【作者】 张冰冰；

【导师】 刘洪章；

【作者基本信息】 吉林农业大学 ， 作物资源学， 2008， 博士

【摘要】 本文对国家果树种质公主岭寒地果树圃保存的抗寒苹果292、梨210份资源进行了表型性状——树姿、树型、树势、一年生枝、叶片、花、果实等基本信息进行描述，同时对这些资源进行了生物学特性——物候期、萌芽率、成枝力、丰产性、矮化性、果实品质、抗寒性、抗病性、染色体倍性等进行鉴定、评价，得到极其丰富的表型遗传多样性。在此基础上，首次建立了抗寒苹果、梨种质资源数据库，提交给国家科技平台建设——E平台，为资源利用提供更广泛的共享。本文应用RAPD技术首次对上述资源进行了分子标记，对其表型遗传多样性进行检测，从分子层面揭示了抗寒苹果、梨的遗传多样性。在此基础上，又结合50余年抗寒苹果、梨育种经验，首次构建了抗寒苹果、梨核心种质。同时应用RAPD分子标记技术首次获得了抗寒苹果、梨种质特异标记。分析研究结果，提出将保存的抗寒苹果资源中的杂交群体分类归属上，应在经典分类学中的真正苹果组中单独列为一系——杂交群系；山楂海棠与陇东海棠在聚类图中，表现出较远的亲缘关系，与山定子亲缘关系较近。实际上山楂海棠和山定子与陇东海棠与陇东海棠野生原始分布也较远。山楂海棠和山定子原产于长白山区，而陇东海棠原产于甘肃，二者有远缘的地理关系。苹果梨、库尔勒香梨因其在聚类图上与白梨聚在一起，表现出与白梨有较近的亲缘关系，因此，将二者在分类学上划分在白梨系统。上述研究为抗寒苹果、梨资源深入研究、加速育种进程——亲本选择、早期鉴定等工作提供了科学依据。主要结果如下：1．本研究对500余份抗寒苹果、梨种质资源的树姿、树型、树势、一年生枝、叶片、花、果实等植物学基本性状进行了较为详细的调查，和对物候期、萌芽率、成枝力、丰产性、矮化性、果实品质、抗寒性、抗病性、染色体倍性等生物学特性进行鉴定、评价的系统研究，掌握了这些资源大量的基本信息：从高大乔木到矮化；从小果型到大果型；从果皮黄白到浓红色；从抱合直立生树姿到开张下垂；果实风味从酸涩苦到甜；从产量极低到丰产；从极抗寒到不抗寒；从极抗病到不抗病，等等这些性状，表现出抗寒苹果、梨种质资源极为丰富的遗传多样性。这些研究为抗寒苹果、梨种质资源数据库建立及初级核心种质构建提供了依据。2．本研究对抗寒苹果、梨种质资源果实中可溶性固形物、糖、酸和维生素C、进行了分析测定。可溶性固形物含量、糖、酸和维生素C含量也表现出丰富的多样性。总的看，苹果资源中小果型果实的可溶性固形物含量、糖、酸和维生素C含量均高于大果型果实，也就是说，山定子高于海棠果高于杂交后代高于大苹果。3．本研究对抗寒苹果、梨种质资源抗寒性鉴定、评价，结果是：苹果资源抗寒能力最强的是山定子(M．baccata Borkh)、毛山定子(M．mandshurica(Maxim．)Kom．)山楂海棠(M．komarovii(sarg)Rehd．)、小酸果等及含有山定子或毛山定子血缘的海棠果(M．prunifolia Brokh．)类；含有苹果(M．pumila)或塞威氏苹果(M．sieversii)血缘的大苹果类抗寒能力弱(如绿香蕉、丹苹一号)；而海棠果与苹果(M．pumila)类的杂种后代趋中变异(如金红、龙冠)，其抗寒能力介于两者之间，并呈连续性正态分布。梨资源抗寒能力强的是秋子梨(P．ussuriensis Maxim．)、白梨(P．bretschneideri Rehd．)次之；洋梨(P．communis L．)抗寒力最差。而秋子梨(P．ussuriensis Maxim．)与白梨(P．bretschneideri Rehd．)、洋梨(P．communis L．)的种间杂种后代趋中变异，平均抗寒能力介于两者之间。同时也有个别后代是超亲遗传。抗寒能力最强的可抗—45℃以上的极端低温。4．本研究对抗寒苹果、梨种质资源抗病性鉴定、评价是：苹果资源抗苹果腐烂病能力最强的是山楂海棠、扎矮山定子、小黄海棠、黄太平、小酸果、大秋等；梨资源抗梨黑星病能力最强的是秋子梨中的酸梨锅子、真红宵梨和西丰京白，他们同时又是抗寒的优良资源。本研究还得出抗寒苹果资源材料抗寒力与抗苹果腐烂病能力二者之间呈正相关，既抗寒能力强的资源材料，其抗苹果腐烂病能力也越强。这对抗寒苹果、梨种质资源的深入研究、构建核心种质和育种利用提供了科学依据和优异的资源。5．本研究对抗寒苹果、梨种质资源染色体倍数性鉴定结果是，所有的供试资源不论分类学上属于哪个种，不论果实、叶片大小均是二倍体，2n=2x=34。6．本研究首次在对抗寒苹果、梨种质资源表型性状描述、鉴定评价研究基础上，建立了抗寒苹果、梨种质资源数据库。7．本研究首次对抗寒苹果、梨种质资源进行RAPD分子标记研究，构建抗寒苹果、梨RAPD反应体系及优化反应程序，筛选出25个引物。用于对供试的266份苹果、107份梨进行RAPD随机扩增，RAPD扩增带的多态性百分率分别为93.47％、96.2％。使用Nei-Li遗传距离，对180份苹果、87份梨资源的RAPD指纹图谱进行分析研究，并计算资源间的遗传距离。其范围苹果在0.08911～1.0000之间，平均0.00506、金红芽变4号与金红芽变7号的遗传距离最小，仅为0.08911；陇东海棠与铃铛果遗传距离最大，为1.0000。梨在0.0400～0.4269之间，平均0.1934。酸梨锅子与龙香梨的遗传距离最小，仅为0.0400；黑龙江红花盖与远东一号遗传距离最大，为0.4269。8．本研究对聚类图进行分析，得出苹果资源中的山定子系、海棠系和陇东海棠系有着相对较近的亲缘关系，这与经典分类系大体一致。然而，供试的苹果属资源材料的表型性状遗传多样性非常丰富。山定子与海棠果表型性状相近，在此可以断定二者亲缘关系最近。同时，也进一步证明前人所做的结论——海棠果是山定子与大苹果的杂交后代。但是，陇东海棠、山楂海棠的表型性状与山定子有着较大差异。前两者叶片与山楂叶片相似。那么，结合聚类图分析，作者推断，陇东海棠、山楂海棠可能是山定子与同属蔷薇科中的山楂远缘杂交后代，因苹果属、梨属、山楂属、花楸属具有共祖关系。得出梨资源材料的表型性状遗传多样性亦非常丰富。其中的苹果梨、库尔勒香梨因其在聚类图上与白梨聚在一起，表现出与白梨有较近的亲缘关系，因此，将二者在分类学上划分在白梨系统。9．本研究首次对抗寒苹果杂交后代资源材料分类归属问题结论是，依照经典分类学，将杂交后代统一划分为杂交群系，在真正苹果组中单独列出一系。10．本研究首次依据对抗寒苹果、梨种质资源植物学、生物学特征特性进行描述及对其抗性鉴定评价、RAPD分子标记分析研究结果，提出并构建了抗寒苹果、梨种质资源的核心种质，苹果46份资源，梨29份资源。11．本研究首次获得抗寒苹果、梨种质资源RAPD特异标记。更多还原

【Abstract】 In the present dissertation, 292 winterhardy apple accessions and 210winterhardy pear accessions conserved in the national fruit germplasm Gongzhulinghardy fruit nursery were employed as the experimental materials to describe theirphenotypic characters including tree posture, tree type and characteristics ofone-year-old branch, leaf, flower and fruit; to evaluate and determine their biologiccharacteristics including phenological period (phenophase), sprouting rate, branchingability, cropping efficiency, dwarfing ability, fruit quality, cold-resistance,disease-resistance and chromosome ploidy; so that the phenotypic genetic diversitywas found.On the base of phenotypic results, the database about winterhardy apple and peargermplasm resources was set up and submitted to E-deck constructed by the nationalscience and technology ministry in order to provide the broad share and utilization.RAPD technique was used to make molecular marker in all experimentalmaterials so that the phenotypic genetic diversity was screened and verified, then thegenetic diversity of winterhardy apple and pear was further revealed in molecularlevel. Combining the result in molecular marker and the experience accumulatedduring the past 50 years in winterhardy apple and pear breeding, the core collectionsin winterhardy apple and pear were first constructed and the particular RAPDmolecular markers were gained from winterhardy apple and pear germplasmresources.By the further analysis of the research results, we proposed that the hybridprogeny (group) in winterhardy apple resources maintained at the repository wasclassified by itself and formed a new serial -- hybrid group serial in Sect. Malus; onthe cluster graph, M. komarovii had long distance with M. kansuensis illustrated thefar genetic relationship, and short distance with M. baccata illustrated the closegenetic relationship. In fact, both M. komarovii and M. baccata originated atChangbai mountain area, but M. kansuensis originated at Gansu province, so thegeographic distance between two origin places is very big. Because Pingguoli andKuerlexiangli were clustered together with Pyrus bretschneideri, so these varietiesillustrated the near relationship with P. bretschneideri, then were assorted into thesystem of P. bretschneideri.By the all research results above, the scientific foundation was made for furthergermplasm resource research, speedup of breeding procedure, parent selection, early stage identification and so on. Main results were as follows:1. The basic information of 502 winterhardy apple and pear accessions wascollected by the detailed investigation of botanic characters including tree posture,tree type and characteristics of 1-year-old branch, leaf, flower and fruit; and theevaluation and determination of their biologic characteristics including phenologicalperiod (phenophase), sprouting rate, branching ability, cropping efficiency, dwarfingability, fruit quality, cold-resistance (hardiness), disease-resistance and chromosomeploidy. Then, substantial genetic diversity was found in many (phenotypic) characters;i. e. from tall arbor to bush, from big to small fruit, from yellowish to strong redpericarp, from upright to drooping tree posture, from sour, astringent, bitter to sweetfruit flavor, from low to high productivity, from strong to weak cold-resistance, fromstrong to weak disease-resistance and so on. All of these results made it possible to setup the database and to construct the preliminary core collections among winterhardyapple and pear germplasm resources.2. By the analysis and measuration of soluble solid content, sugar content, acidcontent and vitamin C content in the fruit of winterhardy apple and pear germplasmresources, the genetic diversity was verified in the characters above. General speaking,the content of soluble solid, sugar, acid and Vitamin C of the small and middle-sizefruit in apple resources were higher than those of big-size fruit, that is, the order fromhigh to low content in fruit was M. baccata > M. asatica > hybrid progeny > M.domestica.3. By the evaluation and determination of cold-resistance about winterhardy appleand pear germplasm resources, the apple resources of strong cold resistance were M.baccata Borkh., M. mandshurica (Maxim.) Kom., M. komarovii (sarg) Rehd.,Xiaosuanguo and M. prunifolia Borkh. which had the genetic relationship with M.baccata Borkh. or M. mandshurica (Maxim.) Kom., but apple resources with big-sizefruit e. g. Lvxiangjiao, Danpingyihao, which had the genetic relationship with M.domestica or M. sieversii, had the weak cold resistance; the hybrid progeny betweenM. prunifolia Borkh. and M. domestica e. g. Jinhong, Longguan, had the moderatecold resistance and displayed the normal distribution. In the winterhardy pearresources, P. ussuriensis Maxim. had the strong cold resistance, then P. bretschneideriRehd. middle; and then P. communis L. had the weak cold resistance. The hybridbetween P. ussuriensis Maxim. and P. bretschneideri Rehd. or P. communis L. had themoderate cold resistance and displayed the central tendency variation, but some individuals displayed super-parent heredity. The resources with the strongest coldresistance could resist minus 45℃.4. By the evaluation and determination of disease-resistance about winterhardyapple and pear resources, the resources with the strong resistance to apple canker wereM. komarovii, Zha’aishandingzi, Xiaohuanghaitang, Huangtaiping, Xiaosuanguo andDaqiu; the resources with the strong resistance to pear scab were Suanliguozi,Zhenhongxiao and Xifengjingbai, which were very good winterhardy pear germplasmresources. It was also found that the resources which had good cold resistance alsohad the good disease resistance; there was the positive correlation between thecharacters. These results could make a good scientific base for further research,construction of core collections and utilization in breeding about winterhardy appleand pear germplasm resources.5. By the determination of chromosome ploidy on winterhardy apple and peargermplasm resources, all of the experimental materials belonged to diploid 2n=2x=34, no matter species, fruit or leaf.6. Based on the results of phenotypic character description, evaluation anddetermination, the database was set up about winterhardy apple and pear germplasmresources.7. Using the winterhardy apple and pear germplasm resources, RAPD reactionsystem and optimizing reaction procedure were established, 25 primer were selectedand used in 266 apple accessions and 107 pear accessions. The percentage of thepolymorphism bands by RAPD amplifying was 93.47% and 96.20% for apple andpear respectively. By the Nei-Li genetic distance, 180 apple accessions and 87 pearaccessions were analysis and calculated the value of the genetic distance, which was0.0891 to 1.000 in apple (average value 0.00506) and 0.040 to 0.4269 in pear (averagevalue 0.1394). In apple resources, Jinhong4hao and Jinhong7hao had the smallestgenetic distance 0.08911, but M. kansuensis and LingDangGuo had the biggestgenetic distance 1.0000. In pear resources, Suanliguozi and Longliangli had theminimum genetic distance 0.040, however, Heilongjianghonghuagai andYuandongyihao had the maximum genetic distance 0.4269.8. By the analysis of cluster graph, it was found that Sect. M. baccata, Sect. M.prunifolia and Sect. M. kansuensis had the close genetic relationship which was samewith the classic taxology. But the phenotypic genetic diversity was very rich(abundant) in the Malus resources. Because the phenotypic character between M. baccata and M. prunifolia was close, they had the nearest genetic relationship. It wasverified that M. prunifolia was the hybrid between M. baccata and M. domestica,which was reported before. But there were obvious differences between M.kansuensis or M. komarovii and M. baccata in the phenotypic characters. Combinedwith the analysis of the cluster graph, the present author proposed that M. kansuensisand M. komarovii were derived from the cross of Malus and Crataegus because Malus,Pyrus, Crataegus and Sorbus had the same ancient species. About pear, there wasalso the abundant phenotypic diversity. Because Pingguoli and Kuerlexiangli wereclustered together with P. bretschneideri, and illustrated the near genetic relationshipwith P. bretschneideri, so they were assorted into the system of P. bretschneideri.9. According to the classic taxology, hybrid progeny in winterhardy appleresources was formed a new series -- hybrid group series in Sect. Malus.10. According to the results in botanic and biologic investigation, evaluation anddetermination, and the result in RAPD molecular marker, core collections inwinterhardy apple and pear germplasm resources was constructed, which were 46accessions in apple and 29 accessions in pear.11. Particular RAPD markers were gained from winterhardy apple and peargermplasm resources.更多还原