Identification of stably expressed QTL for resistance to black shank disease in tobacco(Nicotiana tabacum L.)line Beinhart 1000-1

Yusheng Zhng,Xun Guo,Xingxing Yn,Min Ren,Cihong Jing,Yzeng Cheng,Liuying Wen,Dn Liu,Yu Zhng,Mingming Sun,Qunfu Feng,Aiguo Yng,*,Lirui Cheng,**

aKey Laboratory of Tobacco Improvement and Biotechnology,Tobacco Research Institute,Chinese Academy of Agricultural Sciences,Qingdao 266100,Shandong,China

bCollege of Plant Science and Technology,Qingdao Agricultural University,Qingdao 266109,Shandong,China

1.Introduction

Black shank(BS)is a fungal disease caused by the soil borne oomycete Phytophthora nicotianae(van Breda de Haan)and is one of the most important diseases affecting tobacco(Nicotiana tabacum L.)production in many tobacco-producing areas[1].Phytophthora nicotianae attacks tobacco roots,stems and leaves at all growth stages,leading to wilting,chlorosis,root and stem necrosis,stunting,and even plant death[2].To date,strategies for plant disease prevention and control include genetic resistance,crop rotation and application of fungicides[3–5].Resistance is an economically attractive approach when it can be incorporated into varieties without significantly affecting yield and quality characteristics.

Physiological races 0 and 1[6–9]are the predominant pathogenic races and are widely distributed throughout China and other major tobacco cultivation areas[10,11].Shifts from race 0 to race 1 occurred when the Php or Phl gene was introgressed into N.tabacum[12–14].Other described races such as race 2 and race 3 also cause BS[15–18].The sources of resistance investigated to improve resistance to black shank(BS)exhibit either monogenic resistance or polygenic resistance in cultivated tobacco.Genes Php and Phl introgressed into N.tabacum from Nicotiana plumbaginifolia Viv.and Nicotiana longiflora Cav.,respectively,confer immunity to P.nicotianae race 0.However,these resistance factors do not confer resistance to other pathogen races.Polygenic resistance most likely derived from the cigar variety Florida 301[19]is partial in effect and non-race specific[20,21].This type of resistance is currently present in commercial flue-cured and burley tobacco varieties[14,19].

Cigar tobacco line Beinhart 1000,derived from a selection of tobacco variety Quin Diaz reportedly has the highest reported level of BS resistance[1].This line has been studied genetically[22–24].

Beinhart 1000-1(hereafter referred to as BH)is a selection from Beinhart 1000 that has consistently shown black shank resistance equal to that of Beinhart 1000[25,26],and is considered an important tobacco variety with high resistance to BS[27,28].Heggestad and Lautz[29]reported that BH showed higher resistance than the cigar-wrapper variety Rg and Beltsville breeding lines with resistance derived from Nicotiana plumbaginifolia.Nielsen[30]evaluated BH response to black shank at eight locations in seven countries and it performed with the best resistance at all locations.

The combination of quantitative trait loci(QTL)mapping and molecular marker-assisted selection(MAS)is a good alternative strategy for improvement of complex traits[31].Previous QTL mapping studies of resistance to BS in tobacco focused on the highly resistant resources Beinhart 1000 and Florida 301.Vontimitta and Lewis[23,24]detected two major QTL associated with resistance in a recombinant inbred line(RIL)population derived from a Beinhart 1000×Hicks cross.The QTL located between markers PT61472 and PT30174 was repeatedly detected,explaining 24.7%–54.7%of the phenotypic variance in different environments.The second QTL associated with resistance to BS located between markers PT61373 and PT51164 explained 16.8%–20.4%of the phenotypic variance.Xiao et al.[19]conducted a QTL mapping study of resistance in line Florida 301.The major QTL between PT61472 and PT30174 derived from Beinhart 1000 was also found in Florida 301,suggesting that Beinhart 1000 and Florida 301 shared the same major locus.

Tobacco breeders have usually believed that the genetic bases of resistance to BS in Beinhart 1000 and BH were similar due to their shared origins and similar BS responses.The two sources are often confused and treated as the same material in tobacco breeding,particularly in China[32,33].

The primary objective of this study was to identify the genetic architecture and stable QTL of resistance to BS in a population derived from a Beinhart 1000-1×Xiaohuangjin 1025 cross,and set a preliminary foundation for further study of the underlying mechanisms of BS resistance and for molecular-assisted selection(MAS)in breeding programs.

2.Materials and methods

2.1.Plant materials

Beinhart 1000-1(hereafter referred to as BH,P1),a selection of Beinhart 1000 expressed a high level of resistance to BS and was used as the female parent for this study.The flue-cured variety Xiaohuangjin 1025(hereafter referred to as XHJ,P2)developed by the Tobacco Research Institute,Chinese Academy of Agricultural Sciences,was used as the susceptible male parent and check(CK).An F2population was produced from a single BH×XHJ F1individual and the F1plant was also backcrossed to XHJ to develop a BC1F1population with 100 plants.In 2012,220 F2individuals were used in bulked segregant analysis(BSA)to detect markers associated with BS response.According to the results of QTL mapping,one BC1F1plant was selected by MAS and sown to obtain a BC1F2population with 120 plants.The corresponding BC1F2plants were separately bagged to produce selfed BC1F2:3lines(Fig.1).

2.2.Pathogen isolation and inoculum preparation

The Plant Protection Laboratory of the Chinese Academy of Agricultural Sciences provided an isolate of race 0 of Phytophthora nicotianae for disease response tests.The isolate was maintained on oatmeal glucose agar medium at 28 °C in darkness for 7–10 days.Inoculum was prepared as described by Vontimitta and Lewis et al.[23]with minor modifications.A single agar plug(5 mm diameter)was removed from the actively growing edge of the oatmeal glucose agar culture and placed in a 500 mL glass flask containing sterilized millet(Setaria italica)grain.After 18 days,the millet seeds that were by then completely covered by hyphal growth were used for inoculation.

2.3.Inoculation and phenotyping

The F2population,BC1F1populations and BC1F2populations,along with the parental lines,were planted in BS nurseries at the Jimo Experimental Station,Tobacco Research Institute,Chinese Academy of Agricultural Sciences,Qingdaoin Shandong province(35.4°N,119.3°E,15 m altitude),with a row length of 10 m,row spacing of 1.2 m and plant distance of 0.5 m.Forty days after transplanting,approximately 5 g millet grains colonized by Phytophthora nicotianae 0 race was added to the soil near each plant and then lightly covered with soil for inoculation.

The BC1F2:3populations were tested in the field at Jimo Experimental Station and as seedlings in a growth chamber.The field evaluations were performed in a randomized complete block design with two replications.Each replication consisted of two 10-plant rows with a row length of 10 m,row spacing of 1.2 m and plant distance of 0.5 m.The inoculations and phenotypic evaluations of BC1F2:3in the field were performed as described above.

Fig.1–Summary of procedures used in quantitative trait locus(QTL)mapping of resistance to black shank.

The seedling evaluations were also performed in two replications.Experimental units consisted of25plants contained within a twenty-five-compartment segment of a 25 cm×25 cm square plastic tray.The seeds were sown on Murashige and Skoog(1/2 MS)medium[34]under plastic domes with a 12 h/12 h light/darkness photoperiod at 25°C with 70%–85%relative humidity.After 40 days,the seedlings were transplanted into 25 cm×25 cm square plastic trays.Twenty-one days after transplanting,the roots of the seedlings were inoculated with approximately 1 g of millet grains containing actively growing P.nicotianae and then placed in a chamber with a 12 h/12 h photoperiod at 27 °C with 70%–85%relative humidity.

BS severities(DS,disease scale)were evaluated 40 days postinoculation for the field trial and 10 days post-inoculation for the seedling trial using an empirical 6-point scale(YC/T39-1996,China),where 0 represented a highly resistant response and 9 represented a highly susceptible response.Disease severity scores were used for the assessment of F2and BC1F2individuals.For the two parents and BC1F2:3lines,disease index(DI)scores based on the disease severity were used for assessment and were calculated using the following formula:

2.4.Bulked segregant analysis

BSA was used to identify significant markers associated with BS response.Resistant and susceptible bulks based on the BS severity were constructed by mixing equal amounts of DNA from the 20 most resistant plants and 20 most susceptible plants from the F2population.According to sequences published by Bindler et al.[35],a total of 2156 pairs of simple sequence repeat(SSR)primers were synthesized and scanned for polymorphism between the parents using 8%non-denaturing polyacrylamide gels.PCR and gel staining were performed as described by Vontimitta and Lewis[23]with minor modifications.SSR markers that showed polymorphism between the parents were used to detect polymorphisms between the two bulks.

The SSR markers showing polymorphism between the bulks were used to analyze genotypes of the entire F2population,anda one-way ANOVA using PROC GLM in the SAS statistical software package(SAS Institute 1996)was used to detect significant associations between genotypes and phenotypes at the level of P＜0.05[36].SNPCPS2genotyping of locus NtCPS2[37]was also carried out using the PCR primers forward 5′-CAGAATACAATCTCCTCTCCCCA-3′ and reverse 5′-ATTTTCTGGTGTTTTGATTGGCG-3′in the entire F2population,and fragment results were analyzed by sequence alignment.

2.5.Marker assisted selection in the backcross population

Upon identification of markers associated with the traits,the markers polymorphic between the parents located on the same linkage group as the linked markers were used for marker-assisted selection(MAS)in the BC1F1population.One BC1F1plant that contained heterozygous segments surrounding the specific loci but homozygous in the other loci affecting BS response was selected from the BC1F1population(Fig.1).

2.6.Linkage map and QTL detection

All markers in specific linkage groups and polymorphic between the parents were used to identify genotypes in the entire BC2F2population derived from the selected BC1F1individual.Linkage groups were constructed using JoinMap 4.1 software[38].A recombination frequency≤0.4 and a limit of detection(LOD)value≥3.0 were used as thresholds for groups.The Kosambi function was used to calculate map distances.Genetic maps were drawn using MapChart 2.0 software[39].

Interval mapping(IM)and multiple-QTL model(MQM)mapping,implemented in MapQTL version 6.0,were used to conduct QTL mapping[38].Interval mapping(IM)based on a maximum likelihood approach was performed every 1 cM along linkage groups to scan QTL.Markers closely linked to positions with the highest LOD score were used as cofactors for MQM analysis.The empirical LOD threshold was set at 2.35 for claiming a significant QTL based on 1000 runs and a genome-wide type Ι error rate of 0.05.

3.Results

3.1.Phenotypic distribution of response to BS

The BS responses of the parents were significantly different;BH expressed a very high level of resistance with a score of 0 for both disease severity(DS)and disease index(DI),whereas XHJ was highly susceptible with of DS 9 and DI of 84.50%–100.00%for different environments(Fig.2).The average value of DS for the F2population was 3.20,and for the BC1F2,3.57.For the BC1F2:3population,the mean DI for the field stage and seedling stage tests were 42.02%and 41.86%,respectively.All populations displayed significantly skewed distributions in the direction of resistance,indicating that both major genes and polygenes controlled resistance to BS.As shown in Table 1,the ANOVA detected highly significant differences among lines(P＜0.001)and no significant differences between environments and lines(P=0.289).

Fig.2–Frequency distribution of responses to BS in different environments.A,B,Disease severities for the F2population in 2012(A)and BC1F2population in 2013(B).C,D,Disease indices(%)for the BC1F2:3population in the field in 2014,BC1F2:3population in the greenhouse in 2015.

Table1–ANOVA of disease severity in the BC1F2:3 population.

3.2.Markers associated with resistance to BS from BSA

Two hundred and seventy eight of the 2138 SSR markers were polymorphic between the two parents(Table S1).A total of 156 clear,well amplified and polymorphic SSR markers were employed for further analysis using the BSA method,and seven of those markers showed polymorphism between the two bulks(Table 2).The seven polymorphic markers were located in linkage groups 6,7,17,18,and 22 designated by Bindler et al.[35].The seven markers were used to genotype the entire F2population.Previous studies reported that marker PT61373 and SNPCPS2in linkage group 15 were significantly associated with a QTL for resistance to BS in Beinhart 1000[23,24,37].To avoid missing major QTL the markers were also genotyped in the second study although they showed no significant association between the two bulks and parents.One-way ANOVA results for the F2population confirmed significant markers on linkage groups 6,7,17,18,and 22.Among them,PT30174(Chr.7,12.85 cM)and PT51378(Chr.17,103.44 cM)showed the most significant associations with resistance at the level of P＜3.16×10?10and P＜3.59×10?4,respectively.The results indicated that two QTL affecting response to BS were derived from BH(Table 2).

3.3.Selection of a heterozygous individual for backcrossing

For identification of the major QTL for resistance to BS,85 polymorphic markers between the parents on linkage groups 6,7,15,17,18,and 22 were used in MAS.One BC1F1plant was selected for further identification of major QTL.As shown in Fig.3,four heterozygous segments were located on linkage groups 6,7,and 17 in the BC1F1plant.Among them,one heterozygous segment covered approximately 17.85 cM at the top of linkage group 7 surrounding the PT30174 locus.The second heterozygous region covered approximately 135.69 cM on linkage group 17 surrounding the PT51378 locus.The other two heterozygous regions were located on linkage groups 6 and 7,which had no association with resistance to BS(Fig.3).

3.4.QTL for resistance to BS

The linkage groups 7 and 17 were reconstructed using SSR markers.Linkage group 7 consisted of 6 SSR markers with a genetic length of 13.30 cM;Linkage group 17 consisted of 22 SSR markers with a genetic length of 169.60 cM.

The two major QTL,named qBS7 and qBS17,were repeatedly detected under different conditions in this study(Table 3 and Fig.4).The QTL qBS7 was mapped to the region between PT30174 and PT60621,and explained 17.40%–25.60%of thephenotypic variance under different conditions.The allele from BH increased resistance to BS.QTL qBS17 in the interval PT61564–PT61538 in linkage group 17 was detected in BC1F2in the field and in BC1F2:3in both the field and seedling stage tests,and explained 6.90%–11.60%of the phenotypic variance.The allele from XHJ decreased resistance to BS across all environments.

Table2–Analysis ofvariance forBS resistance for significant markers detected by the BSA method in the F2population.

4.Discussion

The primary objective of this study was to identify stable QTL associated with resistance to BS and to develop molecular markers for use by tobacco breeders.However,response to BS in tobacco is a quantitative trait[23,24,40]that is affected by genetic background and environmental factors[41,42].For rapid and efficient identification of major QTL,we first detected molecular markers associated with resistance by BSA.Subsequently,one introgression line was selected to confirm the locations of the stable QTL by selecting for foreground favorable alleles and againstbackground markers from the donor parent.Utilization of lines with similar genetic background can eliminate the effects of genetic background and increase the sensitivity of QTL mapping.Environmental effects on disease response were estimated across different conditions.Therefore,the precision of QTL detection was improved by using an introgression line population in this study.

Using this method two stable QTL for resistance to BS,designated qBS7 and qBS17,were detected in different environments(Table 3 and Fig.4).

Fig.3–Graphical genotypes in linkage groups(LG)6,7,15,17,18,and 22 of the BC1F1individual selected using MAS.Bluerepresents homozygous genotypes of the recurrent parent,Xiaohuangjin 1025;black represents the heterozygous genotypes.Genetic distances(cM)are according to Bindler et al.[35].

QTL qBS7 with the flanking markers PT30174 and PT60621 on linkage group 7 was mapped to the same adjacent region as reported by Vontimitta and Lewis[23,24]and others[14,19].Several researchers have concluded that this QTL confers resistance to BS in flue-cured and burley tobacco.In the current study,a heterozygous segment covering approximately 17.85 cM at the top linkage group 7 and surrounding the PT30174 locus was selected to confirm and localize the region.The physical distance of the target region was approximately 1.69 Mb,in which two genes encoding a nucleotide binding site and leucine-rich repeats(NBS-LRR)were present(unpublished).The candidate genes of the major QTL could very possibly belong to one or more classical NBSLRR resistance genes.More detailed analyses for the candidate genes are required in the future.

The other stable QTL that mapped to linkage group 17 has not been reported previously.Based on comparative linkage maps,we found that the genomic region harboring qBS17 was not covered using available molecular markers(Fig.5),possibly explaining why this QTL was not identified in previous studies[23,24].Although＞5000 SSR markers were published by Bindler et al.[35]and a high-density SSR genetic map containing 2363 SSR markers had been constructed,the number of markers remains insufficient for genetic research in tobacco and for breeding for resistance to BS.Single nucleotide polymorphisms(SNP)provide an ideal alternative marker system for construction of high-density maps,because they are abundant and evenly distributed in the plantgenome[43–45].Recent developments in next-generation sequencing(NGS)technology allow for rapid discovery of thousands of SNP markers and high-throughput genotyping of large populations[46–50].Therefore,to map the genes controlling quantitative traits in tobacco,high-throughput detection of SNP markers and construction of high-density genetic linkage maps based on NGS technology will be required in the future.

Table 3–Summary of QTL mapping results for field and greenhouse conditions in BC1F2and BC1F2:3populations derived from cross Beinhart 1000-1×Xiaohuangjin 1025.

Fig.4–Likelihood plots and position(cM)of the QTL associated with resistance to black shank in different conditions.Red line:LOD plots for the BC1F2population;Green line:LOD plots for the BC1F2:3population in the field;Black line:LOD plots for the BC1F2:3population in the greenhouse;Dashed lines represent the significant LOD threshold at the level of 2.35.

Fig.5–Comparison of genetic linkage maps anchoring QTL affecting resistance to BS.The left LG7 and LG17 maps were constructed in this study;the middle LG7 and LG17 maps are from Bindler et al.[35];and the LG8 and LG14 maps are from Vontimitta and Lewis[23].

In previous studies,cigar tobacco lines,Florida 301,Beinhart 1000 and BH exhibited partial resistance to all races of P.nicotianae[14,19].Among them,Beinhart 1000 was derived from a selection of tobacco variety Quin Diaz,and BH was derived from Beinhart 1000.Both lines expressed a higher level of partial resistance to BS than that exhibited by Florida 301.Resistance to BS relationship between the cigar lines Beinhart 1000 and Florida 301 were examined in previous studies[19].Those results indicated that two major QTL conferred resistance to BS in Beinhart 1000,and one of these QTL was not present in Florida 301.However,the commonality or otherwise of resistance in BS and its putative source was unknown until the current study was made.Tobacco breeders have usually assumed that the same gene(s)for resistance in BH and Beinhart 1000 were similar based on origin.Thus,the two resistant sources are often confused and considered to be the same in regard to reaction to BS.As previously described,the major QTL that exists in both Florida 301 and Beinhart1000is also present in BH.However,the other major QTL reported previously on LG 4 in Beinhart 1000 was not detected in this study.Thus the results clearly suggest a partial genetic overlap between Beinhart 1000 and BH.

Finally,several reports showed that the major QTL found only in Beinhart 1000 co-segregated with NtCPS2,a major gene controlling Z-abienol biosynthesis in tobacco[23,37].Whether a pleiotropic effect or close linkage between NtCPS2 and the gene affecting resistance remains unclear.In this study,the genotype at the NtCPS2 locus was homozygous T for SNPCPS2in parent XHJ,whereas in BH,the genotype at the NtCPS2 locus was homozygous G.BH produces Z-abienol,whereas XHJ is confirmed to be a non-producer.These results are consistent with those reported by Sallaud et al.[37].However,we found that SNPCPS2was not associated with reaction to BS.The results suggest that the gene(s)affecting response to BS and the gene affecting Z-abienol production are closely linked rather than pleiotropic.

5.Conclusions

In this study,two QTL related to BS response were located on chromosomes 7 and 17 in seedling tests in a greenhouse and in the field.The QTL on chromosome 7 is present in all partial resistance sources and plays an essential role in breeding for BS resistance in both flue-cured and burley tobacco.The other QTL on chromosome 17 was not reported previously.This QTL may be useful for extending the range and level of resistance to BS in tobacco.These results expand our understanding of the inheritance of response to BS and provide information useful for marker-assisted breeding in tobacco.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2017.12.002.

Acknowledgments

This work was supported by grants from the Agricultural Science and Technology Innovation Program(ASTIP-TRIC01)and National Natural Science Foundation of China(31571738).

[1]H.D.Shew,Effect of host resistance on spread of Phytophthora parasitica var.nicotianae and subsequent development of tobacco black shank under field conditions,Phytopathology 77(1987)1090–1093.

[2]A.S.Csinos,N.A.Minton,Control of tobacco black shank with combinations of systemic fungicides and nematicides or fumigants,Plant Dis.67(1983)204–207.

[3]P.D.Legg,M.T.Nielsen,Genetic variation for agronomic characters in a burley tobacco synthetic following recurrent selection for increased black shank resistance,Plant Breed.108(1992)185–189.

[4]D.F.Antonopoulos,T.Melton,A.L.Mila,Effects of chemical control,cultivar resistance,and structure of cultivar root system on black shank incidence of tobacco,Plant Dis.94(2010)613–620.

[5]T.L.Qu,Y.Y.Shao,A.Csinos,P.S.Ji,Sensitivity of Phytophthora nicotianae from tobacco to fluopicolide,mandipropamid,and oxathiapiprolin,Plant Dis.100(2016)2119–2125.

[6]C.C.Litton,G.B.Collins,P.D.Legg,Reaction of Nicotiana tabacum and other N.species to race 0 and 1 of Phytophthora parasitica var.nicotianae,Tob.Sci.14(1970)144–146.

[7]M.J.Sullivan,T.A.Melton,H.D.Shew,Managing the race structure of Phytophthora parasitica var.nicotianae with cultivar rotation,Plant Dis.89(2005)1285–1294.

[8]J.L.Apple,Physiological specialization within Phytophthora parasitica var.nicotianae,Phytopathology 52(1962)351–354.

[9]M.J.Sullivan,T.A.Melton,H.D.Shew,Fitness of races 0 and 1 of Phytophthora parasitica var.nicotianae,Plant Dis.89(2005)93–97.

[10]X.K.Li,F.Y.Kong,X.H.Li,J.Wang,C.S.Zhang,C.Feng,Y.Q.Chen,X.Liu,Z.G.Su,B.L.Li,Preliminary report on physiological race of Phytophthora parasitica in Hubei,China,Tob.Sci.32(2011)84–88(in Chinese with English abstract).

[11]H.Liu,X.Ma,H.Q.Yu,D.H.Fang,Y.P.Li,X.Wang,W.Wang,Y.Dong,B.G.Xiao,Genomes and virulence difference between two physiological races of Phytophthora nicotianae,GigaScience 5(2016)3.

[12]G.W.Stokes,C.C.Litton,Source of black shank resistance in tobacco and host reaction to races 0 and 1 of Phytophthora parasitica var.nicotianae,Phytopathology 56(1966)678–680.

[13]E.S.Johnson,M.F.Wolff,E.A.Wernsman,W.R.Atchley,H.D.Shew,Origin of the black shank resistance gene,Ph,in tobacco cultivar Coker 371-Gold,Plant Dis.86(2002)1080–1084.

[14]K.McCorkle,R.Lewis,D.Shew,Resistance to Phytophthora nicotianae in tobacco breeding lines derived from variety Beinhart 1000,Plant Dis.97(2013)252–258.

[15]E.Van Jaarsveld,M.J.Wingfield,A.Drenth,Evaluation of tobacco cultivars for resistance to races of Phytophthora nicotianae in South Africa,J.Phytopathol.150(2002)456–462.

[16]C.L.Gupton,Occurrence of race 1 of Phytophthora parasitica var.nicotianae in east Tennessee,Plant Dis.Rep.56(1972)593–594.

[17]J.L.Mcintyre,G.S.Taylor,Race 3 of Phytophthora parasitica var.nicotianae,Phytopathology 68(1978)35–38.

[18]C.A.Gallup,H.D.Shew,Occurrence of race 3 of Phytophthora nicotianae in North Carolina,the causal agent of black shank of tobacco,Plant Dis.94(2010)557–562.

[19]B.G.Xiao,K.Drake,V.Vontimitta,Z.J.Tong,X.T.Zhang,M.Y.Li,X.D.Leng,Y.P.Li,R.S.Lewis,Location of genomic regions contributing to Phytophthora nicotianae resistance in tobacco cultivar Florida 301,Crop Sci.53(2013)437–481.

[20]K.E.Drake,J.M.Moore,P.Bertrand,B.Fortnum,P.Peterson,R.S.Lewis,Black shank resistance and agronomic performance of flue-cured tobacco lines and hybrids carrying the introgressed Nicotiana rustica region,Wz,Crop Sci.55(2015)79–86.

[21]S.C.Peng,P.Yang,L.Zheng,H.B.Cai,Y.Guan,Resistance identification of different flue-cured tobacco varieties against black shank,Plant Dis.Pest.4(2015)15–18.

[22]M.T.Nielsen,Sources of resistance to black shank and black root rot diseases,CORESTA Inf.Bull.3(1992)144–150.

[23]V.Vontimitta,R.S.Lewis,Growth chamber evaluation of a tobacco ‘Beinhart 1000'× ‘Hicks'mapping population for quantitative trait loci affecting resistance to multiple races of Phytophthora nicotianae,Crop Sci.52(2012)91–98.

[24]V.Vontimitta,R.S.Lewis,Mapping of quantitative trait loci affecting resistance to Phytophthora nicotianae in tobacco(Nicotiana tabacum L.)line Beinhart-1000,Mol.Breed.29(2012)89–98.

[25]Z.J.Tong,F.C.Jiao,D.H.Fang,X.J.Chen,X.F.Wu,J.M.Zeng,H.Xie,Y.H.Zhang,B.G.Xiao,Construction and genetic evaluation of chromosome segmental substitution lines in tobacco(Nicotiana tabacum L.),Acta Agron.Sin.42(2016)1609–1619(in Chinese with English abstract).

[26]J.R.Stavely,J.F.Chaplin,G.R.Gwynn,Registration of Bel 921 brown spot resistant flue-cured tobacco germplasm,Crop Sci.24(1984)830–831.

[27]J.F.Chaplin,Comparison of tobacco black shank resistance from four sources,Tob.Sci.10(1966)55–58.

[28]J.F.Chaplin,Registration of PD 121 tobacco germplasm,Crop Sci.11(1971)606.

[29]H.E.Heggestad,W.B.Lautz,Some results of studies on resistance to tobacco black shank,Phytopathology 452(1957).

[30]M.T.Nielsen,Sub-group CORESTA black shank collaborative study 1996–1997,CORESTA Inf.Bull.3(1997)51–54.

[31]C.Yin,H.Li,S.Liang,L.Xu,Z.Gang,J.Wang,Genetic dissection on rice grain shape by the two-dimensional image analysis in one japonica×indica population consisting of recombinant inbred lines,Theor.Appl.Genet.128(2015)1969–1986.

[32]J.R.Stavely,T.W.Graham,G.R.Gwynn,J.F.Chaplin,C.E.Main,Resistance to Altenaria alternata in Nicotiana tabacum,Phytopathology 63(1973)806.

[33]X.Guo,X.Yan,C.Jiang,L.Cheng,A.Yang,Q.Feng,Y.Wang,Genetic analysis of Beinhart1000-1 resistance to black shank in tobacco,China,Tob.Sci.38(2017)56–62(in Chinese with English abstract).

[34]T.Murashige,F.Skoog,A revised medium for rapid growth and bioassays with tobacco tissue cultures,Physiol.Plant.15(1962)473–497.

[35]G.Bindler,J.Plieske,N.Bakaher,I.Gunduz,N.Ivanov,R.Van der Hoeven,M.Ganal,P.Donini,A high density genetic map of tobacco(Nicotiana tabacum L.)obtained from large scale microsatellite marker development,Theor.Appl.Genet.123(2011)219–230.

[36]J.L.Xu,H.R.Lafitte,Y.M.Gao,B.Y.Fu,R.Torres,Z.K.Li,QTLs for drought escape and tolerance identified in a set of random introgression lines of rice,Theor.Appl.Genet.111(2005)1642–1650.

[37]C.Sallaud,C.Giacalone,R.Topfer,S.Goepfert,N.Bakaher,S.Rosti,A.Tissier,Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco(Nicotiana tabacum)glandular trichomes,Plant J.72(2012)1–17.

[38]J.W.Van Ooijen,JoinMap 4,Software for the Calculation of Genetic Linkage Maps in Experimental Populations,Kyazma B.V.,Wageningen,The Netherlands,2006.

[39]R.E.Voorrips,MapChart:software for the graphical presentation of linkage maps and QTLs,J.Hered.93(2002)77–78.

[40]M.P.Lamprecht,G.C.Prinsloo,Inheritance of resistance in tobacco to race 1 of the black shank fungus,Phytophthora nicotianae var.nicotianae,Agroplantae 9(1977)19–24.

[41]R.B.Goins,J.L.Apple,Inheritance and phenotypic expression of a dominant factor for black shank resistance from Nicotiana plumbaginifolia in a Nicotiana tabacum milieu,Tob.Sci.14(1970)7–11.

[42]Z.J.Tong,T.L.Jiao,F.Q.Wang,M.Y.Li,X.D.Leng,Y.L.Gao,Y.P.Li,B.G.Xiao,W.R.Wu,Mapping of quantitative trait loci conferring resistance to brown spot in flue-cured tobacco(Nicotiana tabacum L.),Plant Breed.131(2012)335–339.

[43]M.W.Ganal,T.Altmann,M.S.Roder,SNP identification in crop plants,Curr.Opin.Plant Biol.12(2009)211–217.

[44]J.Clevenger,C.Chavarro,S.A.Pearl,P.Ozias-Akins,S.A.Jackson,Single nucleotide polymorphism identification in polyploids:a review,example,and recommendations,Mol.Plant 8(2015)831–846.

[45]R.Singh,V.Bollina,E.E.Higgins,W.E.Clarke,C.Eynck,C.Sidebottom,R.Gugel,R.Snowdon,I.A.P.Parkin,Singlenucleotide polymorphism identification and genotyping in Camelina sativa,Mol.Breed.35(2015)35.

[46]W.J.Ansorge,Next-generation DNA sequencing techniques,New Biotechnol.25(2009)195–203.

[47]R.Q.Li,Y.R.Li,X.D.Fang,H.M.Yang,J.Wang,K.Kristiansen,J.Wang,SNP detection for massively parallel whole-genome resequencing,Genome Res.19(2009)1124–1132.

[48]R.K.Varshney,S.N.Nayak,G.D.May,S.A.Jackson,Nextgeneration sequencing technologies and their implications for crop genetics and breeding,Trends Biotechnol.27(2009)522–530.

[49]M.L.Metzker,Sequencing technologies-the next generation,Nat.Rev.Genet.11(2010)31–46.

[50]L.J.Wei,M.L.Xiao,A.Hayward,D.H.Fu,Applications and challenges of next-generation sequencing in Brassica species,Planta 238(2013)1005–1024.