Genetic Diversity Of Mandarin Investigated By Molecular Markers

Citrus is a very important fruit for agricultural economy in the world,and also is the second largest fruit in China.Mandarin(Citrus reticulata Blanco) is the original species of cultivated citrus.To understand the origin of citrus,it is important to analysis its genetic diversity.Mandarin is one of the most important citrus genotypes in China.Hunan,has abundant citrus native resources existing for over 2000 years.So Hunan is a geographic gene center of citrus.Citrus taxonomy and phylogency are very complicated,controversial and confusing,mainly due to sexual compatibility between citrus and related genera,highly frequency of bud mutation,long history of cultivation and wide dispersion.With the propose to clarify the relationships among the native citrus resources in Hunan,Molecular markers were developed.The main results as follows:1.A appropriate PCR reaction system for detecting gene in Mandarin with SRAP was developed.Each 25ul PCR reaction mixture consisted of 50ng of genemic DNA,1.5mmol/L of MgCl₂,0.3mmol/L of dNTP,0.2umol/L of enemprimer and 1.0 unit of Taq polymerase.The mixture was initially denatured at 94℃for 5min,then cycled 35 times at 94℃for 1min,50℃for 1min,and 72℃for 1min and finally extended at 72℃for 10min.2.A appropriate PCR reaction system for detecting gene in Mandarin with EST-SSR was developed.Each 25ul PCR reaction mixture consisted of 25ng of genemic DNA,2.5mmol/L of MgCl₂,0.2mmol/L of dNTP,0.2umol/L of enemprimer and 1.0 unit of Taq polymerase.The mixture was initially denatured at 94℃for 1min,then cycled 45 times at 94℃for 1min,52℃for 1min,and 72℃for 1min and finally extended at 72℃for 3min.3.Thirty-one pairs of SRAP primers exhibited the polymorphism among 130 SRAP primers combination and eleven pairs of EST-SSR primers exhibited the polymorphism among twenty-three EST-SSR primers combination.The dendrogram was obtained by UPGMA analysis.The 25 accessions were grouped into ten groups at the coefficient of 0.795.It is ascertained that C.ichangensis Swing,Citron, C.grandis Osbeck are among citrus genera.The results showed the closest relationship between C.reticulata Blanco and C.medica L,C.indica Tan.4.Nine pairs of SRAP primers and nine pairs of EST-SSR primers had been adopted in wild mandarin.115 clean and stable bands had been detected.The number of polymorphic bands were 101,and with polymorphic information content values of 0.636.The dendrogram was obtained by UPGMA analysis.The 9 accessions were grouped into five groups at the coefficient of 0.75.The relationships may be esisted that C.mangshanensis S.W.He et G.F.Liu and C.indica Tan→C.reticualta 'Congyi',C.tachibana(Mak.) Tanaka→C. daoxianensis S.W.He et G.F.Liu→C.reticulate Blanco.Nanling Mountains is one of the native centers of the subgenus Eucitrus.5.Eleven pairs of SRAP primers and eleven pairs of EST-SSR primers had been adopted in the 'Guoqing' Satsuma.108 clean and stable bands had been detected.The number of polymorphic bands were 40,and with polymorphic information content values of 0.386.The dendrogram was obtained by UPGMA analysis.The 14 accessions were grouped into four groups at the coefficient of 0.82.1) Gong chuan;2) Wei zhang;3) C.reticulata Blanco 'Bendiguanggan';4) Others nine accessions of C.reticualta Blanco 'C.unshiu Marc',and the various species mutated from C.unshiu Marc 'Gongchuan' had been distinguished.6.Fourteen pairs of SRAP primers and ten pairs of EST-SSR primers had been adopted in the C.ichangensis Swingle and C.junos Sieb.ex Tan.134 clean and stable bands had been detected.The number of polymorphic bands were 90,and with polymorphic information content values of 0.596.Compared with othes C.ichangensis Swing(Such as C.ichangensis Swing 'Huaihua',C.ichangensis Swing ' Guizhou',C.ichangensis Swing '6-3', C.ichangensis Swing 'Gongsun',C.ichangensis Swing '25-86',C.ichangensis Swing '2-3'),C.ichangensis Swing 'Shanye' was more primitive.It may be exsisted that C.ichangensis Swing 'Shanye' is the ancestor of C.ichangensis Swing.7.Eight pairs of SRAP primers and ten pairs of EST-SSR primers had been adopted in the analysis.The results showed the closest relationship between C. reticulate Blanco 'Changsha' and C.reticulate Blanco 'Zhuhong' is 0.825,C. mangshanensis S.W.He et G.F.Liu and Shatangju is 0.82,C.reticulate Blanco 'Chazi' and C.reticulate Blanco 'Bendiguanggan' is 0.81,C.reticulata Blanco 'Xipi' and C.reticulate Blanco 'Dafen' is 0.92.Furthermore,Choupi Gan mandarin was the primary genotype among landraces and wild mandarins,and the various of Citrus Hybird had been distinguished.8.PCR amplified citrus chloroplast DNA was purified,cloned and sequenced in order to identify cpSSRs.These cpDNA microsatellites were usually single nucleotide repeats,mainly A·T repeats.Dinucleotide poly(AT,TC,CT,TA,GA) and A trinucleotide poly(TAA) motify were also found.The microsatellite sequence was "TAATAATAAAA".The chloroplast DNA homology between C.daoxianensis S.W.He et G.F.Liu and C.tachibana(Mak.) Tanaka was very high,between C.reticulate 'Sanxia hong' and C.reticulate 'Ponkan' was as high as 99%bv DNAMAN software.