悬钩子属植物遗传多样性及其特异资源的筛选
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摘要
悬钩子属Rubus L.,又名树莓属,是蔷薇科Rosaceae中的一个大属。全世界有悬钩子属植物750-1000种,中国有200多种,其中有130多种是特有种。该属植物中果实可食的用于园艺栽培生产的种类称为树莓或可食悬钩子,其果实具有较高的营养价值和医疗保健作用,美国人称之为“生命之果”,世界粮农组织(FAO)将其推荐为“世界第三代水果”,在世界范围内得到了广泛种植,已成为继草莓、越桔和醋栗之后发展最快的小果类果树。发展树莓生产,品种是关键。然而,没有优良的种质就不可能有优良的新品种,而了解物种的遗传基础以及物种之间的亲缘关系是合理有效利用种质资源的基础和前提。目前,我国育成的树莓品种极少,而同时我国西南地区是悬钩子属植物的分布和分化中心,类型复杂,变异性大,但对其研究主要以形态描述为主,内在系统学研究较少,遗传基础和遗传关系知之甚少,许多种的分类尚存争议。因此,本研究首次系统地对西南地区尤其是四川分布的悬钩子属Rubus植物中树莓优良野生种质分布集中的空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus的14个亚组的30多个种类以及国外引进的树莓品种进行细胞学、孢粉学和分子生物学研究,探讨种类品种亲缘关系和遗传差异,并对在种质资源调查过程中发现的树莓优良野生种质和18个引进树莓品种进行筛选和评价,以期为该属植物的系统演化规律和合理分类提供依据,为树莓育种种质的有效选择奠定基础。其主要结果如下:
1以红泡刺藤R.niveus、栽秧泡R.ellipticus var.obcordatus和插田泡R.coreanus绿枝或硬枝扦插生根的根尖为材料,通过取材时季与扦插环境气温,8-羟基喹啉、秋水仙素和对二氯苯3种药剂在不同的温度下预处理不同时间,混合酶液和盐酸在不同温度下解离,醋酸洋红、卡宝品红、席夫试剂和铁矾-苏木精各染色剂染色压片等的对比研究,并以细胞中有丝分裂中期分裂相的数目、染色体的清晰性、分散程度和聚缩程度以及染色体的形态为衡量指标,探索出适于悬钩子属植物核型分析的根尖压片方法是:当根尖长至1-2cm、扦插环境气温17-23℃时取材,卡诺氏固定液Ⅰ低温固定24h,用0.002mol·L~(-1)8-羟基喹啉在0-4℃下预处理24h,再用1mol·L~(-1)HCl 60±1℃恒温解离4-7min,最后用卡宝品红染色压片能获得效果好染色体标本,该染色体标本完全适用于核型分析。
2采用根尖压片法对中国悬钩子属Rubus空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus 13个亚组的32个种(变种)45个居群以及15个国外引进的树莓栽培品种进行了细胞学研究。研究结果表明:(1)研究的所有材料的间期核和前期核都分别属于球状前染色体型(Round prochromosome type)和近基型(Proximal type):(2)所有材料均为整倍体,有未发现有非整倍体类型;(3)中国空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus植物染色体均小形,绝对长度均在3μm以下,染色体由中部着丝点染色体(约90%)和近中部着丝点染色体构成,核型分类主要属于“1A”型(85%以上),说明这两组植物在整个系统演化中处于相对原始的地位。但是两个组的染色体数目和核型结构有较大差异。在空心莓组Sect.Idaeobatus的21种(变种)植物中,除拟复盆子R.idaeopsis为2n=3x=21、茅莓R.parvifolius存在2n=2x=14、2n=3x=21、2n=4x=28和2n=2x=14与2n=4x=28的混倍体等4种倍性类型外,其余种(变种)染色体数目均为2n=2x=14。而在木莓组Sect.Malachobatus的11个种(变种)中,除寒莓R.buergeri为2n=8x=56外,其余种的染色体数目和倍性均为2n=4x=28。此外,本研究所涉及的空心莓组Sect.Idaeobatus 11个亚组中的7个亚组的21个种(变种),亚组内的物种间甚至同一物种的不同居群间染色体核型构成差异明显,有些甚至超过了亚组间一些物种的差异;而涉及的木莓组Sect.Malachobatus 13个亚组的6个亚组11个种(变种),亚组间物种染色体核型结构差异较明显,但亚组内物种间染色体核型结构差异较小,依据细胞学资料对其系统演化和一些种的分类进行了讨论;(4)对从国外引进的8个树莓种群品种和7个黑莓种群品种的核型研究表明,它们都为整倍体,未见非整倍体。8个树莓种群的品种染色体数目均为14条,而7个黑莓种群品种有28、42和49三种不同的染色体数目。在树莓种群的8个品种中,‘Algonquin'、‘Reveille'、‘Ploana'和‘Dinkum'4个品种的核型公式皆为2n=2x=14=14m,‘Chilcotin'、‘Nova'和‘Bristol'3个品种核型公式为2n=2x=14=14m(2SAT),‘Chilliwack'的核型公式为2n=2x=14=14m(4SAT)。而黑莓种群的7个品种的核型公式分别如下:‘Arapaho'、‘Shawnee'和‘Navaho'为2n=4x=28=28m,‘Ollalie'为2n=6x=42=38m(6SAT)+4sm,而‘Black Butte'、‘Boysen'和‘Kotata'为2n=7x=49=49m。在核型分类上,除‘Ollalie'由中部着丝粒和近中部着丝粒染色体构成属于“2A”型核型外,其余的14个品种均由中部着丝粒染色体构成,为“1A”型核型。上述研究中拟复盆子R.idaeopsis(2n=3x=21)、紫红悬钩子R.subinopertus(2n=2x=14)、栽秧泡R.ellipticus var.obcordatus(2n=2x=14)、密刺悬钩子R.subtibetanus(2n=2x=14)、刺萼红花悬钩子R.inopertus var.echinocalyx(2n=2x=14)、秀丽莓R.amabilis(2n=2x=14)、直立悬钩子R.stans(2n=2x=14)、光滑悬钩子R.tsangii(2n=2x=14)、乌泡子R.parkeri(2n=4x=28)、峨嵋悬钩子R.faberi(2n=4x=28)和奕武悬钩子R.yiwuanus(2n=4x=28)等11个种(变种)和‘Algonquin'、‘Chilcotin'、‘Reveille'、‘Chilliwack'、‘Nova'、‘Ploana'、‘Dinkum'、‘Bristol'和‘Black Butte'9个树莓品种的染色体数目、30个悬钩子属植物种(变种)和15个树莓品种的核型以及该属植物存在混倍体类型(茅莓R.parvifolius(2n=2x=14与2n=4x=28的混倍体))为首次报道。
3用扫描电镜对中国悬钩子属Rubus空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus 9亚组16种(变种)的20个居群以及从国外引进的分属于黑莓种群和树莓种群的17个栽培品种共37份材料的花粉形态进行了较系统观察和比较分析。结果表明:(1)所有材料的花粉均为长球形或超长球形(P/E为1.67～2.26),大小多为25.52μm×15.35μm～50.06μm×25.65μm;(2)所观察材料花粉的外壁纹饰在组内和栽培种群内有一定的相似性,但变异丰富,在不同种之间、甚至在同一种群的不同品种和同一种的不同居群之间区别明显。其外壁纹饰共有属于脑纹-穿孔型、穿孔网型和条纹型三大类型的脑纹-穿孔状、穿孔状、网状、穿孔-网状、条纹状、条纹-穿孔状和条纹网状7个亚型;(3)对四川有育种潜力的分属于空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus的8个树莓优良野生种质与国外引进的17个树莓品种进行了比较分析发现,空心莓组Sect.Idaeobatus的5个种质和树莓种群的品种的外壁纹饰以条纹-穿孔状为主,黑莓种群的品种主为条纹状和条纹-穿孔状,与木莓组Sect.Malachobatus的3个种的差异较大,聚类分析表明树莓种群品种与空心莓组Sect.Idaeobatus的插田泡和茅莓的亲缘关系较近;而黑莓种群与木莓组Sect.Malachobatus的3个有果用育种潜力的任一种质的亲缘关系较远:(4)绒毛叶亚组Subsect.Stimulantes的椭圆悬钩子R.ellipticus和柔毛叶亚组Subsect.Pungentes的红毛悬钩子R.pinfaensis在花粉形态特征上极为相似,实验结果支持两者归并。
4对中国悬钩子属Rubus空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus14个亚组的36个种(变种)的41个居群以及17个引进树莓品种进行了RAPD分析。结果表明:(1)41份野生种质物种间具有丰富的RAPD变异。从85条引物中筛选出的24条引物产生了636条DNA扩增片断,其中608条(95.6%)具有多态性,每个引物可扩增出14-41条多态性带,平均25.3条。RAPD标记的遗传相似系数(GS)变化范围为0.842-0.976。聚类分析显示RAPD标记能将41份悬钩子属材料完全分开,除秀丽莓R.amabilis外,物种间的聚类关系与植物学分类完全一致。因此,RAPD分析方法可为悬钩子属植物的分类鉴定和系统演化提供依据。(2)用RAPD标记对11个中国树莓优良野生种质和17个国外引进栽培品种共28份材料进行亲缘关系研究表明,RAPD标记能将28份材料完全分开,从85条引物中筛选出的22条引物产生了500条DNA扩增片断,其中490条(98.0%)具有多态性,每个引物可扩增出12-33条多态性带,平均22.3条。RAPD标记的遗传相似系数(GS)变化范围为0.687-0.954。聚类能很好地反映这材料种属的亲缘关系以及两大栽培种群的划分,但不能区分树莓种群品种中的红树莓和双季红树莓。(3)11个中国树莓优良野生种质与国外引进的两大树莓种群栽培品种的亲缘关系较远,而两大栽培种群的各自的品种之间亲缘关系较近的事实说明引进的18个品种的遗传基础较为狭窄。(4)支持栽秧泡R.ellipticus var.obcordatus作为独立的种以及椭圆悬钩子R.ellipticus和红毛悬钩子R.pinfaensis归并,保留椭圆悬钩子的种名,但不支持将归并后的椭圆悬钩子划归柔毛叶亚组Subsect.Pungentes,而应该在原所属的绒毛叶亚组Subsect.Stimulantes中。(5)据其不同形态变异的4个不同倍性的茅莓材料的遗传相似系数和亲缘关系,认为三倍体的茅莓R.parvifolius很可能由同一分布地的二倍体茅莓的减数配子(x)和未减数配子(2x)受精融合产生。
5通过连续三年对引入四川雅安和西充的11个国外树莓种群品种‘Bristol'、‘Reveille'、‘Killarney'、‘Algonquin'、‘Tulameen'、‘Chilcotin'、‘Chilliwack'、‘Nova'、‘Dinkum'、‘Ploana'和‘Kiwigold'和7个国外黑莓种群品种‘Ollalie'、‘Shawnee'、‘Arapaho'、‘Navaho'、‘Boysen'、‘Black Butte'和‘Kotata'的栽培生物学特性、生长结果习性和果实的内外品质进行较系统的观测以及在对四川雅安、峨眉山、天全、西昌、康定、西充、苍溪、内江等地广泛分布的树莓野生种质进行调查的基础上,从中筛选出了红泡刺藤R.niveus、栽秧泡R.ellipticus var.obcordatus、茅莓R.parvifolius、插田泡R.coreanus、山莓R.corchorifolius、光滑高粱泡R.lambertianus var.glaber和川莓R.setchuenensis 7个有育种潜力的优良种质,对它们进行较全面的观测并与优良栽培品种进行比较评价。结果表明:(1)18个树莓品种与引种地(北京)相比,皆早萌芽,晚落叶,个别黑莓品种甚至不落叶,生育期延长近一个月:虽然平均单果重小,抗逆能力较差,但果实品质好。黑莓在引种地的适应性表现较树莓种群品种好,但个别品种产量不高。综合比较,双季红树莓‘Dinkum'和无刺黑莓‘Arapaho'在四川表现较好,红树莓品种‘Chilcotin'、‘Killarney'和黑莓品种‘Shawnee'和‘Black Butte'次之,其余品种表现较差。(2)7个优良树莓野生种质各有优缺点。川莓R.setchuenensis的突出性状是无刺;山莓R.corchorifolius果实品质优良,但丰产性差;果实品质优良同时丰产性好的种质是插田泡R.coreanus和栽秧泡R.ellipticus var.obcordatus,但其受种质遗传多样性及所处生境的影响较大;光滑高粱泡R.lambertianus var.glaber果实品质偏酸,但Vc含量高;红泡刺藤R.niveus可溶固形物含量高,达到14%,丰产性也好,但出汁率低,种子数量极多;而茅莓R.parvifolius果实品质较好,但种子较大。与引入四川的栽培品种相比,这7个种质除平均单果重偏轻外,其它性状如果实风味、丰产性和适应性,均优于我们引进的树莓种群品种。
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.
1以红泡刺藤R.niveus、栽秧泡R.ellipticus var.obcordatus和插田泡R.coreanus绿枝或硬枝扦插生根的根尖为材料,通过取材时季与扦插环境气温,8-羟基喹啉、秋水仙素和对二氯苯3种药剂在不同的温度下预处理不同时间,混合酶液和盐酸在不同温度下解离,醋酸洋红、卡宝品红、席夫试剂和铁矾-苏木精各染色剂染色压片等的对比研究,并以细胞中有丝分裂中期分裂相的数目、染色体的清晰性、分散程度和聚缩程度以及染色体的形态为衡量指标,探索出适于悬钩子属植物核型分析的根尖压片方法是:当根尖长至1-2cm、扦插环境气温17-23℃时取材,卡诺氏固定液Ⅰ低温固定24h,用0.002mol·L~(-1)8-羟基喹啉在0-4℃下预处理24h,再用1mol·L~(-1)HCl 60±1℃恒温解离4-7min,最后用卡宝品红染色压片能获得效果好染色体标本,该染色体标本完全适用于核型分析。
2采用根尖压片法对中国悬钩子属Rubus空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus 13个亚组的32个种(变种)45个居群以及15个国外引进的树莓栽培品种进行了细胞学研究。研究结果表明:(1)研究的所有材料的间期核和前期核都分别属于球状前染色体型(Round prochromosome type)和近基型(Proximal type):(2)所有材料均为整倍体,有未发现有非整倍体类型;(3)中国空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus植物染色体均小形,绝对长度均在3μm以下,染色体由中部着丝点染色体(约90%)和近中部着丝点染色体构成,核型分类主要属于“1A”型(85%以上),说明这两组植物在整个系统演化中处于相对原始的地位。但是两个组的染色体数目和核型结构有较大差异。在空心莓组Sect.Idaeobatus的21种(变种)植物中,除拟复盆子R.idaeopsis为2n=3x=21、茅莓R.parvifolius存在2n=2x=14、2n=3x=21、2n=4x=28和2n=2x=14与2n=4x=28的混倍体等4种倍性类型外,其余种(变种)染色体数目均为2n=2x=14。而在木莓组Sect.Malachobatus的11个种(变种)中,除寒莓R.buergeri为2n=8x=56外,其余种的染色体数目和倍性均为2n=4x=28。此外,本研究所涉及的空心莓组Sect.Idaeobatus 11个亚组中的7个亚组的21个种(变种),亚组内的物种间甚至同一物种的不同居群间染色体核型构成差异明显,有些甚至超过了亚组间一些物种的差异;而涉及的木莓组Sect.Malachobatus 13个亚组的6个亚组11个种(变种),亚组间物种染色体核型结构差异较明显,但亚组内物种间染色体核型结构差异较小,依据细胞学资料对其系统演化和一些种的分类进行了讨论;(4)对从国外引进的8个树莓种群品种和7个黑莓种群品种的核型研究表明,它们都为整倍体,未见非整倍体。8个树莓种群的品种染色体数目均为14条,而7个黑莓种群品种有28、42和49三种不同的染色体数目。在树莓种群的8个品种中,‘Algonquin'、‘Reveille'、‘Ploana'和‘Dinkum'4个品种的核型公式皆为2n=2x=14=14m,‘Chilcotin'、‘Nova'和‘Bristol'3个品种核型公式为2n=2x=14=14m(2SAT),‘Chilliwack'的核型公式为2n=2x=14=14m(4SAT)。而黑莓种群的7个品种的核型公式分别如下:‘Arapaho'、‘Shawnee'和‘Navaho'为2n=4x=28=28m,‘Ollalie'为2n=6x=42=38m(6SAT)+4sm,而‘Black Butte'、‘Boysen'和‘Kotata'为2n=7x=49=49m。在核型分类上,除‘Ollalie'由中部着丝粒和近中部着丝粒染色体构成属于“2A”型核型外,其余的14个品种均由中部着丝粒染色体构成,为“1A”型核型。上述研究中拟复盆子R.idaeopsis(2n=3x=21)、紫红悬钩子R.subinopertus(2n=2x=14)、栽秧泡R.ellipticus var.obcordatus(2n=2x=14)、密刺悬钩子R.subtibetanus(2n=2x=14)、刺萼红花悬钩子R.inopertus var.echinocalyx(2n=2x=14)、秀丽莓R.amabilis(2n=2x=14)、直立悬钩子R.stans(2n=2x=14)、光滑悬钩子R.tsangii(2n=2x=14)、乌泡子R.parkeri(2n=4x=28)、峨嵋悬钩子R.faberi(2n=4x=28)和奕武悬钩子R.yiwuanus(2n=4x=28)等11个种(变种)和‘Algonquin'、‘Chilcotin'、‘Reveille'、‘Chilliwack'、‘Nova'、‘Ploana'、‘Dinkum'、‘Bristol'和‘Black Butte'9个树莓品种的染色体数目、30个悬钩子属植物种(变种)和15个树莓品种的核型以及该属植物存在混倍体类型(茅莓R.parvifolius(2n=2x=14与2n=4x=28的混倍体))为首次报道。
3用扫描电镜对中国悬钩子属Rubus空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus 9亚组16种(变种)的20个居群以及从国外引进的分属于黑莓种群和树莓种群的17个栽培品种共37份材料的花粉形态进行了较系统观察和比较分析。结果表明:(1)所有材料的花粉均为长球形或超长球形(P/E为1.67～2.26),大小多为25.52μm×15.35μm～50.06μm×25.65μm;(2)所观察材料花粉的外壁纹饰在组内和栽培种群内有一定的相似性,但变异丰富,在不同种之间、甚至在同一种群的不同品种和同一种的不同居群之间区别明显。其外壁纹饰共有属于脑纹-穿孔型、穿孔网型和条纹型三大类型的脑纹-穿孔状、穿孔状、网状、穿孔-网状、条纹状、条纹-穿孔状和条纹网状7个亚型;(3)对四川有育种潜力的分属于空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus的8个树莓优良野生种质与国外引进的17个树莓品种进行了比较分析发现,空心莓组Sect.Idaeobatus的5个种质和树莓种群的品种的外壁纹饰以条纹-穿孔状为主,黑莓种群的品种主为条纹状和条纹-穿孔状,与木莓组Sect.Malachobatus的3个种的差异较大,聚类分析表明树莓种群品种与空心莓组Sect.Idaeobatus的插田泡和茅莓的亲缘关系较近;而黑莓种群与木莓组Sect.Malachobatus的3个有果用育种潜力的任一种质的亲缘关系较远:(4)绒毛叶亚组Subsect.Stimulantes的椭圆悬钩子R.ellipticus和柔毛叶亚组Subsect.Pungentes的红毛悬钩子R.pinfaensis在花粉形态特征上极为相似,实验结果支持两者归并。
4对中国悬钩子属Rubus空心莓组Sect.Idaeobatus和木莓组Sect.Malachobatus14个亚组的36个种(变种)的41个居群以及17个引进树莓品种进行了RAPD分析。结果表明:(1)41份野生种质物种间具有丰富的RAPD变异。从85条引物中筛选出的24条引物产生了636条DNA扩增片断,其中608条(95.6%)具有多态性,每个引物可扩增出14-41条多态性带,平均25.3条。RAPD标记的遗传相似系数(GS)变化范围为0.842-0.976。聚类分析显示RAPD标记能将41份悬钩子属材料完全分开,除秀丽莓R.amabilis外,物种间的聚类关系与植物学分类完全一致。因此,RAPD分析方法可为悬钩子属植物的分类鉴定和系统演化提供依据。(2)用RAPD标记对11个中国树莓优良野生种质和17个国外引进栽培品种共28份材料进行亲缘关系研究表明,RAPD标记能将28份材料完全分开,从85条引物中筛选出的22条引物产生了500条DNA扩增片断,其中490条(98.0%)具有多态性,每个引物可扩增出12-33条多态性带,平均22.3条。RAPD标记的遗传相似系数(GS)变化范围为0.687-0.954。聚类能很好地反映这材料种属的亲缘关系以及两大栽培种群的划分,但不能区分树莓种群品种中的红树莓和双季红树莓。(3)11个中国树莓优良野生种质与国外引进的两大树莓种群栽培品种的亲缘关系较远,而两大栽培种群的各自的品种之间亲缘关系较近的事实说明引进的18个品种的遗传基础较为狭窄。(4)支持栽秧泡R.ellipticus var.obcordatus作为独立的种以及椭圆悬钩子R.ellipticus和红毛悬钩子R.pinfaensis归并,保留椭圆悬钩子的种名,但不支持将归并后的椭圆悬钩子划归柔毛叶亚组Subsect.Pungentes,而应该在原所属的绒毛叶亚组Subsect.Stimulantes中。(5)据其不同形态变异的4个不同倍性的茅莓材料的遗传相似系数和亲缘关系,认为三倍体的茅莓R.parvifolius很可能由同一分布地的二倍体茅莓的减数配子(x)和未减数配子(2x)受精融合产生。
5通过连续三年对引入四川雅安和西充的11个国外树莓种群品种‘Bristol'、‘Reveille'、‘Killarney'、‘Algonquin'、‘Tulameen'、‘Chilcotin'、‘Chilliwack'、‘Nova'、‘Dinkum'、‘Ploana'和‘Kiwigold'和7个国外黑莓种群品种‘Ollalie'、‘Shawnee'、‘Arapaho'、‘Navaho'、‘Boysen'、‘Black Butte'和‘Kotata'的栽培生物学特性、生长结果习性和果实的内外品质进行较系统的观测以及在对四川雅安、峨眉山、天全、西昌、康定、西充、苍溪、内江等地广泛分布的树莓野生种质进行调查的基础上,从中筛选出了红泡刺藤R.niveus、栽秧泡R.ellipticus var.obcordatus、茅莓R.parvifolius、插田泡R.coreanus、山莓R.corchorifolius、光滑高粱泡R.lambertianus var.glaber和川莓R.setchuenensis 7个有育种潜力的优良种质,对它们进行较全面的观测并与优良栽培品种进行比较评价。结果表明:(1)18个树莓品种与引种地(北京)相比,皆早萌芽,晚落叶,个别黑莓品种甚至不落叶,生育期延长近一个月:虽然平均单果重小,抗逆能力较差,但果实品质好。黑莓在引种地的适应性表现较树莓种群品种好,但个别品种产量不高。综合比较,双季红树莓‘Dinkum'和无刺黑莓‘Arapaho'在四川表现较好,红树莓品种‘Chilcotin'、‘Killarney'和黑莓品种‘Shawnee'和‘Black Butte'次之,其余品种表现较差。(2)7个优良树莓野生种质各有优缺点。川莓R.setchuenensis的突出性状是无刺;山莓R.corchorifolius果实品质优良,但丰产性差;果实品质优良同时丰产性好的种质是插田泡R.coreanus和栽秧泡R.ellipticus var.obcordatus,但其受种质遗传多样性及所处生境的影响较大;光滑高粱泡R.lambertianus var.glaber果实品质偏酸,但Vc含量高;红泡刺藤R.niveus可溶固形物含量高,达到14%,丰产性也好,但出汁率低,种子数量极多;而茅莓R.parvifolius果实品质较好,但种子较大。与引入四川的栽培品种相比,这7个种质除平均单果重偏轻外,其它性状如果实风味、丰产性和适应性,均优于我们引进的树莓种群品种。
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.
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