Research On Genetic Relationship Among Buckwheat Species In Southwest Of Sichuan

Research on Genetic Relationship among Buckwheat Species in southwest of Sichuan Biochemistry and molecular biology:Yang Xiao-yan Directed by Prof.Chen HuiThe buckwheat belongs to the Fagopyrum of polygonaceae.It origins from China,most of them are planted in northwest,northeast,as well as the arctic-alpine mountain area of southwest.It is considered as one kind of green food material and medicinal plant.It has great values for development. In recent years,because of ecological environment destruction,the buckwheat idioplasmic resources reduced gradually.Meanwhile,not only the research on buckwheat started lately in China especially in southwest of Sichuan province,but also the funds for research were short,the basis reaserch,such as the origin,the evolution and the classification of buckwheat was nearly blank;the applied research,such as tissue culture,cultivation also was smack.So,In this study,10 representative buckwheat species which from Liangshan and Ya'an city were used to study their genetic relationship by using morphological comparison,karyotype analysis,POD,EST and RAPD.The main purposes of the study are establishing the foundations for improving hereditary and the breeding work about buckwheat,simultaneously promoting the research on the origin,evolution and classification of buckwheat.The main results are as following:Morphological features comparsion indicates that:morphological differences of 10 buckwheat materials are mainly embodied in the type of plant,plant height,stem diameter,the number of stem, branches,leaf shape,the type of inflorescence,the number of pedicel,pistil,the luster,size and weight of 1000 seeds.Based on these indicators,10 species are devided into four small groups,the first includes only one specie:F.statice(A'ba);the second includes one specie:F.tataricum;the third includes three species:F.gracilipe,F.gracilipes var.odontopterum and F.gracilipes;the forth group includes five species:F.cymosum(A 'ba),F.gracilipes var.odontopterum(A 'ba),F.cymosum(Xichang), F.esculentum and F.cymosum(Ya'an).The karyotypes of three species,F.densovillosum,F.gracilipes var.odontopterum,and F. gracilipes are reported for the first time.We find that:(1) F.esculentum,F.cymosum,F.tataricum and F. densovillosum are diploid,2n=2x=16,F.gracilipes var.odontopterum and F.gracilipes are tetraploid, 2n=4x=32.(2) In the diploid level,the variation of the buckwheat chromosome karyotypes is bigger,but in the tetraploid level,it presented a higher similarity.(3) The evolutionary trends of karyotype in buckwheat were from asymmetry to symmetry.(4) According to the evolutionary trends,we found that F.densovillosum seemed to be more advanced than F.gracilipes var.odontopterum and F.gracilipes.The POD of 10 species' young leaves separate 15 enzymes bands.Among them,the band of F. densovillosum is the richest,running up to 12.Their ratio of flow ranged from 0.06 to 0.56,the bands concentrate in the range from 0.19 to 0.33.The result of clustering analysis about POD showed that,the Dice similarity coefficients of each interspecies ranged from 0.47 to 0.93.The Dice similarity coefficients of F.cymosum(Ya 'an),and F.cymosum(Xichang) is the highest(0.93),indicated that the relationship between them is the nearest.The Dice similarity coefficients of F.densovillosum and F. tataricum is the lowest(0.29),indicated that the relationship between them is the farthest.The EST of 10 species' young leaves separate 10 enzymes bands.Their ratio of flow ranged from 0.15 to 0.52,the bands mainly concentrate in 0.15～0.33.The result of clustering analysis about EST showed that,the Dice similarity coefficients of each interspecies ranged from 0.44 to 0.92,indicating that the difference of genetic relationship among them is great.The Dice similarity coefficients of F.cymosum(A 'ba) and F. cymosum(Ya'an) is the highest(0.92),indicating that the genetic relationship between them is the nearest.Take primers,dNTPs,Mg²⁺,Taq polymerase,DNA template consistency as five fa.ctors,and each factor set four levels,optimizing the RAPD conditions by orthogonal test.Fourteen random primers were selected from 40 random primers which purchases from the Sai baisheng company.388 bands were obtained,each random primer obtained 27.71 bands;in which 352 bands were polymorphic,each random primer obtained 25.14 polymorphism bands,the ratio of polymorphic bands is 90.72%,and its range of variation is from 62.2%to 100%.According to the expand result,the Dice similarity coefficients of each interspecies ranged from 0.41 to 0.65.The Dice similarity coefficients of F. cymosum(Xichang) and F.cymosum(Ya 'an) is the highest(0.65),indicated that the genetic relationship between them is the nearest.Take 0.48 as the threshold value,10 species are devided into four small groups,the first includes only one spece:F.statice(A 'ha);the second includes one specie:F.tataricum; the third includes three species:F.gracilipe,F.gracilipes var.odontopterum and F.gracilipes;the forth group includes five species:F.cymosum(A 'ha),F.gracilipes var.odontopterum(A'ba),F.cymosum (Xichang),F.esculentum and F.cymosum(Ya'an).According to the special characteristic of F.gracilipe, such as the surface of leaves have a lot of embossments,a small amount of vertical villi are on both sides of leaves;wavy light of a tooth or a small circle round teeth,a small amount of chorionic villi are on petiole,combining the results of karyotypes analysis,isozyme,RAPD markers,we conclude that F. gracilipe is a new specie.