摘要
我国苹果生产中的品种大多数为二倍体品种,但是为数不多的三倍体品种却表现优良。三倍体苹果已经成为苹果育种尤其是多倍体育种工作的重要一部分。目前已经通过杂交获得了一部分多倍体资源,但是对这些资源的研究较少,关于遗传变异的机理报道很少,尤其是通过杂交获得的三倍性苹果的遗传变异的分子机理至今报道甚少。本研究以二倍体苹果‘寒富’、‘富士’和‘金冠’为母本,与‘四倍体嘎拉’苹果为父本进行杂交获得的三倍性杂种后代为试材,在对其生物学性状进行调查研究的基础上,对其遗传变异的分子机理进行了探讨,同时对基因组DNA甲基化水平和甲基化模式变异的表观遗传进行了研究,结果如下:
1.‘寒富’与‘四倍体嘎拉’苹果的三倍性杂种后代的叶片、枝条和花性状变异幅度在26.42%~1.33%之间,花粉平均PE值变异幅度为4.46%。大多数性状平均值小于亲中值。果实性状的变异幅度在5.12%~27.82%之间,除平均单果重低于亲中值以外,果形指数、果实硬度、果实可溶性固形物含量均大于亲中值。这6份杂种后代的特征香气大多数受父母本的共同影响,果实香气倾向于父本遗传。果糖是影响这6份三倍性杂种后代的甜味感的最大因素,而苹果酸则是影响其酸味味感的最大因素。
2.‘寒富’与‘四倍体嘎拉’苹果的三倍性杂种后代经ISSR扩增得到的遗传多样性指数中扩增多态性比率(PPB)为60.00%、观察等位基因数(Na)为1.6000、有效等位基因数(Ne)为1.4204、Nei's基因多样性指数(功为0.2368、Shannon信息指数(Ⅰ)为0.3464,均小于AFLP扩增的遗传多样性指数中的相应数值66.54%、1.6654、1.421、0.2405和0.3570。根据ISSR和AFLP得到的UPGMA聚类图显示这7份三倍性杂种后代倾向于母本‘寒富’。在7份三倍性杂种后代中大部分位点来源于父母本,但也存在一定的缺失位点和特有位点。
3.‘富士’与‘四倍体嘎拉’苹果的三倍性杂种后代ISSR遗传多样性指数中多态性比例为77.42%。观察等位基因数为1.7742,有效等位基因数为1.4004,Nei's基因多样性指数为0.2355,Shannon信息指数为0.3572;‘金冠’与‘四倍体嘎拉’苹果的三倍性杂种后代的ISSR遗传多样性指数中多态性比例为36.55%。观察等位基因数为1.9655,有效等位基因数为1.5322,Nei's基因多样性指数为0.3092,Shannon信息指数为0.4641。在这80份三倍性杂种后代中,子代植株的ISSR条带与母本条带相同占多数,且存在一定的条带增加和缺失;具有母本遗传倾向。
4.‘寒富’与‘四倍体嘎拉’苹果的三倍性杂种后代的总甲基化率(含全甲基化和半甲基化)在14.74%~20.00%之间;全甲基化率在5.41%-10.38%之间,半甲基化率在5.85%~14.59%之间;与亲本相比总甲基化率和全甲基化率均呈高于母本,低于父本的趋势,半甲基化率则均高于亲本。‘富士’与‘四倍体嘎拉’苹果的三倍性杂种后代总甲基化率在14.65%~27.96%之间;全甲基化率在8.25%~17.40%之间;半甲基化率在4.19%-10.82%之间;与母本相比,总甲基化率和半甲基化率大多呈增加趋势,全甲基化率呈下降趋势;与父本相比,均呈下降趋势。‘金冠’与‘四倍体嘎拉’苹果的三倍性杂种后代的总甲基化率在10.74%~28.86%之间,全甲基化率在7.87%~24.22%之间,半甲基化率在2.10%-11.84%之间。与母本相比,总甲基化和全甲基化呈上升趋势,半甲基化呈下降趋势;与父本相比,均呈下降趋势。
5.在这三个杂交组合中DNA甲基化共分5种类型:单态性(A)、去甲基化(B)、过或超甲基化(C)、次甲基化(D)和中间型(E)。在苹果三倍性杂种的基因组加倍和重组过程中,发生了大量过或超甲基化和去甲基化变异,少量发生了次甲基化现象,极少部分的甲基化状态介于双亲之间。通过MSAP分析,共获得10条差异片段,有8条能够在苹果基因组中检测到,且同源性较高,这8条片段分别位于第1条、9条、10条、12条和16条染色体的叠连群上,但是具体功能没有描述。
At present in the apple production of China, diploid varieties play a major part, but a few triploid varieties have good performance. The triploid apple breeding has become an important part in fruit polyploid breeding. But there was very little research on that had obtained polyploidy resources by hybridization, and also very few reports of hybrid mutation mechanism. Especially, the molecular mechanism of genetic variation of triploid apple obtained by crossing has rarely been reported. In this study, molecular mechanism of genetic variation and epigenetic variation in genome including the status and patterns of DNA methylation based on the investigation of the botanical characters of triploid hybrid progenies of 'Hanfu'×'4xGala','Fuji'×'4xGala','Golden Delicious'×'4xGala' were studied, and the results are as follows:
1. The coefficient of variation of leaves, branches and flowers of triploid hybrids of 'Hanfu'×'4xGala' were among26.42%~1.33%and that of pollen PE was4.46%. The averages of most of the characters were lower than that of the mid-parents. Coefficient of varation of fruit characters were among5.12%~27.82%, and the same trend with the botanical characters. Characteristic aromas of6hybrids were effect by both parent, and the genetic trend of fruit aromas was paternal inheritance. The major effect factor on the sweetness of6triploid hybrids was fructose, and that of sour flavor was malic acid.
2. Genetic variation of7triploid hybrids o f'Hanfu'×'4xGala' was investigated based on ISSR and AFLP. The percentage of polymorphic bands (PPB) of ISSR was60.00%, observed number of alleles (Na) was1.6000, effective number of alleles (Ne) was1.4204, Nei's gene diversity (H) was0.2368and Shannon's Information index (I) was0.3464. And these were lower than that of AFLP. The UPMGA cluster showed that most of7triploid hybrids were tend to the female parent'Hanfu'based on ISSR and AFLP. The analysis indicated that ISSR and AFLP both can use to analyze the genetic diversity of apple triploid hybrids, and these triploid hybrids were maternal inheritance.
3. Genetic variation of29triploid hybrids of 'Fuji'×'4xGala' was investigated based on ISSR. The percentage of polymorphic bands (PPB) was77.42%, observed number of alleles (Na) was1.7742, effective number of alleles (Ne) was1.4004, Nei's gene diversity (H) was0.2355and Shannon's Information index (I) was0.3574. Genetic variation of61triploid hybrids of 'Golden Delicious'×'4xGala' were investigated based on ISSR. The percentage of polymorphic bands (PPB) was36.55%, observed number of alleles (Na) was1.9655, effective number of alleles (Ne) was1.5322, Nei's gene diversity (H) was0.3092 and Shannon's Information index (I) was0.4641. In the80triploid hybrid progenies, the bands of hybids were most come from female parent, and there was a certain increase and deletion in the strips, and these triploid hybrids were maternal inheritance.
4. The total methylation ratios of7triploid hybrids of 'Hanfu'×'4xGala' were among14.74%to20.00%, the full methylated ratios were among5.41%to10.38%, and the hemi-methylated ratios of that were among5.85%to14.59%. Compared with 'Hanfu', the total methylation ratios, the full-methylatied ratios and hemi-methylated ratios were mostly tended to increase. But all of that were tended to decrease compared with'4xGala'.There was no obvious trend with that of their parents. The total methylation ratios of29triploid hybrids of 'Fuji'×'4xGala' were among14.65%to27.96%, the full methylated ratios were among8.25%to17.40%, and the hemi-methylated ratios of that were among4.19%to10.82%. Compared with 'Fuji', the total methylation ratios and hemi-methylatied ratios were mostly tended to increase, but full methylated ratios was tended decrease. But all of that were tended to decrease compared with '4xGala'. The total methylation ratios of61triploid hybrids of 'Golden Delicious'×'4xGala' were among10.74%to28.86%, the full methylated ratios were among7.84%to24.22%, and the hemi-methylated ratios of that were among2.10%to11.84%. Compared with 'Golden Delicious', the total methylation ratios and the full-methylatied ratios were mostly tended to increase, but hemi-methylated ratios was tended decrease. But all of that were tended to decrease compared with '4xGala'.
5. There were five patterns of DNA methylatiion in these three hybridized combinations including monomophism (A), demthylation (B), hyermethylation (C), hypomethylation (D) and intermediate methylation (E).During the process of genome recombination and triploidization in apple triploid hybrids, a great number of hypermethylations and demethlation were occurred, a small amout of occurrence methane phenomenon and a very small part of the methylation status were between parents. In total10differential fragments were obtained by MSAP.8can be detected in the apple genome, and the high homology fragments were located in the contig of8,9,10,12and16chromosomes, but the specific function is not described.
引文
1.边禹,汪卫星,韩国辉,梁国鲁.2010.不同倍性“早红3号”枇杷基因组AFLP分析.山西农业大学学报(自然科学版),30(3):209-211
2. 柴倩倩,王利军,吴本宏,范培格,段伟,李绍华.2011.中国李和樱桃李及其种间杂种果实香气成分分析.园艺学报,38(12):2357-2364
3. 党志国.2012.苹果(Malus domestica Borkh.)大群体杂种F1代若干性状变异特征及预先选择的研究.西北农林科技大学博士论文
4. 邓大君,邓国仁,吕有勇,周静,辛慧君.2001.变性高效液相色谱法检测CpG岛胞嘧啶甲基化.中华医学杂志,81(3):158-161
5. 邓秀新,Gimitter FG, Grosser JW.1995b.柑桔同源及异源四倍体花粉育性研究.园艺学报,22(1):16-20
6. 邓秀新,Grosser JW, Gmitter FC.1995a.柑桔种间体配融合及培养研究.遗传学报,22(4):316-321
7. 丁立华.2000.苹果梨杂种后代果实主要经济性状遗传规律初探.吉林农业科学,25(6):38-43
8. 段永红,王铭,孙毅,杨武德.2012.高粱甲基化连锁群A、B的构建及甲基化位点、甲基化模式的分析.中国农业科学,45(18):3699-3708
9. 方从兵.2002.我国果树孢粉学研究进展.安徽农业大学学报,29(2):154-157
10.郭广平,顾小平,袁金玲,吴晓丽.2011.不同生理年龄毛竹DNA甲基化的MSAP分析.遗传,33(7):794-800
11.郭文武,伊华林,邓秀新.1997.离体培养与柑桔三倍体育种.中国南方果树,26(6):11-13
12.郭文武,陈春丽,邓于洋,邓秀新.2007.柑橘种间六倍体体细胞杂种开花及其花粉育性.园艺学报,4(1):71-74
13.过国南,郭兆年,阎淑芝,张顺妮,潘兴.1994.苹果杂交后代经济性状的遗传变异分析.果树科学,11(2):71-75
14.韩柏明,赵恺,李贺,高秀岩,张志宏.2010.组织培养导致的草莓DNA甲基化变异.植物生理学通讯,40(8):797-802
15.韩继成,赵胜建,郭紫娟.2005.三倍体葡萄品种花粉形态及其坐果率的研究.果树学报,22(5):561-563
16.胡宝全,王春国,方成泉,宋文芹,林盛华.2011.不同倍性(2x、3x、4x)鸭梨基因组DNA甲基化水平与模式分析.南开大学学报(自然科学版),44(2):32-37
17.洪柳,邓秀新.2005.应用MSAP技术对脐橙品种进行DNA甲基化分析.中国农业科学,38(11):2301-2307
18.雷家军,谭昌华,代汉萍,庞兰.2010.草莓五倍体种间杂种的获得及回交研究.吉林农业大学学报,32(3):284-288
19.李宝江,景士西,丁玉英,张景娥.1994.苹果糖酸遗传和选择研究.遗传学报,21(2):147-154
20.李林光,何平,欧春青,李慧峰,张志宏.2007.金冠苹果成熟胚离体诱导四倍体的研究.园艺学报,34(5):1129-1134
21.李林光.2008.苹果多倍体种质创新及鉴定评价研究.沈阳农业大学博士论文
22.李锁平,张大乐,王秀娥,亓增军,刘大钧.2012.节节麦和黑麦杂种F1及双二倍体中基因组变异分析.作物学报,38(6):996-1002
23.李象松,魏丽华,李炫丽,汪艳杰,王冰,吴江生,龙鸿.2010.萝卜-芥蓝异源四倍体F4和F1o世代DNA甲基化变异的MSAP分析.华中农业大学学报,29(1):96-100
24.刘庆忠,苑克俊,张力思,赵红军,刘鹏,韩振海.2006.同源四倍体皇家嘎拉苹果的生长结果特性.果树学报,23(1):102-104
25.刘志,伊凯,王冬梅,杨巍,杨锋,张景娥.2004a.富士苹果果实外观品质性状的遗传.果树学报,21(6):505-511
26.刘志,伊凯,王冬梅,杨巍,杨锋,张景娥.2004b.富士苹果果实内在品质性状的遗传.果树学报,21(2):95-102
27.罗向东,戴亮芳,钱春桃,娄群峰,陈劲枫.2008.黄瓜与酸黄瓜异源多倍体3种同工酶表达基因组剂量效应.园艺学报,35(3):377-382
28.马跃,张蕾,李元源,东中东,张志宏.2012.‘寒富’苹果二倍体及其同源四倍体叶片超微结构和叶绿素荧光参数特征.西北植物学报,32(3):477-483
29.孟海军.2006.柑橘胚胎发生过程中DNA甲基化/去甲基化研究及SSR标记开发.华中农业大学博士论文
30.聂丽娟,王子成,王一帆,李国申,何艳霞.2009.二倍体和同源四倍体西瓜的DNA甲基化差异分析.核农学报,23(1):80-84
31.祈传磊,靳春莲,李开隆,李志新,赵慧.2010.不同倍性大青杨的光合特性及叶片解剖结构比较.植物生理学通讯,46(9):917-922
32.饶静云,刘义飞,黄宏文.2012.中华猕猴桃不同倍性间杂交后代倍性分离和遗传变异分析.园艺学报,39(8):1447-1456
33.沈德绪.2001.果树育种学,北京:中国农业出版社:157
34.沈红香,赵天田,宋婷婷,姚允聪,高俊平.2011.观赏海棠‘王族’自然杂交后代的遗传多样性分析.园艺学报,38(11):2157-2168
35.石庆华,刘平,刘孟军.2012.果树倍性育种研究进展.园艺学报,39(9):1639-1654
36.宋健坤,郭文武,伊华林,刘继红,陈春丽,邓秀新.2005.以异源四倍体体细胞杂种为父本与二倍体杂交创造柑橘三倍体的研究.园艺学报,32(4):594-598
37.宋健坤,朱世平,谭美莲,郭文武,邓秀新.2012.沙田柚(2x)×柑橘异源体细胞杂种NS(4x)的三倍体后代遗传分析.园艺学报,39(6):1021-1026
38.孙俊,房经贵,王飞,孙其宝,章镇,王昆.2010.苹果Ty1-copia类逆转座子LTR1o序列及其在 苹果属植物中的遗传多样性分析.南京农业大学学报,33(1):43-48
39.孙晓荣.2012.天然三倍体枇杷基因表达倍性效应的cDNA-AFLP研究.西南大学硕士论文
40.汪卫星,李春艳,向素琼,郭启高,李晓林,梁国鲁.2008c.番木瓜四倍体的诱导及形态学分析.果树学报,25(1):115-118
41.汪卫星,李晔,李晓林,向素琼,郭启高,何桥,梁国鲁.2011.天然三倍体枇杷与其二倍体植株的形态学比较.果树学报,38(6):1090-1092
42.汪卫星,向素琼,陈瑶,郭启高,李晓林,梁国鲁.2008a.‘红江橙’天然多倍体的45S rDNA荧光原位杂交分析.园艺学报,35(1):103-106
43.汪卫星.2008b.天然与人工合成三倍体枇杷基因组变异及DNA甲基化分析.西南大学博士论文
44.王春国,古瑜,陈成彬,焦定量,薛振毅,宋文芹.2009.不同倍性西瓜基因组DNA甲基化水平与模式的MSAP分析.分子细胞生物学报,42(2):118-126
45.王海波,李林光,陈学森,李慧峰,杨建明,刘嘉芬,王超.2010.中早熟苹果品种果实的风味物质和风味品质.中国农业科学,43(11):2300-2306
46.王力荣,朱更瑞,方伟超.2005.桃(Prunus persica L.)种质资源果实数量性状评价指标探讨.园艺学报,32(1):1-5
47.王茜龄,余茂德,鲁成,吴存容,敬成俊.2011.果叶兼用多倍体新桑品种的选育及其光合特性研究.中国农业科学,44(3):562-569
48.王文国,李锐,朱珈仪,王胜华,陈放.2010.水稻愈伤组织形成过程中甲基化对OsMAPK2的表达调控.遗传,32(12):1275-1280
49.位芳,张改生.2010.利用荧光原位杂交技术分析新合成异源四倍体拟南芥.作物学报,36(7):1216-1220
50.吴绍华,薛晶晶,张红宇,徐培洲,吴先军.2011.双胚苗水稻单倍体及其杂交后代基因组DNA甲基化特异位点的分析及功能探讨.中国水稻科学,25(3):249-255
51.向素琼,何建,何波,汪卫星,李晓林,郭启高,何桥,梁国鲁.2009.沙田柚多倍体遗传差异的SSR分析.果树学报,26(3):382-385
52.向素琼,梁国鲁,李晓林,汪卫星,郭启高,何桥,陈瑶.2008.沙田柚多倍体的获得与基因组原位杂交(GISH)分析.中国农业科学,41(6):1749-1754
53.谢玥,潘美玲,庄启国,董官勇,廖明安,王丽华,李明章.2013.猕猴桃红肉新种质'FXJK-CK-04137'与‘红阳’生物学特性及染色体倍性差异分析.果树学报,30(1):110-114
54.宣继萍,章镇.2002.适合于苹果的ISSR反应体系的建立.植物生理学通讯,38(6):549-550
55.颜菱,刘义飞,黄宏文.2012.猕猴桃倍性混合居群基因组遗传和表观遗传变异.植物学报,47(5):454-461
56.杨晓伶,程舟,李珊,北岛宣,长谷川耕二郎.2006.秋水仙素诱导文旦柚2n配子获得自交三倍体.园艺学报,33(5):1045-1047
57.仪治本,孙毅,牛天堂,梁小红,刘龙龙,赵威军,李炳林.2005.高粱基因组DNA胞嘧啶甲基化在杂交种和亲本间差异研究.作物学报,31(9):1138-1143
58.郁香荷,章秋平,刘威生,孙猛,刘宁,张玉萍,徐铭.2011.中国李种质资源形态性状和农艺性状的遗传多样性分析.植物遗传资源学报,12(3):402-407
59.张小猜,赵政阳,樊红科,党志国,张志敏.2009.苹果杂种F1代叶片性状分离及早期选择研究.西北农业学报,18(5):228-231
60.周广芳,王成强.1997.果树多倍体育种途径与成就.落叶果树,(增刊):50-52
61.朱玉贤,李毅,郑晓峰.1997.现代分子生物学.高等教育出版社
62. Adams KL, Cronn R, Percifield R, Wendel JF.2003. Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing. Proceeding of the National Academy of Sciences of USA,100:4649-4654
63. Adams KL, Percifield R, Wendel JF.2004. Organ-specific silencing of duplicated genes in a newly synthesized cotton allotetraploid. Genetics,168:2217-2226
64. Adams KL, Wendel J F.2005. Polypioidy and genome evolution in plants. Current Opinion in Plant Biology,8(2):135-141
65. Adams RLP.1995. Eukaryotic DNA methyltransferases:structure and function. Bioessays.17: 139-145
66. Akimoto K, Katakami H, Kim HJ, Ogawa E, Sano CM, Wada Y, Sano H.2007. Epigenetic inheritance in rice plants. Ann. Bot. (Lond) 100:205-217
67. Albertin W, Brabant P, Catrice O, Eber F, Jenczewski E, Chevre AM, Thiellement H.2005. Autopolyploidy in cabbage (Brassica oleracea L.) does not alter significantly the proteomes of green tissues. Proteomics,5:2131-2139
68. Baumel A, Ainouche M, Kalendar R, Schulman AH.2002. Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica C. E. Hubbard (Poaceae). Molecular Biology and Evolution,19:1218-1227
69. Baurens FC, Bonnot F, Bienvenu D, Causse S, Legavre T.2003. Using SD-AFLP to assess CCGG methylation in the banana genome. Plant Molecular Biology Reporter,21:339-348
70. Ben TH, Baraket G, Ben MS, Mohamed M, Mokhtar T, Amei SH.2009. Genetic relatedness among Tunisian plum cultivars by random amplified polymorphic DNA analysis and evaluation of phenotypic characters. Scientia Horticulturae,121(4):440-446
71. Blanc G, Wolfe KH.2004. Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution. Plant Cell,16:1679-1691
72. Brenet F, Moh M, Funk P, Feierstein E, Viale AJ, Socci ND, Scandura JM.2011. DNA methylation of the first exon is tightly linked to transcriptional silencing. Plosone.6(1):1-12
73. Causevic A, Gentil MV, Delaunay A, El-Soud WA, Garcia Z, Pannetier C, Brignolas F, Hage'ge D, Maury S.2006. Relationship between DNA methylation and histone acetylation levels, cell redox and cell differentiation states in sugarbeet lines. Planta,224(4):812-827
74. Chen L, Chen J.2007b. Allopolyploid-induced sequence elimination. Gene Genome Genomics,1: 113-116
75. Chen L, Chen J.2008. Changes of cytosine methylation induced by wide hybridization and allopolyploidy in Cucumis. Genome,51:789-799
76. Chen L, Lou Q, Zhuang Y, Chen J, Zhang X, Wolukau JN.2007a. Cytological diploidization and rapid genome changes of the newly synthesized allotetraploids Cucumis×hytivus. Planta,225: 603-614
77. Chen ZJ, Ni Z.2006. Mechanisms of genomic rearrangements and gene expression changes in plant polyploids. BioEassays,28(3):240-252
78. Chen ZJ.2007c. Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. Annual Review of Plant Biology,58:377-406
79. Chen ZJ.2010. Molecular mechanisms of polyploidy and hybrid vigor. Trends Plant Science,15(2): 57-71
80. Chu CC.2002. Contributions of Chinese Botanists to Plant Tissue Culture in the 20th Century. Acta Botanica Sinica,44(9):1075-1084
81. Cisneros A, Garcia RB, Tel-Zur N.2011. Ovule morphology, embryogenesis and seed development in three Hylocereus species (Cactaceae). Flora,206:1076-1084
82. Claudia K, Ortrun MS, Aleksandra E.2010. The impact of the triploid block on the origin and evolution of polyploidy plants. Trends in Genetics,26(3):142-148
83. Comai L, Tyagi AP, Winter K, Holmes-Davis R, Reynolds SH, Stevens Y, Byers B. 2000a.Phenotypic instability and rapid gene silencing in newly formed arabidopsis allotetraploids. Plant Cell,12(9):1551-1568
84. Comai L.2000b. Genetic and epigenetic interactions in allopolyploid plants. PMB,43:387-399
85. Coser SM, Ferreira MF, Ferreira A, Mitre LK, Carvalho CR, Clarindo WR.2012. Assessment of genetic diversity in Psidium guajava L. using different approaches. Scientia Horticulturae,148: 223-229
86. Curriel AJ, Ganeshanandam S, Noiton DA, Garrick D, Shelbourne CJA, Oraguzie N.2000. Quantitative evaluation of apple (Malusxdomestica Borkh.) fruit shape by principal component analysis of fourier descriptors. Euphytica,111:219-227
87. Dahl C, Guldberg P.2003. DNA methylation analysis techniques. Biogerontology,4:233-250
88. Dai X, Yang X, Huang Q, Qin G.2009. Observation on Double Fertilization and Early Embryonic Development in Autotetrap loid Polyembryonic Rice. Rice Science,16(2):124-130
89. Dajoz I, Bottraud IT, Gouyon PH.1993. Pollen aperture polymorphism and gametophyte performance in Viola diversifocia. Evolution,47(4):1080-1093
90. Davey MR, Anthony P, Power JB, Lowe KC.2005. Plant protoplast technology:Current status. Acta Physiologiae Plantarum,27 (1):117-129
91. Dewitte A, Eeckhaut T, Huylenbroeck JV, Bockstaele EV.2010. Induction of 2n pollen formation in Begonia by trifluralin and N2O treatments. Euphytica,171:283-293
92. Dillmann C, Bar-Hen A, Guerin D, Charcosset A, Murigneux A.1997. Comparison of RFLP and morphological distances between maize Zea mays L. inbred lines. Consequences for germplasm protection purposes. Theoretical and Applied Genetics,95:92-102
93. Dixon J, Hewett EW.2000. Factors affecting apple aroma/flavor volatile concentration:A review. New Zealand Journal of Crop and Horticultural Science,28:155-173
94. Dong YZ, Liu ZL, Shan XH, Qiu T, He MY, Liu B.2005. Allopolyploidy in wheat induces rapid and heritable alterations in DNA methylation patterns of cellular genes and mobile elements. Russion Journal of Genetics,41 (8):1089-1095
95. Echeverria G, Fuentes T, Graell J, Lara I, Lopez M L.2004. Aroma volatile compounds of'Fuji' apples in relation to harvest date and cold storage technology:A comparison of two seasons. Postharvest Biology and Technology,32:29-44
96. Echeverria G, Graell J, Lara I, Lopez M L.2008. Physicochemical measurements in'Mondial Gala@, apples stored at different atmospheres:Influence on consumer acceptability. Postharvest Biology and Technology,50:135-144
97. Echeverria SP, Valencia V AH, Kay AJ.2001. Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism (MSAP). Plant Science,161:359-367
98. Finnegan EJ, Kovac KA.2000. Plant DNA methyltransferase. Plant Molecular Biology,43: 189-201
99. Ge Y, Zhang F, Shen XL, Yu YM, Pan XY, Liu XJ, Liu JX, Pan GM, Tian DQ.2012. Genetic variations within a collection of anthuriums unraveled by morphological traits and AFLP markers. Biochemical Systematics and Ecology,45:34-40
100. Goubely C, Arnaud P, Tatout C, Heslop-Harrison JS, Deragon JM.1999. S1 SINE retroposons are methylated at symmetrical and non-symmetrical positions in Brassica napus:identifieation of a preferred target site for asymmetrical methylation. Plant Molecular Biology,39(2):24-55
101.Grigg G, Clark S.1994. Genes and genomes:Sequencing 5-methylcytosine residues in genomic DNA. Bioessays,16(6):431-436
102. Grosser JW, Gmitter FG.2011. Protoplast fusion for production of tetraploids and triploids: Applications for scion and rootstock breeding in Citrus. Plant Cell, Tissue and Organ Culture,104 (3):343-357
103.Guggisberg A, Mansion G, Kelso S, Conti E.2006. Evolution of biogeographic patterns, ploidy levels, and breeding systems in adiploid-polypioid species complex of Primula. New Phytologist,171 (3):617-632
104. Han FP, Fedak G, Ouellet T, Liu B.2003. Rapid genomic changes in interspecific and intergenetic hybrids and allopolyploids of Triticeae. Genome,46:716-723
105. He Q, Wang WX, Guo QG, Xiang SQ, Li XL, Liang GL.2012. Genetic diversity and utilization of triploid loquats (E.japonica Lindl). Genetic Diversity in Plants,14:103-116
106. He YC, Wei Q, Ge J, Jiang AM, Gan L, Song ZJ, Cai DT.2010. Genome duplication effects on pollen development and the interrelated physiological substances in tetraploid rice with polyploid meiosis stability. Planta,232:1219-1228
107. Herman JG, Graff JR., Myohanen S, Nelkin BD, Baylin SB.1996. Methylation-specific PCR:a novel PCR assay for methylation status of CpG islands. Proceedings of the National Academy of Sciences of USA,93(18):9821-9826
108. Hisato K, Kohji N, Kayo T, Haruki K.2002. Molecular and cytogenetic characterization of triploid somatic between 'Shogun' mandarin and grape fruit. Plant Biotechnology,19 (5):345-352
109. Hodkinson TR, Chase MW, Renvoize SA.2002. Characterization of a Genetic Resource Collection for Miscanthus (Sac charinae, Andropogoneae, Poaceae) using AFLP and ISSR PCR. Annals of Botany,89(5):627-636
110. Kakutani T, Munakata K, Richards EJ Hirochika H.1999. Meiotically and mitotically stable inheritance of DNA hypomethylation induced by ddml mutation of Arabidopsis thaliana. Genetics,151:831-838
111. Khan H, Sivalingam N, Chauhans S, Awasthi OP, More TA.2013. Improved crossing technique and identification of true F1 hybrids of Ziziphus mauritiana Lam. by molecular markers. Scientia Horticulturae,150(4):164-171
112. King B J, Lee L S, Scott P T.1996. Identification of triploid Citrus by sozyme analysis. Euphytica, 90:223-231
113. Kuo KC, McCune RA, Gehrke CW, Midgett. Rehrlich M.1980. Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucle-osides in DNA. Nucleic Acids Research,8(20):4763-4776
114. Lai J, Ma J, Swigonova Z, Ramakrishna W, Linton E, Llaca V, Tanyolac B, Park YJ, Jeong OY, Bennetzen JL, Messing J.2004. Gene loss and movement in the maize genome. Genome Research,14:1924-1931
115. Lee HS, Chen ZJ.2001. Protein-coding genes are epigenetically regulated in Arabidopsis polyploisd. Proceedings of the National Academy of Sciences USA,98(12):6753-6758
116. Li A, Hu BQ, Xue ZY, Chen L, Wang WX, Song WQ, Chen CB, Wang CG.2011. DNA methylatiion in genomes of several annual herbaceous and woody perennial plants of varying ploidy as detected by MSAP. Plant Molecular Biology Reporter,29:784-793
117. Li A, Song WQ, Chen CB, Zhou YN, Qi LW, Wang CG.2013. DNA methylation status is associated with the formation of heterosis in Larix kaempferi intraspecific hybrids. Molecular Breeding,31(2):463-475
118. Li XL, Guo WW, Wang B, Li XS, Chen HG, Wei LH, Yang YJ, Wu JS, Long H.2010. Instability of chromosome number and DNA methylation variation induced by hybridization and amphidiploids formation between Raphanus sativus L. and Brassica alboglabra Bailey. BMC Plant Biology,10:207
119. Li XQ, Xu ML, Korban SS.2002. DNA methylation profiles differ between field- and in vitro-grown leaves of apple. Journal of Plant Physiology,159:1229-1234
120. Lira-Medeiros CF, Parisod C, Fernandes RA, Mata CS, Cardoso MA, Ferreira PCG.2010. Epigenetic variation in Mangrove plants occurring in contrasting natural environment. PLoS One, e10326,5(4):1-8
121. Liu B, Brubaker C L, Mergeai G, et al.2001. Polyploid formation in cotton is not accompanied by rapid genomic changes. Genome,44:321-330
122. Liu B, Wendel JF.2003. Epigenetic phenomena and the evolution of plant allopolyploids. Molecular Phylogenetics and Evolution,29:365-379
123. Liu JX, Chen JG, Chen DL, Song ZJ, Dai BC, Cai DT.2009. Study on floral and pollen characters of tetraploid rice. Agricultural Sciences in China,8(10):1149-1158
124. Liu LQ, Gu ZJ.2011. Genomic in situ hybridization identifies genome donors of Camellia reticulate (Theaceae). Plant Science,180:554-559
125. Long L, Lin X, Zhai J, Kou H, Yang W, Liu B.2006. Heritable alteration in DNA methylation pattern occurred specifically at mobile elements in rice plants following hydrostatic pressurization. Biochemical and Biophysical Research Communications,340:369-376
126. Longley AE.1926. Triploid citrus. Wasington Acad Science,16:543-545
127. Lopes L, Olga S, Pereira JA, Baptista P.2012. Genetic diversity of Portuguese Arbutus unedo L. populations using leaf traits and molecular markers:An approach for conservation purposes. Scientia Horticulturae,142:57-67
128. Lopez M L, Villatoro C, Fu Fuentes T, Graell J, Lara I, Echeverria G.2007.Volatile compounds, quality parameters and consumer acceptance of 'Pink Lady(?)' apples stored in different conditions. Postharvest Biology and Technology,43:55-66
129. Lu BB, Pan XZ, Zhang L, Huang BB.2006. A genome-wide comparison of genes responsive to autopolyploidy in Isatis indigotica using Arabidopsis thaliana Affymetrix Genechips. Plant Molecular Biology Reporter,24:197-204
130. Lu YL, Rong TZ, Cao MJ.2008. Analysis of DNA methylation in different maize tissues. Journal of Genetic Genomics,35:41-48
131. Lukens LN, Pires JC, Leon E, Vogelzang R, Oslach L, Osborn T.2006. Patterns of sequence loss and cytosine methylation within a population of newly resynthesized Brassica napus allopolyploids. Plant Physiology,140(1):336-348
132. Maccaferi M, Stefanelli S, Rotondo F, Tuberosa R, Sanguineti MC.2007. Relationships among durum wheat accessions. Ⅰ. Comparative analysis of SSR, AFLP, and phenotypic data. Genome,50 (4):373-384
133.Madlung A, Masuelli RW, Watson B, Reynolds SH, Davision J, Comai L.2002. Remolding of DNA methylation and Phenotypic and transcriptional changes in synthetic Arabidopsis allotetraploids. Plant Physiology,29(2):733-746
134. Maghuly F, Schmoellerl B, Temsch EM, Laimer M.2010. Genome size, karyotyping and FISH physical mapping of 45s and 5s genes in two cherry rootstocks:Prunus subhirtella and Prunus incise×serrula. Journal of Biotechnology,149:88-94
135. Mehinagic E, Royer G, Symoneaux R, Jourjon F, Prost C.2006. Characterization of odor-active volatiles in apples:influence of cultivar and maturity stage. Journal of Agricultural and Food Chemistry,54:2678-2687
136. Messing J, Bharti AK, Karlowski WM, Gundlach H, Kim HR, Yu Y, Wei F, Fuks G, Soderlund CA, Mayer KFX, Wing R A.2004. Sequence composition and genome organization of maize. Proceedings of the National Academy of Sciences of the United States of America,101(40): 14349-14354
137. Meyer P.2011. DNA methylation systems and targets in plants. FEBS Letters,585:2008-2015
138. Moricova P, Ondrej V, Navratilova B, Luhova L.2013. Changes of DNA methylation and hydroxymethylation in plant protoplast cultures. Acta biochimica Polonica,60(1):33-36
139. Nakamura S, Hosaka K.2010. DNA methylation in diploid inbred lines of potatoes and its possible role in the regulation of heterosis. Theor Appl Genet,120(2):205-214
140. Nguyen TT, Shi J, Do DC.2011. Study on the meiosis of pollen mother cells and mechanism producing 2n pollens in Dien and Phuc Trach pummel. Science and technology journal of agriculture and rural development,4:23-27
141. Nybom T.1959. On the inheritance of acidity in cultivated apples. Heriditas,45:332-350
142. Osborn TC, Pires JC, Birchler JA, Auger DL, Chen ZJ, Lee H-S, Comai L, Madlung A, Doerge RW, Colot V, Martienssen RA.2003. Understanding mechanisms of novel gene expression in polyploids. Trends in Genet,19:141-147
143. Ou X, Long L, Zhang Y, Xue Y, Liu J, Lin X, Liu B.2009. Spaceflight induces both transient and heritable alterations in DNA methylation and gene expression in rice (Oryza sativa L.). Mutation Research,662:44-53
144. Ozkan H.2001. Genomic changes in newly synthesized amphiploids of Aegilops and Triticum. Ph.D. Thesis (Adana, Turkey:University of Cukurova)
145. Papa CM, Springer NM, Muszynski MG, Meeley R, Kaeppler SM.2001. Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation. Plant cell,13:1919-1928
146. Patzak J.2001. Comparison of RAPD, STS, ISSR and AFLP molecular methods used for assessment of genetic diversity in hop (Humulus lupulus L.). Euphytica,121:9-18
147. Paun O, Bateman RM, Fay MF, Hedren M, Civeyrel L, Chase MW.2010. Stable epigenetic effects impact adaptation in allopolyploid orchids (Dactylorhiza:Orchidaceae). Molecular Biology and Evolution,27(11):2465-2473
148.Pellicer J, Garcia S, Valles J, Kondo K, Garnatje T.2013. FISH mapping of 35S and 5S rRNA genes in Artemisia subgenus Dracunculus (Asteraceae):changes in number of loci during polyploid evolution and their systematic implications. Botanical Journal of the Linnean Society,171,655-666
149.Pontes O, Neves N, Silva M, Lewis MS, Madlung A, Comai L, Viegas W, Pikaard CS.2004. Chromosomal locus rearrangements are a rapid response to formation of the allotetraploid Arabidopsis suecica genome. Proceedings of the National Academy of Sciences of the United States of America,101(52):18240-18245
150. Portis E, Acquadro A, Comino C, Lanteri S.2004. Analysis of DNA methylation during germination of pepper(Capsicum annuum L.) seeds using methylation-sensitive amplification polymorphism (MSAP). Plant Science,166:169-178
151.Reyna-Lopes GE, Simpson J, Ruiz-Herrera J.1997. Differences in DNA methylation patterns are detectable during the dimorphic transition of fungi by amplification of restriction polymorphisms. Molecular and General Genetics.253(6):703-710
152. Rho IR, Hwang YJ, Lee HI, Lee CH, Lim KB.2012. Karyotype analysis using FISH (fluorescence in situ hybridization) in Fragaria. Scientia Horticulture,136:95-100
153. Rival A, Ilbert P, Labeyrie A, Torres E, Doulbeau S, Personne A, Dussert S, Beule T, Durand-Gasselin T, Tregear JW, Jaligot E.2013. Variations in genomic DNA methylation during the long-term in vitro proliferation of oil palm embryogenic suspension cultures. Plant Cell Reports, 32(3):359-368
154. Roth E, Berna A, Beullens K, Yarramraju S, Lammertyn J, Schenk A, Nicolai B.2007. Postharvest quality of integrated and organically produced apple fruit. Postharvest Biology and Technology,45:11-19
155. Salmon A, Ainouche ML, Wendel JF.2005. Genetic and epigenetic consequences of recent hybrid-dization and polyploidy in Spartina (Poaceae). Molecular Ecology,14(4):1163-1175
156. Salmon A, Clotault J, Jenczewski E, Chable V, Dauleux M J M.2008. Brassica oleracea displays a high level of DNA methylation polymorphism. Plant Science,174(1):61-70
157. Sarilar V, Palacios PM, Rousselet A, Ridel C, Falque M, Eber F, Chevre AM, Joets J, Brahant P, Alix K.2013. Allopolyploidy has a moderate impact on restructuring at three contrasting transposable element insertion sites in resynthesized Brassica napus allotetraploids. New Phytologist,198(2):593-604
158. Schaal BA, Leverich WJ, Bendich AJ.1991. Comparison of methods for assessing genetic variaiton in plant conservation biology. Falk DA, holsinger KE. Genetics and conservation of rare plants. New York:Oxford University Press,123-134
159. Sedov EN, Sedysheva GA, UI'yanovskaya EV, Serova ZM.2009. Donors of diploid gametes in apple breeding at the polyploidy level. Russian Agricultural Sciences,35(3):156-159
160. Shaked H, Kashkush L. Ozkan H, Feldman M, Levy AA.2001. Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome of wide hybridization and allopolyploid in wheat. The Plant Cell,13:1749-1759
161. Soltis, DE, Soltis PS, Pires JC, Kovarik A, Tate JA, Mavraodiev E.2004. Recent and recurrent polyploidy in Tragopogon (Asteraceae):cytogenetic, genomic and genetic comparisons. Biological Journal of the Linnean Society.82(4):485-501
162. Stupar RM, Bhaskar PB, Yandell BS, Rensink WA, Hart AL, Ouyang S, Veileux RE, Busse JS, Erhardt RJ, Buell CR, Jiang J.2007. Phenotypic and transcriptomic changes associated with potato autopolyploidization. Genetics,176:2055-2067
163. Sugawara K, Oowada A, Moriguchi T, Omura M.1995. Identification of Citrus Chimeras by RAPD markers. HortScience,30 (6):1276-1278
164. Swapna M, Sivaraju K, Sharma RK, Singh NK, Mohapatra T.2011. Single-strand conformational polymorphism of EST-SSRs:A potential tool for diversity analysis and varietal identification in Sugarcane. Plant Molecular Biology Reporter,29(3):505-513
165. Tantasawat P, Trogchuen, Prajongjai T, Seehalak W, Jittayasothorn Y.2010. Variety identification and comparative analysis of genetic diversity in yardlong bean (Vigna unguiculata spp. Sesquipedalis) using morphological characters, SSR and ISSR analysis. Scientia Horticulturae, 124(2):204-216
166. Tatum TC, Stepanovic S, Biradar DP, Rayburn AL, Korban SS.2005. Variation in nuclear DNA content in Malus species and cultivated apples. Genome,48:924-930
167. Town CD, Cheung F, Maiti R, Crabtree J, Haas BJ, Wortman JR, Hine EE, Althoff R, Arbogast TS, Tallon LJ, Vigouroux M, Trick M, Bancroft I.2006. Comparative genomics of Brassica oleracea and Arabidopsis thaliana reveal gene loss, fragmentation, and dispersal after polyploidy. Plant Cell, 18(6):1348-1359
168. Trap-Gentil MV, Hebrard C, Lafon-Placette C, Delaunay A, Hagege D, Joseph C, Brignolas F, Lefebvre M, Barnes S, Maury S.2011. Time course and amplitude of DNA methylation in the shoot apical meristem are critical points for bolting induction in sugar beet and bolting tolerance between genotypes. Journal of Experimental Botany,62(8):2585-2597
169. Troy EW, Naoki T, Michael SB, Mayrose I, Philip BG, Loren HR.2009. The frequency of polyploidy speciation in vascular plants. National Acad Science,106 (33):1387-13879
170. Tsaftaris AS, Kafka M.1997. Mechanisms of heterosis in crop plants. Journal of Crop Production, 1(1):95-111
171. Tsaftaris AS.1995. Molecular aspects of heterosis in plants. Physiologia Plantarum,94:362-370
172. Vandenhout H, Ortiz R.1995. Effect of ploidy on stomatal and other quantitative traits in plantain and banana hybrids. Euphytica,83:117-122
173.Vanyushin BF, Genger RK, Peacock WJ, Dennis ES.2006. DNA methylation in plants. Plant Physiology and Plant Molecular Biology,301(49):67-122
174. Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar S, Troggio M, Pruss D, et al.2010.The genome of the domesticated apple (Malus×domestica Borkh.).Nature Genetics,42:833-839
175. Vieira J, Santos R, Ferreira SM, Vieira CP.2008. Inferences on the number and frequency of S-pollen gene specificities in the polyploidy Prunus spinosa. Heredity,101:351-358
176. Visser TT.1978. Inheritance and selection of some fruit characters of apple. Euphytica,27:753-765
177. Vos P, Hogers R, Bleeker M, Reijans M, Lee T, Homes M, Friters A, Pot J, Paleman J, Kuiper M, Zabeau M.1995. AFLP:a new technique for DNA finger printing. Nucleic Acids Research,23 (21): 4407-4414
178. Walsh JB, Lynch.1996. Mapping and characterizing QTL. Fundament of quantitative Genetics, Chap 14. Sinauer Associates, Inc.6(2):221-225
179. Wang H, Feng QZ, Zhang MS, Yang CW, Sha W, Liu B.2010. Alteration of DNA methylation level and pattern in sorghum (Sorghum bicolor L.) pure-lines and inter-line F1 hybrids following low-dose laser irration. Journal of Photochemistry and Photobiology B:Biology,99:150-153
180. Wang J, Tian L, Madlung A, Lee HS, Chen M, Lee JJ, Watson B, Kagochi T, Comai L, Chen ZJ. 2004. Stochastic and epigenetic changes of gene expression in Arabidopsis polyploids. Genetics, 167:1961-1973
181. Wanger I, Capesius I.1981. Determination of 5-methylcytosine from plant DNA by HPLC. Biochim Biophy Acta,654(1):52-56
182. Wei J, Fan H, Li T, Wang W, Guo N, Li Z, Cai Y, Lin Y.2012. The effects of reciprocal cross on inheritance of DNA methylation in cotton (Gossypium hirsutum). African Journal of Biotechnology, 11(23):6383-6388
183. Wu CT, Morris JR.2001. Genes, genetics and epigenetics:A correspondence. Science,293 (5532): 1103-1105
184. Xiong LZ, Xu CG, Saghai Maroof MA, Zhang QF.1999. Patterns of cytosine methylation in an elite rice hybrid and its parental lines detected by a methylation-sensitive amplification polymorphism technique. Molecular and General Gentics,261:439-446
185. Xue ZH, Liu P, Liu MJ.2011. Cytological mechanism of 2n pollen formation in Chinese jujube (Ziziphus jujuba Mill. 'Linglingzao'). Euphytica,182:231-238
186. Yang CY, Huang YB, Tang ZX, Liu L.2011. Analysis of DNA methylation variation in sibling tobacco (Nicotiana tabacum) cultivars. African Journal of Biotechnology,10(6):874-881
187. Yang TJ, Kim JS, Kwon SJ, Lim KB, Choi BS, Kim JA, Jin M, Park JY, Lim MH, Kim HI, Lim YP, Kang JJ, Hong JH, Kim CB, Bhak J, Bancroft I, Park BS.2006b. Sequence-level analysis of the diploidization process in the triplicated FLOWERING LOCUS C region of Brassica rapa. Plant Cell, 18(6):1339-1347
188. Yang XM, Gao ZY, An LZ, Wang YM, Fang XW.2006a. In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine (Vitis vinifera L.). Euphytica, 152:217-224
189. Yang H, Guo X.2006c. Polyploidy induction by heat shock-induced meiosis and mitosis inhibition in the dwarf surfcalm, Mulinia lateralis Say. Aquaculture.252:171-182
190. Zhang C, Li J, Zhu Z, Zhang YD, Zhao L, Wang CL.2010a. Cluster analysis on Japonica rice (Oryza sativa L.) with good eating quality based on SSR markers and phenotypic traits. Rice Science,17(2):111-121
191. Zhang HY, Peng H, Li Y, Xu PZ, Wang XD, Wu XJ.2006. Patterns of DNA cytosine methyaltion between haploids and corresponding diploids in rice. Chinese Science Bulletin,51 (14):1721-1728
192. Zhang L, Li Z, Wang Y, Jiang Z, Wang S.2010b. Vitamin C, flower color and ploidy variation of hybrids from a ploidy-unbalanced Actinidia interspecific cross and SSR characterization. Euphytica, 175:133-143
193. Zhang X, Ge X, Shao Y, Sun G, Li Z.2013. Genomic change, retrotransposon mobilization and extensive cytosine methylation ateration in Brassica napus introgressions from two intertribal hybridizations. Plos one, e56346,8(2):1-10
194. Zhang XF, Liu XD, Qi B, Liu B.2009. Characterization of copia retrotransponsons in Zizania latifolia shows atypical cytosine methylation patterns and differential occurrence from other species of the grass family. Aquatic Botany,90:213-221
195. Zhang XY, Hu CG, Yao JL.2010c. Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance. Journal of Plant Physiology,167 (12): 88-94
196. Zhang YT, Wang GX, Dong J, Zhong CF, Kong J, Li TZ, Han ZH.2009. Analysis of volatile components in strawberry cultivars Xingdu 1 and Xingdu 2 and their parents. Agricultural sciences in China,8(4):441-446
197. Zhao X, Chai Y, Liu B.2007. Epigenetic inheritance and variation of DNA methylation level and pattern in maize intra-specific hybrids. Plant Science,72(5):930-938