Study On Genetic Diversity Of Rubus And Selection Of Its Specific Excellent Germplasm

The genus Rubus is one of the largest genera in the Rosaceae,consisting of 750-1000 species in many parts of the world,of which about 200 species with more than 130 native have been recorded in China.Those Rubus plants used for horticultural plantations are generally named bramble or edible Rubus.Due to its nutritional values and medical uses, Rubus fruits arc named Fruits of Life by Americans and brambles were recommended as The Third Generation Fruits by FAO.Until today,brambles have been widely planted in many parts of the world and has increasingly become the most rapidly developing small fruit tree just next to strawberry,blueberries and currants.Appropriate cultivars is the key to develop planting,which are decided by the quantity and quality of genetic resources owned and the depth and width studied,so is brambles industry.The southwest part of China is now not only the center of distribution of Rubus,but it is the center of its differentiation,where exceptionally abundant in wild bramble resources.However,there is almost no deep study on them.Therefore,it is quite necessary to study systematically the genetic diversity of Rubus distributed in the southwest region,especially Sichuan province and bramble cultivars introduced abroad based on cytological,palynological and molecular methods.At the same time,It should also be done for selection and evaluation of excellent Rubus resources.Thus it will bc helpful for better understanding biosystematic relationship of Rubus,and it also will be helpful for further ultilizing effectively germplasm resources of Rubus.Based on these considerations,the present study had been done and its main results showed as follows:1 Using the root-tips of Rubus niveus,R.ellipticus var.obcordatus and R.coreanus as materials,the key factors such as the sampling time and environmental temperature when sampling materials,pretreatment agents(8-hydroxyquinoline,8-hydroxyquinoline plus colchicine and saturated p-dichlorobenzene),temperatures and times,dissociating agents(25 g·L^-1 cellulase plus 25 g·L^-1 pectic enzyme and hydrochloric acid),processes and times and staining agents(acetocarmine,carbol fuchsin,schiff's solution and ferriammonium sulfate-haematoxylin) and methods,which influenced the preparation of chromosome,had been studied.Based on the contrasts of numbers of mitosis metaphase cells,the difficulty of techniques,the degree of treatment complications and the fact that chromosomes were distinct and countable from their background under microscope,the suitable root-tip squashing method for chromosome preparation of Rubus was as follows:sampling materials when the roots were 1-2 cm in length and the environmental temperature was 17-23℃and fixating with Carnoy's I,pretreating with 0.002mol·L^-1 8-hydroxyquinoline at 0-4℃for 24 h, dissociating with 1mol·L^-1 HCl at 60±1℃for 4-7 m,and staining with carbol fuchsin solution.The chromosomes obtained by the preparation method described above could be used for karyotype analysis.2 Rubus is a taxonomically difficult group and cytological data are expected hopefully to gain insight into the relationships of the genus.In this study the chromosome numbers and karyotypes of 45 populations belonging to both 21 taxa from sect.Idaeobatus and 11 taxa from sect.Malachobatus were investigated,and those of 17 bramble cultivars with 8 Raspberry and 7 Blackberry introduced abroad were investigated too.The results showed: (1) These taxa were characterized by the round prochromosome type of the interphase nuclei and the proximal type of the prophase chromosomes in somatic cells.(2) No aneuploids were found in all the materials studied.(3) The chromosomes were small in size with a length of less than 3μm and metacentric(about 90%) or submetacentric.All taxa have karyotypes of"1A" except R.cockburnianus,R.innominatus and R.ellipticus var.obcordatus which had karyotypes of "2A".Plants of sect.Idaeobatus have diploids with 2n=2x=14, except R.idaeopsis(2n=3x=21) and R.parvifolius(four various ploidys of 2n=2x=14, 2n=3x=21,2n=4x=28 and a mixoploid of 2n=2x=14 and 2n=4x=28).However,Plants of sect. Malachobatus had tetraploids with 2n=4x=28,except R.buergeri with 2n=8x=56.In addition, conspicuous karyotype differences existed within the 21 taxa belonging to 7 of 11 subsections in sect.Idaeobatus,and the differences between some species within subsections were greater than that between subsections,while uniform karyotypes within subsections and variable karyotypes between subsections were observed in 6 of 13 subsections in sect.Malachobatus represented by 11 taxa.Systematic values of the cytological data were discussed for some cases when applicable to the genus.(4) To provide and accumulate cytological data for bramble breeding in China,karyotypes of 8 Raspberry cultivars and 7 Blackberry cultivars introduced from foreign countries were investigated in the article.The results suggested that all the 15 bramble cuitivars were of euploids and no aeuploids were found.Chromosome numbers of 14 were found in all the 8 Raspberry cultivars,and the karyotype formulae of 2n=2x=14=14m,2n=2x=14=14m(2SAT) and 2n=2x=14=14m(4SAT) were found respectively in the 4 cultivars 'Algonquin','Reveille','Dinkum' and 'Ploana',in the other 3 cultivars 'Chilcotin','Nova',and 'Bristol' and in the cultivar 'Chilliwack'.While three various chromosome numbers of 28,42 and 49 were found in the 7 Blackberry cultivars,their karyotypes formulae were as follows:'Arapaho','Shawnee' and 'Navaho',2n=4x=28=28m; 'Ollalie',2n=6x=42=38m(6SAT)+4sm;'Black Butte','Boysen' and 'Kotata', 2n=7x=49=49m.The karyotypes of all the bramble cultivars,except that of'Ollalie',which was composed of metacentric chromosomes and submetacentric chromosomes and had a karyotype asymmetry type 2A,were composed of metacentric chromosome with type 1A asymmetry.(5) In the study,the chromosome numbers of 11 taxa R.idaeopsis(2n=3x=21),R subinopertus(2n=2x=14),R.ellipticus var.obcordatus(2n=2x=14),R.subtibetanus (2n=2x=14),R.inopertus var.echinocalyx(2n=2x=14),R.amabilis(2n=2x=14),R.stans (2n=2x=14),R.tsangii(2n=2x=14),R.parkeri(2n=4x=28),R.faberi(2n=4x=28) and R. yiwuanus(2n=4x=28) and karyotypes of 30 taxa were reported firstly and mixoploidy was observed new in the genus.At the same time,chromosome numbers of 9 bramble cultivars 'Algonquin','Chilcotin','Reveille','Chilliwack','Nova','Ploana','Dinkum','Bristol' and 'Black Butte' and karyotypes of all the 15 bramble cultivars were firstly reported too.3 Pollen morphology of totally 37 materials including 20 populations belonging to 16 taxa from Rubus Sect.Idaeobatus and Sect.Malachobatus and 17 cultivars introduced abroad were examined with scanning electron microscopy.The result showed that:(1) The pollen grains were prolate or perprolate,oval or long oval was in equatorial view,and 3-lobed circular in polar view,25.52μm×15.35μm～50.06μm×25.65μm in size.The pollen exine ornamentations could be divided into cerebroid-perforate,perforate-reticulate and striate types,which included 7 subtypes:cerebroid-perforate,perforate,reticulate, perforate-reticulate,striate,striate-perforate and striate-reticulate.(2) The exine ornamentation of these taxa in the same Subsection and these cultivars in the same cultivated populations was of some similarity,while it was distinctly different among even in species and among cultivars.(3) The results of cluster analysis on pollen submicroscopic morphology of 8 wild resources with potential breeding capabilities from Sect.Idaeobatus and Sect.Malachobatus and the 17 bramble cultivars indicated that the size and exine ornamentation characters of pollen could reflect the traditional taxonomy of Rubus.A close genetic relationship between those raspberry cultivats and the wild species R.coreanus or R. niveus in Sect.Idaeobatus and far genetic relationship between those blackberry cultivars and those 3 wild species in Sect.Malachobatus were found.(4) The combination of R.ellipticus Smith(Subsect.Stimulantes Yü& Lu) and R.pinfaensis Levl.et Vant.(Subsect.Pungentes (Focke) Yü& Lu) were supported by their great similarity in pollen morphology in the study.4 The genetic relationships of totally 41 accessions with 20 taxa from sect.Idaeobatus and 16 taxa from sect.Malachobatus was analyzed by using RAPD markers.The results showed:(1) Distinct genetic differences and abundant RAPD variation were present among the accessions.A total of 636 bands were produced by 24 arbitrary primers that selected out from 85 primers,and 608 out of 636 bands(95.60%) were polymorphic.Thus 14-41 polymorphic bands could be amplified from each primer,with an average of 25.3 bands.The genetic similarity coefficients ranged from 0.842-0.976.The results of cluster analysis showed that 41 accessions could be distinguished by RAPD markers and be consistent with botanical classification except R.amabilis.Therefore,RAPD assay is an effective additional method in the systematic study in Rubus.(2) R.pinfaensis Lévl.et Vant.combinated into R.ellipticus Smith was also supported by the study.However,the results indicated that combinated R. ellipticus Smith should be reduced to Subsect.Stimulantes Yü& Lu rather than to Subsect. Pungentes(Focke) Yü& Lu.(3) According to their larger genetic similarities in the 6 R. parvifolius accessions with four various ploidys,it was concluded that the accession R04-2 (3x) probably originate from nuclear fusion of n and 2n gamete generated by the accession R04-1(2x) which was in the same distribution. 5 11 wild excellent Rubus germplasms in China and 17 bramble cultivars introduced abroad were analyzed by using RAPD markers.The results showed:(1) a total of 500 bands were amplified from 22 arbitrary primers,and 490 out of 500 bands(98.00%) were polymorphic.Thus 12-33 polymorphic bands could be amplified from each primer,with an average of 22.3 and the genetic similarity coefficient ranged from 0.687-0.964.(2) 28 materials could be distinguished by RAPD markers and the genetic relationship among taxa and the two cultivated populations Raspberry and Blackberry could be completely reflected. However,it was failed to distinguish red raspberry with even red raspberry.(3)There was a far genetic relationship between excellent bramble wild germplasms in China and any one of two cultivated populations introduced abroad,while quite close relationship among cultivars in the same cultivated population was found,which indicated that the genetic diversity of these cultivars introduced was narrow.6 Three-year continuous systematical observation has been made on 18 introduced bramble cultivars,distributed to 11 Raspberry cultivars 'Bristol,'Reveille','Killarney', 'Algonquin','Tulameen','Chilcotin','Chilliwack','Nova','Dinkum','Ploana' and 'Kiwigold' and 7 Blackberry cultivars 'Ollalie','Shawnee','Arapaho','Navaho','Boysen','Black Butte' and 'Kotata',which were planted in Ya'an and Xichong regions in Sichuan prinvice.At the same time,based on investigation of Rubus distributed in regions as Yaan,Emei Mountain,Tianquan,Xichang,Kangding,Xichong,Cangxi,Neijiang of Sichuan province,7 bramble excellent wild germplasms with potential breeding capabilities, which were R.niveus,R.ellipticus var.obcordatus,R.parvifolius,R.coreanus,R.corchorifolius, R.lambertianus var.glaber and R.setchuenensis,has been selected and evaluated.The results were as follows:(1) Compared with those introduced to Beijing,due to their early budding while late or even fail to leaf-falling for some Blackberry population cultivars,the18 brambles cultivars lasted longer in the growth period and showed better in fruit quality.However,they all were smaller in fruit size and poor in drought resistance and waterlogging tolerance.(2) Although some of which were poor in fruit yield,totally the Blackberry population cultivars showed better adaptability than the Raspberry ones.(3) The results of comprehensive evaluation on the 18 bramble cultivars were that 'Dinkum' and 'Arapaho' were the best, 'Chilcotin','Killarney','Shawnee' and 'Black Butte' were better and the others were relatively poor.(4) Advantanges and disadvantages happened on any one of the elected 7 bramble excellent wild germplasms.There was prominent spine-free character while poor quality for R.setchuenensis.It was excellent in fruit quality but poor in productivity for R.corchorifolius.For R.coreanus and R.ellipticus var:obcordatus,both fruit quality and productivity were good but greatly impacted by germplasm diversity and inhabits.There was relatively high acid content while Vc abundant in fruit for R.lambertianus var.glaber.For R.niveus,there were good productivity and abundant TTS content(up to 14%) but poor in Juice ratio due to too much seeds in fruits.They both were good in both fruit quality and productivity of R.parvifolius,but its seeds were a little big in size.Compared with those of the cultivars introduced,it was small obviously in fruit size for the 7 bramble wild germplasms. However,their other economic traits as fruit flavors,productivity and adaptability,were superior to those cultivars introduced.