An AFLP marker linked to the leaf rust resistance gene LrBi16 and test of allelism with Lr14a on chromosome arm 7BL

Peipei Zhang，Huixin Zhou，Caixia Lan，Zaifeng Li*，Daqun Liu*

aDepartment of Plant Pathology，College of Plant Protection，Hebei Agricultural University，Baoding 071001，Hebei，China

bCollege of Plant Science and Technology，Huazhong Agricultural University，Wuhan 430070，Hubei，China

cInternational Maize and Wheat Improvement Center（CIMMYT），Apdo.Postal 6-641，06600 Mexico D.F.，Mexico

An AFLP marker linked to the leaf rust resistance gene LrBi16 and test of allelism with Lr14a on chromosome arm 7BL

Peipei Zhanga，1，Huixin Zhoua，1，Caixia Lanb，c，Zaifeng Lia，*，Daqun Liua，*

aDepartment of Plant Pathology，College of Plant Protection，Hebei Agricultural University，Baoding 071001，Hebei，China

bCollege of Plant Science and Technology，Huazhong Agricultural University，Wuhan 430070，Hubei，China

cInternational Maize and Wheat Improvement Center（CIMMYT），Apdo.Postal 6-641，06600 Mexico D.F.，Mexico

A R T I C L E I N F O

Article history：

6 November 2014

Accepted 19 November 2014

Available online 10 January 2015

Amplified fragment length polymorphism

Genetic linkage

Molecular mapping

Puccinia triticina

Leaf rust（LR），caused by Puccinia triticina，is one of the most widespread diseases of common wheat（Triticum aestivum L.）worldwide.The LR resistance gene LrBi16 has been mapped on chromosome arm 7BL in Chinese wheat cultivar Bimai 16 and was closely linked to SSR loci Xcfa2257 and Xgwm344 with genetic distances of 2.8 cM and 2.9 cM，respectively.In the present study，a total of 304 AFLP primer pairs were used to screen Bimai 16 and Thatcher and resistant and susceptible DNA bulks.The polymorphic AFLP marker P-ATT/M-CGC173bpwas used to genotype F2and F3populations to identify markers more closely linked to LrBi16.Marker P-ATT/M-CGC173bpwas tightly linked to LrBi16 with a genetic distance of 0.5 cM.As LrBi16 was mapped near the Lr14a locus，809 F2plants from the Bimai 16/RL6013（Lr14a）cross were inoculated with the Pt pathotype FHNQ to test the allelism of Lr14a and LrBi16.All of the F2plants were resistant to FHNQ（IT between；and 2），suggesting that Lr14a and LrBi16 are allelic.

?2015 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license

（http://creativecommons.org/licenses/by-nc-nd/4.0/）.

1.Introduction

Leaf rust（LR），caused by Puccinia triticina（Pt），is one of the most important and widespread diseases in wheat（Triticum aestivum L.）.It occurs in a wide range of climates wherever wheat is grown and causes yield losses up to 65%under favorable conditions［1］.In China LR has historically been important only in the southwest and northeast regions［2］，but with increased planting densities and changing management practices it has become increasingly important in most major wheat-producing areas.Destructive epidemics of LR occurred in 1969，1973，1975，and 1979 in China［2］，and yield losses occurred in regions of Gansu，Sichuan，Shaanxi，Henan and Anhui provinces in 2012［3］.Resistant cultivars are the most efficient，economical，and environmentally safe way to manage LR.

Resistance to LR can be classified into two types，qualitative resistance conferred by single resistance genes（also termed as major，seedling，or race specific resistance）and quantitative resistance，mediated by multiple genes or quantitative trait loci（QTL）（alsotermed asadult plantresistance，race nonspecific，or slow rusting resistance）.To date，more than 100 LR resistance genes have been described in wheat，with 72 formally designated genes［4］.Most of these genes are qualitative and interact with the pathogen in a gene-for-gene fashion［5］.This type of gene is rapidly overcome by the pathogen.Only Lr9，Lr19，Lr24，and Lr38 are currently effective against the prevalent Chinese Pt pathotypes［6，7］.Thus，it is important to identify and use new resistance resources to control the dynamic and rapidly evolving pathogen population［8］.

Molecular markers，including RFLP，RAPD，SSR，AFLP，and EST，have been widely used to map LR resistance genes in different genetic populations.To date，46 LR genes have been mapped on various chromosomes using molecular markers［9］.However，most markers are too far from resistance genes for reliable use in breeding programs.It is thus important to identify more closely linked or gene-based molecular markers for marker-assisted selection（MAS）.The AFLP technique has been widely used in the construction of high-density genetic maps，enabling several LR genes to be finely mapped in wheat using this type of marker combined with bulked segregant analysis（BSA）［10-15］.

Bimai 16，released in 2004 by the Bijie Agricultural Science Research Institute，Guizhou province，has high levels of resistance to leaf rust，stripe rust（caused by Puccinia striiformis f.sp.tritici）and powdery mildew（caused by Blumeria graminis f.sp.tritici）under field conditions［16，17］.A dominant LR resistance gene，LrBi16，on chromosome arm 7BL is flanked by SSR markers Xcfa2257 and Xgwm344 at genetic distances of 2.8 cM and 2.9 cM，respectively［18］.LrBi16 is very close to the Lr14 locus，with named resistance alleles Lr14a and Lr14b that were combined in a rare recombination event［19］.The objectives of the present study were to identify molecular markers more closely linked to LrBi16 using AFLP markers and to determine the allelic relationship between LrBi16 and Lr14a.

2.Materials and methods

2.1.Plant materials and Pt pathotypes

Atotalof359F2plantsand298F3lineswerederivedfromacross of the resistant parent Bimai 16（pedigree:8513-1624/Ji 1002）withthesusceptibleparentThatcher.TheChinesePtpathotype，F(xiàn)HTT，was used to inoculate the genetic materials.Phenotypic and genotypic data for the F2and F3populations were derived from our previous study［18］.A total of 809 F2plants from Bimai 16/RL6013（Lr14a）and the Chinese P.triticina pathotype FHNQ were employed to test the allelism of LrBi16 with Lr14a.The Thatcher near-isogenic line，RL6013 with Lr14a，was kindly provided by the USDA-ARS Cereal Disease Laboratory，University of Minnesota，St.Paul，USA.The two pathotypes were designated based on the coding system of Long and Kolmer［20］with addition of a fourth letter for the reactions of a fourth set of differentials（http://www.ars.usda.gov/SP2UserFiles/ad_hoc/ 36400500Cerealrusts/pt_nomen.pdf）.All the wheat germplasm and Pt pathotypes are maintained at the Biological Control Center for Plant Diseases and Plant Pests of Hebei，Hebei Agricultural University，Baoding，China.

Table 1-AFLP adapters developed in this study.

2.2.Allelism analysis between LrBi16 and Lr14a

Pathotype FHNQ（avirulent to both LrBi16 and Lr14a）was used to inoculate the F2population from the Bimai 16/RL6013（Lr14a）cross for the allelism test between Lr14a and LrBi16. The F2plants were grown in a growth chamber.Inoculation was performed when the first leaves were fully expanded，by brushing urediniospores from fully infected susceptible plants of Zhengzhou 5389 onto the F2plants.Inoculated plants were placed in plastic-covered cages，incubated at 15°C and 100% relative humidity（RH）for 24 h in darkness，and then transferred to a growth chamber programmed for 12 h light/12 h darkness at 18 to 22°C and 70%RH.Infection types（ITs）were scored 10 to 14 days after inoculation according to the Stakman scale，modified by Roelfs et al.［21］.

2.3.AFLP analysis

A total of 304 AFLP primers were used to screen the parents and resistant（Br）and susceptible DNA bulks（Bs）.Markers with consistent polymorphism between the parents and the bulks were used to analyze the entire F2and F3populations. AFLP analysis was performed following Zhang et al.［14］. Genomic DNA was digested with Pst I and Mse I（Table 1），ligated with adapters，and then pre-amplified with primers containing one selective nucleotide（Table 2）.The samples were diluted 20-fold with ddH2O and stored at 4°C.Selective amplification was achieved by primers with three selective nucleotides（Table 3）.Amplification was performed in a T-gradient thermal cycler PCR（Bio-Metra，G?ttingen，Germany）. PCR-amplified AFLP products（5 μL）were mixed with 1 μL of loading buffer（98%formamide，10 mmol L-1EDTA，0.25% bromophenol blue，0.25%xylene cyanol，pH 8.0），and loaded on 6%denaturing polyacrylamide gels.The gels were run at 80 W for approximately 3 h and visualized by silver staining［22］.

2.4.Linkage analysis

Linkage analysis using the combined previous SSR and current AFLP results was performed using the software MapManager QTXb20［23］and recombination values converted to centiMorgans using the Kosambi mapping function［24］.

Table 2-Pre-amplification primers.

Table 3-Primers used for selective amplification.

2.5.Cloning of PCR fragments amplified from the AFLP markers

The isolation of PCR fragments followed Xu et al.［25］.Briefly，the PCR products from the AFLP marker were excised from dried gels.Spliced gels containing the amplification products were transferred to a PCR tube and eluted twice with 200 μL of 1×TE buffer（pH 8.0）for 30 min and once with 200 μL of ddH2O for 30 min.The gel was then soaked in 50 μL of ddH2O and kept in a PCR thermocycler at 95°C for 10 min to release the DNA from the gel.After the gel debris was spun down by centrifuging at 3000 r min-1for 10 min，the supernatant was used as template DNA for PCR amplification，using the same AFLP primers and PCR conditions.

3.Results

3.1.Seedling allelism test

Both Bimai 16 and RL6013 were resistant to pathotype FHNQ with IT 2.A total of 809 F2plants derived from Bimai 16/RL6013（Lr14a）were inoculated with pathotype FHNQ.Both parents and all the F2plants were resistant to FHNQ with ITs ranging from fleck（；）to 2，indicating that Lr14a and LrBi16 were allelic or closely linked genes.

3.2.AFLP marker of LrBi16

Among 304 AFLP markers，only P-ATT/M-CGC173bpwas polymorphic between the parents as well as Br and Bs.This marker was then used to genotype the entire F2population.Marker P-ATT/M-CGC173bpwas closely linked to LrBi16 at a genetic distance of 0.5 cM（Figs.1，2）and was more closely linked to LrBi16 than was marker Xgwm344 reported in our previous study［18］.The marker was also used to genotype the 298 F3lines，and was found to lie 0.7 cM from LrBi16.

3.3.Cloning and sequencing of the AFLP specific DNA fragment

The size of the re-amplified band was the same as that of the AFLP-specific DNA fragment after recovery and a specific band of 173 bp was sequenced（Fig.3）.Although STS primers were designed，no polymorphism was found between the parents and the bulks（Fig.3）.This AFLP could not be converted to an STS/SCAR marker，owing to the small fragment size.

4.Discussion

4.1.Comparison between LrBi16 and other wheat leaf rust resistance genes on chromosome arm 7BL

Fig.1-Electrophoresis of PCR products amplified with AFLP marker P-ATT/M-CGC173bpon a polyacrylamide gel.M：PBR322/ MspI marker；P1：resistant parent Bimai 16；P2：susceptible parent Thatcher；Br：resistant bulk；Bs：susceptible bulk；R：resistant F2plants；and S：susceptible F2plants.

Fig.2-Deletion bin map of chromosome 7B［30］（left）and linkage maps of leaf rust resistance gene region and 8 loci based on 359 F2plants of Bimai 16/Thatcher（right）.Locus names and corresponding locations are indicated on the right and map distances in centiMorgans are shown on the left（center panel）and compared with previously［31］published map（right panel）.

Fig.3-Specific band sequence of P-ATT/M-CGC173bp.Primer sequences are shown in shadow part.

Leaf rust resistance genes located on chromosome arm 7BL includeLrBi16，Lr14a，Lr14b［19］，Lr68［26］，and LrFun［9］.Lr68isan adult plant resistance gene closely linked to SSR marker Xgwm146［26］，which is also linked to LrBi16［18］，whereas LrBi16confersseedlingresistancewithITsfrom1to2［18］.Lr14a，originating in T.turgidum，was mapped on 7BL［27］，and like LrBi16，is linked to SSR marker Xgwm344-7B［18］.However， RL6013（Thatcher+Lr14a）was susceptible to most pathotypes，including two that are avirulent to LrBi16［18］，indicating that LrBi16 is different from Lr14a.A test of allelism involving 809 F2plants revealed no recombinants between Lr14a and LrBi16，indicating that the genes are allelic or tightly linked.In an earlier study Lr14b was very closely linked rather than allelic to Lr14a［19］.Another seedling resistance gene，LrFun，was also mapped near LrBi16 on chromosome arm 7BL，but LrBi16 differed from LrFun in reactions to a panel of Pt cultures［9］. However，the relationship between LrBi16 and LrFun awaits confirmation by further allelic studies.Several LR resistance genes have been mapped in this region，suggesting that it is an important region for leaf rust resistance.

4.2.Development of STS/SCAR markers from the AFLP marker

Although AFLP technology can detect many genetic loci，it has limitations when applied to MAS，owing to the production of many nonspecific bands and corresponding difficulty in scoring. STS markers developed from AFLP products are more specific and more easily used in MAS.However，not all AFLP can be successfullyconverted toSTS markers，owing to the small size of DNA fragments amplified by AFLP and a lack of polymorphisms in restriction sites between genotypes［28］.In addition，different AFLP fragments with the same size may co-migrate on a gel，and a target polymorphic band may contain contaminating fragments from different bands［11，29］，again complicating the development of an STS marker from an AFLP product.In the present study，the specific fragment linked to LrBi16 was cloned and sequenced，and primers were designed according to the sequence，but no polymorphism was found between resistant and susceptible bulks.This result was attributed to either the small size of the AFLP fragment or a lack of polymorphism at the restriction site.

Acknowledgments

This study was supported by the National Natural Science Foundation of China（31361140367 and 31301309）and the Natural Science Foundation of Hebei Province（C2014204113）.

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27 June 2014

in revised form

s.

E-mail addresses:lzf7551@aliyun.com（Z.Li），ldq@hebau.edu.cn（D.Liu）.

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science，CAAS.1These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.cj.2014.11.004

2214-5141/?2015 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.