Qi SUN，Kang LlAO，Wenjuan GENG，Juan LlU，Mansur NASlR，Huan LlU，Yang JlA，Qian CAOResearch Centre of Characteristic Fruit Tree，Xinjiang Agricultural University，Urumqi 830052，China

Analysis of Genetic Relationships in Prunus domestica L.in Xinjiang Using lSSR Markers

Qi SUN，Kang LlAO*，Wenjuan GENG，Juan LlU，Mansur NASlR，Huan LlU，Yang JlA，Qian CAO
Research Centre of Characteristic Fruit Tree，Xinjiang Agricultural University，Urumqi 830052，China

［Objective］This paper aimed to explore the genetic relationships among different types of Prunus domestica L.in Xinjiang. ［Method］The inter-simple sequence repeat（ISSR）markers were applied to analyze the genetic diversity and relationships among 30 individual plants from 5 types of P.domestica germplasm resources in Xinjiang.［Result］A total of 317 bands were amplified by 16 selected ISSR primers.Among the amplified bands，there were 246 polymorphic bands，accounting for 77.60%of the total.The Nei’s gene diversity index （H）of individuals was 0.266 6.The average Shannon’s information index（I）of individuals was 0.399 1. And most of the genetic similarity coefficients ranged from 0.555 2 to 0.996 8.It is indicated that the P.domestica germplasm resources have a certain genetic diversity in Xinjiang.The cluster analysis showed that，at the genetic similarity coefficient of 0.719，the 30 individuals were divided into 3 major groups，including a group of introduced European plum varieties，a group of Tacheng Smoked Plum and Tacheng Binzi and a group of Jiashi Smoked Plum and Wild European Plum.While at the genetic similarity coefficient of 0.949，Jiashi Smoked Plum and Wild European Plum were divided into two small groups.［Conclusion］There are very close genetic relationships between Jiashi Smoked Plum and Wild European Plum，and among the introduced European plum varieties，Tacheng Smoked Plum and Tacheng Binzi.

Xinjiang；Prunus domestica L.；ISSR；Genetic relationship

ISSR is considered an efficient，reliable and highly polymorphic molecular marker［15］.But so far，there has been no research available on the genetic relationships among different types of P.domestica germplasm resources in Xinjiang by using ISSR markers.Therefore，this study selected Jiashi Smoked Plum，introduced European plum varieties，Wild European Plum，Tacheng Smoked Plum and Tacheng Binzi as the test materials and studied the genetic characteristics and relationships of representative types of P.domestica germplasm resources in Xinjiang using ISSR markers.

Materials and Methods

Materials

The 30 tested individual plants of P.domestica，including 6 individuals of Jiashi Smoked Plum，4 varieties of introduced European plum，8 individuals of Tacheng Binzi，6 individuals of Tacheng Smoked Plum and 6 individuals of Wild European Plum were growing in 4 regions （Jiashi，Luntai，Tacheng and Ili）of Xinjiang.The specific distribution sites and types of sampled individuals were listed in Table 1.

Methods

Sampling After leaves were expanded in spring，30 to 50 pieces of tender leaves without petioles were collected from each of the individual plants，respectively.The sampled leaves were placed into sealed pockets with silica until they were dried completely.Subsequently，the dried leaves were fully grinded in liquid nitrogen，and the leaves powders were collected into centrifuge tubes and stored at-80℃.

Extraction of DNA and establishment of ISSR polymerase chain reaction procedure The DNAs of P.domestica individual plants were extracted by using modified CTAB method［16］.The purities and concentrations of extracted DNAs were examined with 0.8% agarose gel electrophoresis and ultraviolet spectrophotometer.The values of OD230，OD260and OD280of each sample were recorded.Then the extracted DNA was diluted into concentrations of 50 to 80 ng/μl.Referring to the orthogonal and single factor experimental design methods of Bai et al.［17］，Wang［18］and He et al.［19］，the 20 μl ISSR-PCR procedures were optimized.The finally optimized amplification system （20 μl）was as follows:1.0 U Taq polymerase，0.75 μmol/L primer，0.25 mmol/L dNTPs，2.0 mmol/L Mg2+，50-80 ng DNA，2 μl 10×Buffer and double distilled water.The optimized amplification conditions were as follows:predenaturation at 94℃for 4 min；denaturation at 94℃ for 4 s，annealing at 50-63℃ （depending on primers）for 1min，extension at 72℃for 2 min，33 cycles；final extension at 72 for 7 min. The PCR amplifications would proceed in a gradient thermal cycler（TProfessional，Biometra）.The amplified products were examined by 2.0% agarose gel electrophoresis in 1×TAE buffer at 120 V and 165 mA for 35 min（DYY-7C，Liuyi（Beijing））.The gels were stained with 10 mg/ml ethidium bromide for 15 min and then photographed under the ultraviolet light gel imaging system （Alphalmager HP）. The used molecular weight standard was Trans2K DNA Marker.The Taq polymerase，dNTPs，Mg2+，Buffer and Marker were all purchased from the Beijing Transgen Biotech Co.，Ltd.

Selection of primers and their annealing temperatures Referring to related researches［5，11，20-23］，the 59 ISSR primers for amplifying Prunus sequences were selected and then synthesized by the Beijing Genomics Institute.The individuals of JS-1 and YE-1，which had big differences in appearance，were used to select primers. To optimize the annealing temperature，a total of 8 gradient annealing temperatures（recommended annealing temperature-3，-2，-1，0，+1，+2，+3，+4℃）were designed for each of the primers.The primers and its optimum annealing temperatures which had richer and brighter bands，cleaner background and more stable amplification were finally selected.

ISSR markers The ISSR markers of genomic DNAs of the 30 individual plants were performed with selected primers and their optimum annealing temperatures under conditions of optimum amplification system，optimum amplification conditions and optimum cycle index.The amplified bands of each primer for each individual were counted，respectively.At certain site of certain lane，if there is one band，it is recorded as ‘1’；if there is no band，it is recorded as ‘0’.At final，a matrix composed of 0 and 1 was obtained for each sample.

Data statistics The obtained data was first processed with Excel 2010 and then analyzed by using SPSS 20.0.The total numbers of amplified bands and polymorphic bands were counted，and then the ratios between them were calculated.POPGEN3.2 was used to calculate the Nei’s gene diversity index （H），Shannon’s information index （I）and other kinds of similarity coefficients and genetic indexes.NTSYSPC2.1 was used to draw the dendrogram.

Results and Analysis

Selection of primers and their optimum annealing temperatures

At final，a total of 16 primers，along with their optimum annealing temperatures which had richer and clearer bands，cleaner background and more stable amplification were screened out from 59 ISSR primers（Table 2）.There were 11 primers of which the optimum annealing temperatures were higher than the recommended annealing temperatures；there were 2 primers of which the optimum annealing temperatures were as same as the recommended annealing temperatures；there were 3 primers of which the optimum annealing temperatures were lower than the recommended annealing temperatures.The results showed that there were only 2 primers of which the optimum annealing temperatures were same with the recommended annealing temperatures.It is indicated the exploration of optimum annealing temperatures is necessary and essential.

Table 1 Types and distribution sites of sampled individuals

Table 2 Annealing temperatures and amplification results of primers

YE-1 was used as the template.It was amplified with UBC836and UBC840respectively （Fig.1）.As shown in Fig.1，the bands amplified by UBC836 were more than those amplified by UBC840.So UBC836 was selected.Subsequently，the amplification results of UBC836 were compared among different annealing temperatures.As shown on electrophoresis，the bands amplified at 54 and 55℃were more and brighter than others.At the same time，the two lanes had cleaner backgrounds，and had no nonspecific amplifications.Therefore，54.5℃ was selected as the optimum annealing temperature for UBC836. The annealing temperatures of other primers were determined as above.

Polymorphism analysis of primer amplification

ISSR-PCR amplification of genomic DNAs of the 30 individual plants of P.domestica in Xinjiang was performed with 16 selected primers（Table 2）.A total of 317 bands，distributed between 150 to 2 600 bp，were obtained from the amplification. Among them，there were 246 polymorphic bands， accounting for 77.60%.It was suggested the genetic diversity of P.domestica was abundant in Xinjiang.The band numbers of types differed among the 16 primers. The numbers of bands amplified by UBC857 and UBC873 were all 29，ranking highest among the primers. The number of bands amplified by UBC812 was 13，ranking last.The average number of bands amplified by the total 16 primers was 19.8.On the other hand，the numbers of polymorphic bands amplified by UBC857 and UBC873 were all 24，ranking first.But the number of polymorphic bands amplified by UBC812 was only 8，ranking last.The average number of polymorphic bands amplified by the total 16 primers was 15.4.The ratio of polymorphic bands for UBC811 was the highest（86.67%），while for UBC861 was the lowest（58.82%）.

Genetic diversity analysis of P.domestica individuals

The calculation results of genetic diversity indexes of each amplification site which came from the genomic DNA of 30 tested individuals of P.domestica in Xinjiang showed that the average observed number of alleles（Na）was 1.776 0；the average effective number of alleles （Ne）was 1.459 5；the average Nei’s gene diversity index （H）was 0.266 6；the average Shannon’s information index（I）was 0.399 1.The results above suggested that there was a certain genetic diversity among the 30 tested individuals （Fig.2）.When the 30 individuals were divided into 5 germplasm types of P.domestica，the total gene diversity of the 30 individual plants（Ht）was 0.255 3；the average gene diversitywithin the 5 germplasm types of P.domestica （Hs）was 0.090 1；the Nei’s genetic differentiation coefficient（Gst）was 0.647 1；the gene flow among different germplasm types （Nm）was 0.272 7.The results above showed that there was 64.71%of genetic variation existing among the 5 different germplasm types of P.domestica，and there was only 35.29%of genetic variation existing within the 5 germplasm types.So it could be concluded that the genetic variation of P.domestica in Xinjiang mainly exists among different germplasm types，and the genetic differentiation among different germplasm types of P.domestica is greater than that within germplasm types.The gene flow among different germplasm types was only 0.272 7 that was less than 1，indicating that the gene flow among the 5 germplasm types of P.domestica was not high enough to resist the genetic drift among different germplasm types which resulted in the genetic differentiation.

Similarity analysis of P.domestica individuals

The 317 loci obtained from the amplification of 30 individual plants of P.domestica in Xinjiang by 16 primers were used to calculate the 435 genetic similarity coefficients between each two individuals by software.The genetic similarity coefficient between JS-13 and JS-15，as well as between YE-1 and YE-2，was the maximum.They were all up to 1.000 0.So the four individuals were probably two pairs of materials which were collected repeatedly.In addition，the genetic similarity coefficients among JS-12，JS-13 and JS-15，among YE-1，YE-8 and YE-9 and among YE-2，YE-8 and YE-9 all reached 0.996 8，ranking second. These materials were the most similar individuals except the same individuals.However，the genetic similarity coefficient between L1 and JS-6，as well as between L1 and YE-3 and between L1 and YE-9，was only 0.555 2，ranking last.It was also suggested that the similarities among those materials were the lowest.

Cluster analysis of P.domestica individuals

The cluster analysis of genetic similarity coefficients was conducted by unweighted pair-group method with arithmetic means （UPGMA）（Fig.3）. Among the 5 germplasm types of P.domestica in Xinjiang，all the tested individuals of Wild European Plum，Jiashi Smoked Plum and introduced European plum varieties were clustered in their own groups obviously. But the individuals of Tacheng Smoked Plum and Tacheng Binzi were clustered in a same group without obvious boundary.It was indicated that Wild European Plum，Jiashi Smoked Plum and introduced European plum varieties all have their own special and obvious genetic characteristics of germplasm types，respectively.Although there were not genetic characteristics that could be used to distinguish between Tacheng Smoked Plum and Tacheng Binzi，both of them had common genetic characteristic which could distinguish themselves from the other three germplasm types.

The 30 Tested individuals of P.domestica were divided into 3 large groups at about similarity coefficient of 0.719（Line 1）.The 4 introduced European plum varieties were clustered into the first group；the second group consisted of all the varieties of Tacheng Smoked Plum and Tacheng Binzi；the third group consisted of all the varieties of Jiashi Smoked Plum and Wild European Plum.In addition，the third large group was further divided into two small groups，Jiashi Smoked Plum and Wild European Plum，at about similarity coefficient of 0.949（Line 2）.Above all，there was a close genetic relationship between Wild European Plum and Jiashi Smoked Plum；and there were also close genetic relationships among introduced European plum varieties，Tacheng Smoked Plum and Tacheng Binzi.However，the similarity betweenthe first two germplasm types was higher than those among the latter three types.In addition，all individuals of Wild European Plum were classified into one group at similarity coefficient of 0.990；all individuals of Jiashi Smoked Plum were classified into one group at similarity coefficient of 0.983；all individuals of introduced European plum varieties were classified into one group at similarity coefficient of 0.836；all individuals of Tacheng Smoked Plum and Tacheng Binzi were classified into one group just at similarity coefficient of 0.757.The differences in similarity coefficient above reflected that the genetic diversity of Wild European Plum was lowest among the 5 germplasm types of P.domestica in Xinjiang.The genetic diversity of Jiashi Smoked Plum was slightly higher than that of Wild European Plum.Moreover，the genetic diversity of introduced European plum varieties was much higher than those of both of the germplasm types above.The genetic diversities of Tacheng Binzi and Tacheng Smoked Plum were most abundant.

Discussion

Genetic characteristics of germplasm resources reflected by diversity and similarity

The study results showed that the genetic variation among different germplasm types of P.domestica in Xinjiang was much higher than that within germplasm types，which was similar or mostly close to the study results of Feng［11］on a few cultivars of P.domestica and of Geng［24］on Wild European Plum.It is indicated that the genetic variation within germplasm types is limited，and each of the germplasm types all has its own unique genetic characteristics.ISSR markers can be applied to distinguish different types of P. Domestica germplasm resources.In addition，the gene flow among different types of germplasm was only 0.272 7，indicating that there is a certain intension of genetic drift existing within germplasm types［25］.What’s more，the 5 germplasm types of P.domestica were lacking in gene flow，which was mainly resulted from the long distances among major germplasm types［2］.It is also reflected that people has done litter on exploiting germplasm resources of P.domestica.In addition to the 4 materials in pairs，there were all 3 plants，among which the similarity coefficients were up to 0.996 8，in each of the Wild European Plum group and the Jiashi Smoked Plum group.Compared with other germplasm resources of P.domestica，Wild European Plum and Jiashi Smoked Plum showed a much higher similarity within groups.It was also showed that the two types of germplasm resources have more stable genetic characteristic，as well as stronger genetic drift and greater chances of inbreeding.However，the similarity coefficient between the introduced European plum varieties and the individuals of Jiashi Smoked Plum was only 0.555 2.Despite the same characteristics shared by the germplasm types，it could be deduced that there are big differences in genetic characteristics between introduced European plum varieties and Jiashi Smoked Plum.Similarly，there are also big differences in genetic characteristics between introduced European plum varieties and Wild European Plum.Moreover，the previous study on the appearance characteristics of P.domestica also showed the similar results.

Discussion of genetic relationship and evolution based on cluster result

In the clustering，the individuals of Wild European Plum grew in Birsay，Xinyuan and grew in Yiligedai，Gongliu were distributed in two different small groups alternately，which were all included in the large group of Wild European Plum.It was suggested that there might be certain varieties of Wild European Plum，at the beginning of their formation，existing in both of the two places simultaneously.In other words，the genetic relationship between the Wild European Plum grew in Birsay，Xinyuan and those grew in Yiligedai，Gongliu is very close.The clustering results indicated that Jiashi Smoked Plum had the closest relationship with Wild European Plum in Xinjiang.Lin et al.［26］thought that Wild European Plum is the ancestor of cultivated European Plum.So the Jiashi Smoked Plum might be the cultivated type which is the closest one to the original type.In addition，it was also indicated that Tacheng Smoked Plum and Tacheng Binzi were difficult to divide，indicating similar，complicated and abundant relationships within P.domestica resources in Tacheng.

The research on parts of introduced European plum varieties conducted by Chen et al.［27］showed that introduced varieties of plum were likely to contain the gene of P.salicina.In this study，the clustering results showed that the introduced European plum varieties had a closer genetic relationship with Tacheng Smoked Plum and Tacheng Binzi than with the other local P.domestica germplasm types. Therefore，it could be speculated that there are certain P.domestica varieties derived from the hybridization between Tacheng Smoked Plum and Tacheng Binzi and introduced European plum varieties.Although there is almost no difference in appearance between Tacheng Smoked Plum planted in Jiashi and Jiashi Smoked Plum，all the individuals of Tacheng Smoked Plum， except the individuals of Tacheng Binzi，were divided into one group according to cluster analysis. This not only proved the ability of ISSR markers to identify the essence of different germplasm types of P.domestica，but also showed the relatively strong heritability of Tacheng Smoked Plum［25］.Moreover，it was also indicated that there is no significant correlation between the genetic relationships among samples and distances among cultivated locations，which is same with the research results of Song et al.［28］on P.cerasifera.

There are studies showing that in the clustering，the greater the variation range of similarity coefficient becomes，the more abundant the genetic diversity of the germplasm type is，and the more complicated the genetic background of the germplasm type is［29-30］. In this research，the genetic diversity of Wild European Plum was the lowest，followed by Jiashi Smoked Plum. However，the genetic diversity of the Tacheng Smoked Plum and Tacheng Binzi was most abundant，followed by introduced European plum varieties.In other words，the genetic backgroundsof Wild European Plum and Jiashi Smoked Plum are simple，so they are both simplex germplasm types.However，the genetic backgrounds of the Tacheng Smoked Plum，Tacheng Binzi and the introduced European plum varieties are complex，so they are all genetic variation-abundant germplasm types.In conclusion，the evolutions of introduced European plum varieties and P.domestica germplasm resources in Tacheng are more complex than those of Wild European Plum and Jiashi Smoked Plum.

Conclusions

The results of ISSR markers on 30 individuals of 5 types of P.domestica germplasm resources in Xinjiang showed that 317 bands were amplified by 16 primers in total.The polymorphic percentage of bands reached 77.60%. There is a certain genetic diversity among all the tested P.domestica germplasm resources in Xinjiang.And the genetic variation among different germplasm types of P.domestica in Xinjiang is much greater than that within germplasm types.The cluster analysis indicated that the tested individuals of P.domestica were divided into 4 groups in total at similarity coefficients of 0.719 and 0.949.The 4 groups included the group of introduced European plum varieties，the group of Tacheng Smoked Plum and Tacheng Binzi，the group of Jiashi Smoked Plum and the group of Wild European Plum.Among all the groups，the group of Wild European Plum had the lowest genetic diversity，while the group of Tacheng Smoked Plum and Tacheng Binzi had the most abundant genetic diversity.There is a very close genetic relationship between Jiashi Smoked Plum and Wild European Plum.In addition，there are also close genetic relationships among introduced European plum varieties，Tacheng Smoked Plum and Tacheng Binzi.

References:

［1］ZHANG JY（張加延），ZHOU E（周恩）. China’s Fruit Trees:Plum（中國果樹志·李卷）［M］.Beijing:China Forestry Press（北京:中國林業(yè)出版社），1997.

［2］LIAO K（廖康）.Study on wild fruit tree resources in Xinjiang（新疆野生果樹資源研究）［M］.Urumqi:Xinjiang Science and Technology Press（新疆:新疆科技出版社），2013.

［3］LIN PJ（林培鈞），LIAO MK（廖明康），SHI L（施麗），et al.Finding and distribution of wild Prunus domestica L（P.communis Fritsch）in Yili，Xinjiang（first report）（新疆伊犁野生歐洲李Prunus domestica L（P.communis Fritsch）的發(fā)現(xiàn)與分布（第一報））［J］.Liaoning Fruit Trees（北方果樹），1986，1:1-3.

［4］WANG L（王磊）.Wild Hawthorn，Wild European Plum，Wild Rose and other wild resources of fruit trees in Xinjiang（新疆野山楂、野歐洲李、野薔薇等野生果樹資源）［J］.Xinjiang Agricultural Sciences（新疆農(nóng)業(yè)科學(xué)），1990，3:78-79.

［5］LIU WS（劉威生）.Studies on the genetic diversity among plum germplasm resources and the phylogenetic relationship of main plum species（李種質(zhì)資源遺傳多樣性及主要種間親緣關(guān)系的研究）［D］.Beijing:China Agricultural University（北京:中國農(nóng)業(yè)大學(xué)），2005.

［6］WANG YZ（王玉柱），YANG L（楊麗），YAN AL（閻愛玲），et al.Progress of plum breeding in the world（李品種選育研究進(jìn)展）［J］.Journal of Fruit Science（果樹學(xué)報），2002，19（5）:243-244.

［7］ZOHARY D.Is the European plum，Prunus domestica L，a P cerasifera EHRH×P spinosa L allopolyploid［J］. Euphytica，1992，60:75-77.

［8］ERYOMINE GV.New data on origin of Prunus domestica L.［J］.Acta Horticulturae，1991，283:27-29.

［9］HOU B（侯博），XU Z（許正）.Study on wild fruit trees and their kindred species in the Yili Valley，China（中國伊犁野生果樹及近緣種研究）［J］.Arid Zone Research（干旱區(qū)研究），2006，23（3）:455.

［10］GENG WJ（耿文娟），LIAO K（廖康），DIAO YQ（刁永強(qiáng)），et al.SSR analysis on Wild European Plum genetic relationship of different populations（野生歐洲李不同居群親緣關(guān)系的SSR分析）［J］.Acta Horticulturae Sinica（園藝學(xué)報），2012，39（supplement）:2602.

［11］FENG CJ（馮晨靜）.Phylogenetic relationships and genetic diversity revealed by RAPD，SSR，ISSR markers in Prunus（李種質(zhì)資源RAPD、SSR、ISSR親緣關(guān)系鑒定及遺傳多樣性研究）［D］.Baoding:Hebei Agricultural U-niversity（保定:河北農(nóng)業(yè)大學(xué)），2005.

［12］LIANG M（梁敏），DONG WX（董文軒），ZHEN R（甄睿），et al.ISSR analysis of genetic relationship of Peach，Plum and Apricot resources（桃李杏資源親緣關(guān)系的ISSR分析）［J］.Guizhou Agricultural Sciences（貴州農(nóng)業(yè)科學(xué)），2011，39（5）:27-31.

［13］DECROOCQ V，HAGEN LS，F(xiàn)AVé M G，et al.Microsatellite markers in the hexaploid Prunus domestica species and parentage lineage of three European plum cultivars using nuclear and chloroplast simple-sequence repeats［J］.Molecular breeding，2004，13: 135-138.

［14］SHIMADA T，HAYAMA H，HAJI T，et al.Genetic diversity of plums characterized by random amplified polymorphic DNA（RAPD）analysis［J］.Euphytica，1999，109:143.

［15］ZHANG QL（張青林），LUO ZR（羅正榮）. ISSR technology and its applications in fruit trees（ISSR及其在果樹上的應(yīng)用）［J］.Journal of Fruit Science（果樹學(xué)報），2004，21（1）:54-58.

［16］WANG XL（王小利），ZHANG GS（張改生），LI HX（李紅霞）.Plant molecular and cytogenetic experiment（植物分子細(xì)胞遺傳學(xué)實驗）［M］.Shanghai: Shanghai Science and Technology Press（上?？茖W(xué)技術(shù)出版社），2010，8: 60-63.

［17］BAI JJ（白錦軍），WEI AZ（魏安智），WANG J（王佳），et al.Optimization of an ISSR analysis system of Apricot of kernel-consuming based on orthogonal design（杏仁用ISSR分析體系的正交優(yōu)化）［J］.Molecular Plant Breeding（分子植物育種），2009，7（6）:1237-1224.

［18］WANG HN（王會娜）.The researches on blossom biology and the analysis on genetic relationship using ISSR of the series Prunus varieties of‘Ziguiqiulu’（秭歸秋露李系列品種開花生物學(xué)研究及ISSR分析）［D］.Changsha: Central South University of Forestry and Technology（長沙:中南林業(yè)科技大學(xué)），2012.

［19］HE JW（何建文），YANG WP（楊文鵬），HAN SY（韓世玉），et al.Single major influencing factor and it is orthogonal optimized design of SRAP-PCR in Hot Pepper（Capsicum annum L.）（辣椒SRAP-PCR反應(yīng)體系主要影響因素的單因素和正交設(shè)計優(yōu)化）［J］.Seed（種子），2009，28（9）:25-27.

［20］WANG J（王進(jìn)），HE Q（何橋），OU Y（歐毅），et al.Germplasm identification and phylogenetic analysis of Plum using ISSR marker（李種質(zhì)資源ISSR鑒定及親緣關(guān)系分析）［J］.Journal of Fruit Science（果樹學(xué)報），2008，25（2）:182-187.

［21］QIAO YS（喬玉山）.Establishment of RAPD，ISSR and SSR reaction system and analysis of genetic diversity of Japanese Plum cultivars （中 國 李RAPD、ISSR和SSR反應(yīng)體系的建立及其品種資源遺傳多樣性分析）［D］. Nanjing:Nanjing Agricultural University（南京:南京農(nóng)業(yè)大學(xué)），2003.

［22］GOUL?O L，CORVO LM，OLIVEIRA CM.Phenetic characterization of Plum cultivars by high multiplex ratio markers:amplified fragment length polymorphisms and inter-simple sequence repeats［J］.J Amer Soc Hort Sci，2001，126（1）:72-74.

（Continued on page 503）

基于ISSR分子標(biāo)記的新疆歐洲李親緣關(guān)系分析

孫琪，廖康*，耿文娟，劉娟，曼蘇爾·那斯?fàn)?，劉歡，賈楊，曹倩 （新疆農(nóng)業(yè)大學(xué)特色果樹研究中心，新疆烏魯木齊830052）

［目的］探究不同新疆歐洲李種質(zhì)類型間的親緣關(guān)系。［方法］運(yùn)用ISSR分子標(biāo)記對5個類型共30個單株的新疆歐洲李種質(zhì)資源進(jìn)行遺傳多樣性及親緣關(guān)系分析。［結(jié)果］篩選獲得的16條引物共擴(kuò)增出317條帶，其中有多態(tài)性條帶246條，占擴(kuò)增總數(shù)的77.60%。供試單株的Nei’s遺傳多樣性指數(shù)（H）為0.266 6，平均Shannon信息多樣性指數(shù)（I）為0.399 1，遺傳相似系數(shù)多在0.555 2～0.996 8之間，表明新疆歐洲李資源具有一定的遺傳多樣性。聚類分析顯示，在遺傳相似系數(shù)0.719處30株供試歐洲李被劃分為3個大組，國外引進(jìn)歐洲李品種一組、塔城酸梅和塔城檳子一組、伽師酸梅和野生歐洲李一組，而在相似系數(shù)0.949處，伽師酸梅和野生歐洲李又分為兩個小組。［結(jié)論］伽師酸梅與野生歐洲李的親緣關(guān)系很近，國外引進(jìn)歐洲李品種與塔城酸梅及塔城檳子的親緣關(guān)系較近。

新疆；歐洲李；ISSR；親緣關(guān)系

A t the taxonomy，Prunus domestica L.belongs to Prunus，Rosaceae［1］.There are several types of P.domestica germplasm resources across Xinjiang.For example，there are Jiashi Smoked Plum and introduced European plum varieties in Kashgar，Wild European Plum in Ili，and Tacheng Smoked Plum and Tacheng Binzi in Tacheng，etc［2］.Xinjiang is the only region where Wild European Plum has been discovered［3-4］.The origin of P.domestica is still controversial.Someone considers that P.domestica is the offspring formed by crossing between Prunus cerasifera L.and Prunus spinosa L.；while someone considers that Wild European Plum which is only distributed in Xinjiang is the true progenitor of cultivated P.domestica［5-9］.This study clarified the genetic relationships among different types of P.domestica germplasm resources in Xinjiang so as to help to explore the genetic characteristic of P.domestica germplasm resources in depth and to lay a foundation for future studies on origin and new variety breeding of P.domestica.

There are rare previous studies on P.domestica at the molecular level. Geng et al.［10］analyzed the genetic relationships of P.domestica by SSR markers.They found the genetic diversity in the internal of Wild European Plum was limited，and the one who had closest genetic relationship with Wild European Plum was local varieties.Liu［5］，F(xiàn)eng［11］and Liang et al.［12］all involved parts of P.domestica materials when they studied Prunus and its related species，and they speculated that there was a closer genetic relationship between P.domestica andP.spinosa.Decroocq et al.［13］studied the genetic relationship between parent and offspring of introduced cultivated P.domestica by using chloroplast cDNA-SSR markers.Shimada et al.［14］investigated the genetic relationships among Prunus plants by using RAPD markers，and hypothesized that there might exist a possibility of crossing between P.domestica and P.salicina.

野生歐洲李親緣關(guān)系研究；國家自然科學(xué)基金項目（31160387）；新疆維吾爾自治區(qū)果樹重點學(xué)科基金。

孫琪（1989-），女，新疆烏魯木齊人，在讀碩士研究生，研究方向：果樹種質(zhì)資源，E-mail：1056441863@qq.com。*通訊作者，教授，博士生導(dǎo)師，E-mail：13899825018@163.com。

2014-12-22

2015-02-03

Supported by Study on Genetic Relationships of Wild European Plum；National Natural Science Foundation of China （31160387）；Financial Aid from Xinjiang Uygur Autonomous Region Fruit Trees Key Subject.

.E-mail:13899825018@163.com

December 22，2014Accepted:February 3，2015