朱 云，郭 曉，孫小磊，馬偉清，董道峰，郭寶太，楊 煜，楊曉慧，李廣存

(1.山東省農(nóng)業(yè)科學(xué)院 蔬菜花卉研究所，山東省設(shè)施蔬菜分子生物學(xué)重點(diǎn)實(shí)驗(yàn)室，山東 濟(jì)南 250100；2.青島農(nóng)業(yè)大學(xué) 生命科學(xué)學(xué)院，山東 青島 266109)

二倍體馬鈴薯SSR遺傳圖譜的構(gòu)建

朱 云1，郭 曉1，孫小磊1，馬偉清1，董道峰1，郭寶太2，楊 煜1，楊曉慧1，李廣存1

(1.山東省農(nóng)業(yè)科學(xué)院 蔬菜花卉研究所，山東省設(shè)施蔬菜分子生物學(xué)重點(diǎn)實(shí)驗(yàn)室，山東 濟(jì)南 250100；2.青島農(nóng)業(yè)大學(xué) 生命科學(xué)學(xué)院，山東 青島 266109)

為了豐富馬鈴薯分子標(biāo)記，定位青枯病抗性基因位點(diǎn)，利用具有抗青枯病遺傳背景的二倍體馬鈴薯親本USW5337.3(C)和772102.37(E)及其123份雜交一代無性系，進(jìn)行了馬鈴薯SSR遺傳圖譜的構(gòu)建。根據(jù)馬鈴薯全基因組序列設(shè)計(jì)了864對(duì)SSR引物，其中187對(duì)在親本間表現(xiàn)出差異，135對(duì)(72.2%)能夠在雜交一代中擴(kuò)增出清晰的條帶，最終構(gòu)建了一張由12個(gè)連鎖群組成，包含135個(gè)SSR標(biāo)記的馬鈴薯分子標(biāo)記遺傳圖譜。12個(gè)連鎖群總長(zhǎng)度為948.4 cM，標(biāo)記間平均間距7.03 cM，含有5個(gè)標(biāo)記偏分離區(qū)域。不僅為馬鈴薯飽和分子遺傳圖譜的構(gòu)建提供新的SSR標(biāo)記，對(duì)于馬鈴薯抗青枯病QTL定位、基因克隆和分子標(biāo)記輔助選擇也具有重要的參考意義。

二倍體；馬鈴薯；SSR；連鎖圖譜

Potato (SolanumtuberosumL.) is the fourth most important food crop of the world behind wheat，rice，and maize and is roughly half the world′s annual output of all root and tuber crops[1].Potato exhibits a considerable amount of variability for morphological traits and for resistance to insects and diseases.However，cultivated potato is an autotetraploid (2n=4x=48) which complicates both genetic/genomic studies as well as breeding efforts to improve important traits such as disease/pest resistance，processing quality and nutritional value[2].Multiple linkage maps of potato have been developed to better understand the potato genome，develop markers for marker assisted breeding，and facilitate map-based cloning[3-5].Most potato linkage maps have been generated from diploid populations which can simplify genetic segregation and incorporate polymorphism from wild species and primitive cultivars[6-8].

Potato linkage maps have been developed from many types of markers，including isozymes，restriction fragment length polymorphisms (RFLPs)，amplified fragment length polymorphisms (AFLPs)，simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs)[9-11].DNA simple sequence repeats，also known as microsatellites，are tandem repeats of 2-6 bp DNA core sequences，which are widely distributed in both non-coding and transcribed sequences.With the advantages of being PCR-based，reliable，co-dominant，multi-allelic，chromosome specific，and highly informative，SSRs are useful for many applications in plant genetics and breeding such as construction of high-density linkage maps，genetic diversity analysis，cultivar identification，and marker-assisted selection.Recent studies have shown that SSRs can detect more polymorphism in plant species than RFLP，RAPD and AFLP[12-14].There is a growing need for saturating the genetic map with SSR.Early development of SSRs was hampered by the high cost of library screening and clone sequencing.

Currently，large public SSR datasets exist for many crop species，but the number of publicly available，mapped SSRs for potato was relatively low.With the release of potato genome and advances of techniques to enrich SSRs，the development of genomic SSR markers in potato could be accelerated[15-16].In this study，a set of new SSR markers were designed and a SSR-based potato linkage map was constructed using a diploid potato population which harbored the resistance toRalstoniasolanacearum.This may be useful for further map saturation which could more facilitates the QTL mapping，identification and cloning of resistance genes，and in marker assisted breeding for resistance against disease.

1 Materials and Methods

1.1Plantmaterials

An F1(n=123) progeny developed from a cross between two diploid (2n=2x=24) potato clones USW5337.3(C) and 772102.37(E) was used as the mapping population in this study.Clone C was a hybrid betweenSolanumtuberosumL.Group Phureja PI 225696.1 and theS.tuberosumGroup Tuberosum dihaploid USW42.Clone E was obtained from a cross between clone C and theS.vernei-S.tuberosumbackcross clone VH34211[17].Previous research showed C and E clones were resistant to bacterial wilt[18].Both clones C and E were used for the polymorphism analysis of SSR markers.Genomic DNA was extracted from young leaf tissues of 123 F1progeny individuals together with C and E using CTAB method[19].

1.2SSRmarkersandpolymorphismanalysis

SSR markers were designed from the potato genome assembly using PRIMER3 online (http：//www.simgene.com/Primer3).A total of 864 primer pairs were selected.All primer pairs were initially screened against two clones C and E and a small offspring screening panel for polymorphism.The polymorphic markers were then used to genotype 123 individuals of the F1population for map construction.

Assessment of genetic polymorphism was performed by separation of PCR products using non-denaturing polyacrylamide gel electrophoresis.PCR reactions were prepared in a total volume of 15 μL containing 10×Taqpolymerase Buffer (500 mmol/L KCl，200 mmol/L Tris-HCl，pH 8.4)1.5 μL ，3.0 mmol/L MgCl20.9 μL，2.5 mmol/L dNTPs 0.3 μL，5 μmol/L each of forward and reverse primers 1.0 μL，1 UTaqpolymerase (Invitrogen Corporation，USA)，and 20 ng of DNA template.The final volume was adjusted with sterile distilled water.PCR was performed in 96-well plates in GeneAmp PCR 9700 thermocycler (Applied Biosystems) programmed as follows：94 ℃ for 3 min，followed by 37 cycles of 94 ℃ for 30 s，55 ℃，53 ℃，50 ℃，48 ℃ for 50 s，72 ℃ for 45 s and a final extension step at 72 ℃ for 10 min.Amplified products were separated on 6% non-denaturing polyacrylamide gels (PAGE) in 0.5 Tris-borate EDTA Buffer (TaKaRa) and visualized by silver (1 mg/mL) staining.

1.3Mapconstruction

Data were collected in the F1mapping population for all segregating marker alleles，regardless of whether segregation was from one parent or both.The segregation types were divided into biparental (genotype：ab×cd，ef×eg and hk×hk)，maternal (genotype：lm×ll) and paternal (genotype：nn×np) datasets.A cross-pollinated (CP) population type was used to develop the linkage map in Joinmap 4.0[20].The goodness-of-fit between observed and expected Mendelian ratios was analyzed for each marker locus using a χ2test.Markers that deviated from the theoretical expected ratios (1∶1) were considered distorted and were marked to indicate different significance levels (P<0.05，P<0.01，P<0.001，andP<0.000 1).Markers assigned to linkage groups had a minimum LOD score of 3 and a maximum of 10.

2 Results and Analysis

2.1Identificationofpolymorphicmolecularmarkers

A total of 864 SSR primers pairs from potato were screened for polymorphisms，and 187 out of 864 SSR primers showed to be polymorphic between the parents C and E.After amplifying them on the entire map population individuals，52 gave monomorphic amplifications (no segregation) or produced unclear amplifications，resulting in difficulties in band scoring and thus were subsequently discarded.The remaining 135 SSR markers (Tab.1) were used for the linkage map construction.Four separation types were generated as nn×np，lm×ll，ef×eg，and hk×hk respectively (Fig.1).

1-21.Eighteen potato individuals of F1 progeny.The different segregation types of SSR114，SSR096，SSR087 andSSR034 are nn×np，lm×ll，ef×eg，hk×hk，respectively.

2.2Mapconstruction

One hundred and thirty-five loci had been assigned to positions on 12 linkage groups (LGs)，with between 6 and 20 loci per group (Tab.1).The length of each linkage group varied from 27.8 to 132.8 cM，with three linkage groups (LG1，LG9，and LG11) having a size over 100 cM.The total map distance was 948.4 cM，with an average marker interval of 7.03 cM between adjacent marker loci (Tab.1).

A total of nine gaps between two adjacent markers 20 cM existed on the maps and varied from 20.1 to 42.2 cM，with one gap on LG1 (20.4 cM between SSR069 and SSR072)，LG4 (35.1 cM between SSR162 and SSR116)，LG8 (20.3 cM between SSR033 and SSR090)，and LG12 (42.2 cM between SSR006 and SSR005)，2 gaps on LG11(32.7 cM between SSR173+and SSR163+，36.5 cM between SSR137 and SSR089)，and 3 gaps on LG9 (27.7 cM between SSR049 and SSR001，20.1 cM between SSR156+and SSR134，and 20.2 cM between SSR134 and SSR154)，respectively.The largest gap was 42.2 cM for SSR006-SSR005 on LG12 (Fig.2).

Tab.1 The characteristics of 12 linkage groups constructed by SSR markers in potato linkage group

Marker positions(cM)are in the left of the linkage group; The marker names are in the right.

2.3Segregationdistortion

Distorted segregation ratios of genetic markers were often observed in progeny of inter- and intra-specific hybrids and might result from competition among gametes or from abortion of the gamete or zygote.In this study，135 polymorphic markers on the linkage map were surveyed for distorted segregation ratios.Among those，40 markers (29.6%) showed a significant segregation distortion (P<0.05)，favoring either the marker alleles of female parent C (52.5%) or male parent E (35.0%) or both parents (12.5%) (Tab.2).

Tab.2 Chi-square test for segregation distortion of markers in F1 population

Tab.2(Continued)

Note：*at 0.05 significant level;More than* at 0.01 extremely significant level.

Segregation distortion marker distribution along the present molecular maps of potato was far from uniform，with clusters of tightly linked loci and single marker.In this linkage map，five segregation distortion regions (SDRs，clusters of 3-7 markers) were detected on the linkage groups; LG1，LG3，and LG10 (Fig.3).Among these five SDRs，SDR-1 skewed to parent C，SDR-2 skewed to parent E，and SDR-3，SDR-4，SDR-5 skewed to C and E.The segregation distortion ratio of LG1，LG3，LG10 are 68%，100%，50%，separately.

3 Discussion

This manuscript reported the construction of an SSR-based map of diploid potato.The primary goal for the construction of this map was to develop a framework for future improvement.The application value of a genetic linkage map depends on its marker numbers，saturation，accuracy and the uniformity of marker distribution.Except for an elementary chromosome draft，the marker intervals of <20 cM are necessary for major gene location，and 1-10 cM for QTL positions and gene cloning.In this study，the total map spanned 948.4 cM，with an average marker interval of about 7.03 cM between two adjacent marker loci.This new SSR-based linkage map contained almost twice as many loci as did the previous linkage map which was constructed using 86 SSR markers[21].Moreover，the map population was derived from the cross of C and E clones conferring high resistance to bacterial wilt disease.This map will be a useful resource and tool，in which SSR markers may be used for future map saturation，QTL studies and other potato improvement.

Marker positions(cM)are in the left of the linkage group;The marker names are in the right.*Marker are declared with distorted segregation at the 0.05 level of significance and more than** at 0.01 significant level.

Allele distorted segregation is a common phenomenon in organisms.It is considered to be one of the driving forces of biological evolution.Distorted segregation ratios of genetic markers are often observed in progeny of inter- and intra-specific hybrids and might result from competition among gametes or from abortion of the gamete or zygote[22].Additionally，the mapping population，environmental factors，non-homologous chromosome recombination，gene jumping，hitchhiking，transposons，the genetic purity of parents，etc.could be the reasons of distorted segregation[23].Segregation distortion marker distribution along the present molecular maps of potato was far from uniform，with clusters of tightly linked loci and single marker.In this linkage map，five segregation distortion regions (SDRs，clusters of 3-7 markers) were detected on 3 linkage groups，indicating that the possible causes for segregation deviation of molecular markers were based on gametic selection.

Acknowledgments：This work was supported by the Natural Science Funds of Shandong Province (ZR2014YL014，ZR2016CM27)，and the Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences (2014QNZ03).Special thanks to Professor Richard G F Visser (Wageningen University) for kindly providing the potato materials used in this research.

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ConstructionofanSSR-basedGeneticLinkageMapofDiploidPotato

ZHU Yun1，GUO Xiao1，SUN Xiaolei1，MA Weiqing1，DONG Daofeng1，GUO Baotai2，YANG Yu1，YANG Xiaohui1，LI Guangcun1

(1.Vegetable and Flower Institute，Shandong Academy of Agricultural Sciences，Shandong Key Laboratory for Biology of Greenhouse Vegetables，Jinan 250100，China;2.College of Life Sciences，Qingdao Agricultural University，Qingdao 266109，China)

In order to enrich molecular markers and position the bacterial wilt resistant gene loci in potato,an SSR-based genetic linkage map was constructed from a diploid population consisting of 123 individuals derived from a cross between two potato diploid clones (C and E) which harborer resistance toRalstoniasolanacearumin this study.A total of 864 simple sequence repeat primer pairs were designed according to the potato genome sequence and used to screen parents C and E for polymorphisms.Of 187 polymorphic loci，72.2% showed clear amplifiable and scorable bands in the F1population.Then a total of 135 locus were mapped to 12 linkage groups，of which the cumulative length was 948.4 cM，with an average marker interval of 7.03 cM,and 5 segregation distortion regions in three linkage groups.This study provided a set of new SSR markers for genetic map construction and increased saturation of map in potato.The relatively high density map may provide a useful reference for further identification，mapping of quantitative trait loci (QTL) and cloning of potato bacterial wilt resistance genes and marker-assisted breeding.

Diploid;Potato;Simple sequence repeat;Linkage map

2017-08-09

山東省自然科學(xué)基金項(xiàng)目(ZR2014YL014；ZR2016CM27)；山東省農(nóng)業(yè)科學(xué)院青年科研基金項(xiàng)目(2014QNZ03)

朱 云(1987-)，女，安徽馬鞍山人，碩士，主要從事馬鈴薯抗病分子生物學(xué)研究。朱云、郭曉為同等貢獻(xiàn)作者。

楊曉慧(1980-)，女，山東聊城人，助理研究員，博士，主要從事馬鈴薯分子育種研究。

S532.03

A

1000-7091(2017)05-0117-07

10.7668/hbnxb.2017.05.018