, were obtained from the NCBI database (http://www.ncbi.nlm.nih.gov/). Protein domains were examined by using the SMART (Letunicet al. 2015). Clustal X was used to conduct protein multiple sequence alignments with default parameters (Larkinet al. 2007). The phylogenetic tree was built by the neighbor-joining methodviaMEGA6.0 Software with 1 000 bootstraps (Tamuraet al. 2013). ThePmU-RGAs ofT.urartuwere blasted against the annotated protein sequences in wheat,Aegilops tauschii, rice, andBrachypodium distachyonto obtain homologous sequences in the species mentioned above.

2.4. Material treatment and microscopic examination

T.urartuUR206 and UR203 were planted in a growth chamber at 20°C with a photoperiod of 14 h light/10 h dark per day and inoculated withBgtrace E09 at the two-leaf stage. Subsequently, the leaf tissues collected from the two materials at 0, 12, and 24 h post inoculation were stained by Coomassie brilliant blue solution (containing 0.15% (w/v)trichloroacetic acid in water and 0.6% (w/v) Coomassie brilliant blue R-250 in methanol, with a rate of 1:1) for 4 h and aniline blue solution (0.05% (w/v) aniline blue in phenol oil, pH=6.8) for 8 h as detailed in previous studies (W?spiet al. 2001; Zhanget al. 2016). The stained leaf materials were examined by using a compound microscope (Leica DMRE, Wetzlar, Germany).

2.5. qRT-PCR validation

The leaves of UR206 and UR203 were harvested at 0,4, 12, and 24 h after being inoculated with the conidia of E09 at two-leaf stage. Total RNA was extracted by RNA Extraction Kit (Sangon Biotech, Shanghai, China) and then reverse-transcribed into cDNA using M-MLV Reverse Transcription Kit (Thermo Scientiflc, Waltham, MA, USA).RT-qPCR was performed on ABI StepONE using SYBR?Green Master Mixes (Thermo Scientiflc, Waltham, MA, USA)with GADPH as the internal control and each reaction was repeated three times; data were calculated by the 2–ΔΔCtmethod and all the primers were listed in Appendix B. The expression level changed over two-fold after inoculation at 4, 12, and 24 h than that at 0 h was deflned as differential expression.

3. Results

3.1. Chromosome distribution of NBSs, PKs, and ABCs in T. urartu

In total, 463 complete NBS-, 1 046 PK-, and 141 ABC-containing protein sequences were obtained by retrieving theT.urartudatabase using HMM proflles, and the conserved domains were subsequently examined. All of the above sequences were assigned to chromosomes 1Au–7AuofT.urartu(Fig. 1) based on chromosomal location information of the annotated sequences and the blast results against the wheat A genome (Appendix C). The results showed that chromosome 2Aucontained the most RGAs(446 in total) while only 163 RGAs, the least, were included on chromosome 5Au. On the long arm of chromosome 7AuwherePmUis located, there were 126 RGAs, including 57 PKs, 60 NBSs, and 25 ABCs.

3.2. ldentification of PmU-RGAs

The analysis showed that thePmUflanking markersXgwm63,Xcfa2019,Xcfa2293, andXwmc525were located in 7AL16-0.86-0.90 on wheat chromosome 7A,andXcfa2040, the closest marker toPmU,was located at the end at 7AL18-0.90-1.00 (Fig. 2-A and B, Appendix D).Consequently, the correspondingPmUregion on the wheat chromosome 7A genetic map was 168.99–184.91 cM, while that on the genomic map was 177 081 025–182 007 620 (from 7AL_4467022 to the end of the long arm) (Fig. 2-A and C,Appendix D). Moreover, 19 RGAs-containing Tu-scaffolds on chromosome 7AuL could accordingly flnd homologous Ta-Scaffolds in this region (Fig. 2-C and D), and seven of these scaffolds had physical position information, which corresponded to the 7AL18-0.90-1.0 region in wheat(Fig. 2-B and D); therefore, 7AL18-0.90-1.00was presumed as thePmUregion.

Fig. 1 Chromosome distribution of nucleotide binding site(NBS), protein kinase (PK), and ATP-binding cassette (ABC)gene families in Triticum urartu.

The 19 Tu-Scaffolds, within thePmUregion, contained one ABC, flve TuPKs, and 24 TuNBSs. These 30 TuRGAs show homology to RGAs on the homologous group 7 chromosomes of wheat, the 7D chromosomes ofAe.tauschiiand chromosome 11 of rice (Appendix E). There was a good collinearity between thePmUregion and a certain region onB.distachyonchromosome 1, consistent with the results of previous studies (Linget al. 2013). Collinearity analysis showed that thePmUregion was well conserved,thus the informatics analysis conducted in this region was considered reliable. In addition, an NBS gene cluster, which may have been caused by tandem duplication events,was found in this region, whose location corresponded to 181.24 cM on wheat chromosome 7A (Appendices D and E).Pm60andPmR1have high sequence similarity with TRIUR3_00770 and TRIUR3_00771 of TuSca7153 on thisNBS-cluster.

3.3. Expression analysis of PmU-RGAs in transcriptome database

The expression levels of 30PmU-RGAs, after inoculation with E09, were retrieved from the transcriptome database of sixT.urartuaccessions with resistance to powdery mildew.The results revealed only 14PmU-RGAs with expression data (Fig. 3), among which the expression level of threeNBSsequences remarkably changed after inoculation:TRIUR3_06270 was down-regulated in four accessions;TRIUR3_14879 was up-regulated in all accessions, except PI428196; and the transcripts of TRIUR3_00450 were decreased in all six accessions. Moreover, no signiflcant changes in expression level were observed for the remaining genes after inoculation.

3.4. Cluster analysis of 14 PmU-RGAs and other proteins encoded by R genes

Fig. 3 Expression analysis of 14 PmU-RGAs based on transcriptome database of six Triticum urartu resistant accessions at 4 and 24 h after inoculation of Bgt race E09. The PmU-RGAs with signiflcant changes in expression levels after inoculation were labeled by ＊.

FourteenPmU-RGAs with expression data, including 11PmU-NBSs and threePmU-PKs, were selected and clustered with proteins encoded by 15 cloned R genes from wheat as well as 50 R genes fromA.thaliana, rice, barley,and other species. As shown in Fig. 4, the PKs fell into two groups: Groups I and II (Fig. 4-A). Group I included the L-PK type (I-1) and PK type (I-2, I-3) and the former contains a leucine-rich repeat (LRR) domain, which is mainly involved in the speciflc recognition of pathogenic proteins (Elmoreet al. 2011), and TRIUR3_19375 belonged to this group.TRIUR3_07971 together withPm21, a cloned powdery mildew resistance gene, were grouped into the I-2 type and TRIUR3_30359 was grouped into type I-3. Group II consisted of only one member,Yr36(Fuet al. 2009),which is a wheat stripe rust resistance gene with a START domain in its PK protein. The NBSs were also divided into two groups (Fig. 4-B). Group I included the TIR-NBS-LRR(TNL) type, suggesting that in this group, the LRR domain is fused to the C-terminus and a TOLL/interleukin 1 receptor(TIR) domain is linked to the N-terminus. This TNL type is speciflc to dicotyledonous plants (Hammond-Kosack and Jones 1997), and noPmU-NBS was included in this group.Group II included a non-TNL type, in which the N-terminus of NBS in Group II-1 contains a coiled-coil (CC) domain(Hammond-Kosack and Jones 1997) that mainly binds to pathogen-derived ligands, and all 11PmU-NBSs were included in this group, while Group II-2 comprised two CNL and NL structures. TRIUR3_00772, TRIUR3_01038,TRIUR3_09136, and TRIUR3_09139 from Group II-1(Fig. 4-B) were clustered closely with the cloned wheat R genes; TRIUR3_00772 was located in the same branch withSr35, a wheat stem rust resistance gene. Nevertheless, the other sevenPmU-NBSs were clustered far away, among which the TRIUR3_06270 and TRIUR3_00450 were situated in a separate branch.

3.5. Reactions to Bgt of UR206 and UR203

After infecting with powdery mildew, the cells in Triticeae crops typically form papillae to prevent further invasion of the appressorium germ tube (Collingeet al. 2009), which is a main reaction phenotype for plant basal resistance and some resistance genes, such asmlo(Wolteret al. 1993)andPm2(Liet al. 2005). In the present study, papillae were observed on the leaves of UR206 at 12 and 24 h post inoculation (Fig. 5-B and C), but not in UR203 (Fig. 5-E),instead of multiple hyphae at 24 h post inoculation (Fig. 5-F).Furthermore, after inoculation for one week, there were no powdery mildew conidia and necrotic spots on UR206 leaves, while the incidence of UR203 was sufflcient. Thus,the reinforcement of the cell wall is one of the disease resistance phenotypes ofPmUrather than hypersensitive reaction.

3.6. The qRT-PCR analysis of 14 PmU-RGAs and Pm60

The expression level changes of 14PmU-RGAs, before and after E09 inoculation, were identifled in UR206 bearingPmUand the susceptible accession UR203, respectively. The results revealed that with the exception of TRIUR_14879,which showed no expression in UR203, the remaining 13PmU-RGAs were all expressed in both UR206 and UR203 (Fig. 6). Two NBS sequences, TRIUR_14879 and TRIUR_00450, presented differential expression after inoculation. TRIUR_14879 was up-regulated in UR206 but showed no expression in UR203; the transcripts of TRIUR_00450 were decreased in UR206, while no change was observed in UR203. Thus, TRIUR_14879, in UR206,may play an important role in the process of disease resistance, in which TRIUR_00450 may also participate.

Fig. 5 Reaction phenotypes of UR206 (A–C) and UR203 (D–F) following Bgt inoculation. A and D, 0 h post inoculation. B and E,12 h post inoculation; C–F, 24 h post inoculation. c, condium. PA, papilla; AGT, appressorium germ tube; Hy, hyphae.

Fig. 6 Relative expression levels of 14 PmU-RGAs, Pm60, and PmR1 in UR206 and UR203 at 0, 4, 12, and 24 h after inoculation of Bgt race E09. Signiflcant changes in expression level after inoculation were labeled by ＊ with P-value as 0.05. The error bars indicate the SD.

The qRT-primers ofPm60,PmR1and three otherNBSsequences on TuSca7153, except for TRIUR_00772,were also used for expression level veriflcation. The results showed that TRIUR_00770, TRIUR_00771, and TRIUR_00773 were expressed in neither UR206 nor UR203. However,Pm60andPmR1were only expressed in UR206, and the latter showed a signiflcant difference in the expression level after infection, revealing thatPm60andPmR1are carried by UR206.

4. Discussion

4.1. The PK and NBS families in PmU section

During the evolutionary process against fungi, bacteria,viruses, and other invading pathogens, plants have gradually developed a set of sophisticated defense mechanisms.The mechanism, by which a pathogen can be speciflcally recognized by corresponding R genes, is of particular importance (Hammond-Kosack and Jones 1997; Jones and Dangl 2006). The structures of these recognition proteins are conserved, among which the PK and NBS resistant proteins are the largest and most important families in plants.Within the cloned wheat R genes, except the powdery mildew R genes, the stripe rust resistance geneYr36(Wheeler and Eddy 2013) is aPKand another seven genes,includingYr10,Lr1,Lr10,Lr21,Sr22,Sr33,Sr35,andSr45,areNBSs(Huanget al. 2003; Cloutieret al. 2007; Fuet al.2009; Selaet al. 2012; Periyannanet al. 2013; Saintenacet al. 2013; Liuet al. 2014; Steuernagelet al. 2016). Gene prediction based on the draft genome ofT.urartushowed that the number of members in thePKandNBSfamilies was signiflcantly higher than that in rice and maize, which suggested that these two gene families may play more important roles in the disease resistance ofT.urartu.

In the present study, oneTuABC, threeTuPKs, and 11TuNBSswith expression data were identifled from thePmUregion inT.urartu. Transcriptome database analysis and qRT-PCR analysis showed that twoPmU-NBSs:TRIUR3_14879 and TRIUR3_00450 were signiflcantly changed in expression levels during the process against E09 invasion, and Os11g41540 (The Rice Chromosomes 11 and 12 Sequencing Consortia 2005) and Os11g47780 (Dinget al. 2007), the orthologous proteins of TRIUR3_14879 and TRIUR3_00450 in rice both played important roles in disease resistance. Hence, further validations, such as transgenic experiments, are favored to verify the functions of TRIUR3_14879 and TRIUR3_00450. Furthermore,twoPmU-NBSs, TRIUR3_00770 and TRIUR3_00771 on TuSca7153 were not expressed in either UR206 or UR203, while their highly similar sequencesPm60andPmR1were speciflcally expressed in UR206, revealing that TRIUR3_00770 and TRIUR3_00771 have been mutated toPm60andPmR1in the UR206 genome.

4.2. Possible disease resistance mechanism of PmU

Pm60exhibited no signiflcant change in expression post inoculation in the present study, consistent with the description of Zouet al. (2017).PmUis likelyPm60with a complex disease resistance mechanism. In addition,TRIUR3_14879 and TRIUR3_00450 may also be involved in the defense of UR206 againstBgt. TRIUR3_14879 showed up-regulation but was not expressed in the susceptible accession UR203, indicating that this sequence may participate in the powdery mildew resistance process through the ETI signaling pathway. The expression of TRIUR3_00450 in UR203 showed no signiflcant change but was down-regulated in UR206. The down-regulation ofNBSgene expression after inoculation was also reported in some studies (Aryaet al. 2014; Yuet al. 2014; Zhanget al.2016);NBSgenes may function in pathogen monitoring or other plant physiological processes, while the expression of a speciflcNBSgene, such as TRIUR3_14879, was up-regulated and accompanied by the down-regulation of otherNBSgenes (like TRIUR3_00450) in response to the invasion ofBgt(Bonardiet al. 2011; Mukhtaret al. 2011).In the present study, the down-regulation of TRIUR3_00450 may contribute to the proper function of TRIUR3_14879 inBgtresistance, which requires follow-up validation.

Many studies on cloned wheat R genes have indicated that one single R gene is sufflcient to provide resistance to pathogens. However, a growing number of studies have revealed that disease resistance is typically conferred by the interaction between different R genes located in the same region. For example, the rice bacterial blight resistance geneXa21, belonging to thePKfamily, is regulated by anotherPKgeneRox1located near its locus(Leeet al. 2011); the defense process ofArabidopsisagainst certain bacteria or fungi requires a combination of two NBS proteins, RPS4 and RRS1, to initiate the resistance signal transduction (Sarriset al. 2015); barley resistance to powdery mildew is regulated by multiple genes, includingPKandNBS(Douchkovet al. 2014).Thus, we propose that the resistanceof UR206 may be subjected to the regulation of multiple genes.

4.3. Utilization of PmU

Selecting and breeding disease-resistant wheat cultivars by using powdery mildew R genes plays a pivotal role in reducing yield loss (Mwaleet al. 2014). Some R genes that originated from close relatives of wheat confer excellent and durable resistance to powdery mildew and have been broadly used in wheat production. However, due to the various races ofBgtand the rapid mutation of virulence genes, most resistant cultivars begin to lose their resistance after 3–5 years of application. For example,Pm8, a widely used powdery mildew R gene derived from rye, has lost resistance (Paillardet al. 2000). Therefore, the continuous exploration and utilization of novel wheat powdery mildew R genes from its close relatives is of great importance.Unlike other distant relatives,T.urartu, the ancestor of the wheat A genome, can produce high levels of chromosome pairing and genome exchange, by which the R genes carried by disease-resistant accessions can be introgressed into cultivated wheat to achieve resistance improvement (Maet al. 1997; Santraet al. 2013). UR206 showed resistance toBgtraces in major wheat producing regions in China(Qiuet al. 2005), which renders this cultivar an excellent source of resistance. The F1population, derived from the cross between UR206 and a common wheat cultivar Chinese Spring, had 28 chromosomes and an average of 5.32 bivalents and 17.36 univalents in meiotic pollen mother cells; after backcrossing with Chinese Spring, the derivative lines with 42 chromosomes and similar powdery mildew resistance to that of the donor UR206 were successfully obtained (Qiuet al. 2005), demonstrating that the exogenous resistance genes can be steadily introduced into common wheat. In the present study, we analyzed three types of RGAs in thePmUregion of UR206. TheNBSsequencePm60was identifled as a candidate gene,with the functional marker M-Pm60 (Zouet al. 2017). Two otherNBSsequences, TRIUR3_14879 and TRIUR3_00450,may also be involved in disease resistance. Molecular markers developed based on these sequences can be used for improving the efflciency of molecular marker-assisted selection in wheat breeding.

5. Conclusion

Thirty RGAs, including one ABC, flve TuPKs, and 24 TuNBSs, as well asPm60were found in thePmUregion. Two TuNBSs among them, TRIUR3_14879 and TRIUR3_00450, were up- and down-regulated,respectively in UR206 carriedPmUafter inoculation withBgt; andPm60was speciflcally expressed in UR206 but not in susceptible UR203, indicating thatPmUisPm60.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31601307), the Key Scientiflc and Technological Innovation Platform of the Main Crop Germplasm Innovation and Molecular Breeding in Shanxi Province, China (201605D151002) and the Youth Foundation of Institute of Crop Science, Shanxi Academy of Agricultural Sciences (ZZQ1701). We would like to acknowledge Prof.Jia Jizeng of Chinese Academy of Agricultural Sciences for providingT.urartuUR203 and UR206.

Appendicesassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm