摘要
石榴(Punica granatum L.)属石榴科(Punicaceae)石榴属(Punica L.)植物,又名安石榴、若榴、天浆等。原产中亚,是一种古老的温带落叶果树。全世界栽培的石榴都属于石榴属中的石榴(Punica granatum L)这个种,但由于不同栽培石榴品种对生态环境适应能力的差异性与选择性,加上驯化栽培和人为选择,形成了不同地理生态条件下的栽培群体。本文利用分子系统学的原理和方法,采用荧光AFLP标记技术,分析了山东主要栽培石榴品种之间的遗传多样性,为山东优良石榴品种选育提供分子依据。主要研究结果如下:
1、山东25个石榴品种,即‘大马牙甜’、‘青皮软籽’、‘三白甜’、‘大红皮酸’、‘重瓣玛瑙’、‘牡丹石榴’、‘白皮酸’、‘冰糖冻’和‘重瓣白石榴’、‘小红皮甜’、‘青皮谢花甜’、‘青皮岗榴’、‘墨石榴’、‘月季石榴’、‘红皮马牙甜’、‘枣庄短枝红’、‘巨籽蜜’、‘鲁峪酸’、‘小青皮甜’、‘大青皮甜’、‘大红皮甜’、‘大青皮酸’、‘泰山三白石榴’、‘泰山红’和‘大红袍’,的形态特征进行初步的统计。结果表明,山东石榴品种的果皮颜色和花瓣颜色差异较大,并且无显著相关性;果实风味以甜味为主(占78%),花瓣以单瓣花为主,同时有部分复瓣花;
2、利用荧光AFLP标记技术,从64对EcoRI/MseI引物中选出8对引物,25个石榴品种共扩增出1728条带,其中多态性位点数为722条,占总数的41.78%。不同的引物组合扩增的多态性位点数目和多态位点百分比明显不同,8对引物组合扩增的多态性位点数从70(E-AAG/M- CAT)到126(E-AAG/M- CTA)条不等,平均多态性位点数为90.25条;E-AAG/M-CTA引物组合的多态性百分比最高P=58.33%,E-AAG/M- CAT引物组合的多态性百分比最低P=32.41%,平均多态性百分比为41.78%。说明供试品种在DNA水平上酶切位点的分布存在广泛的变异。
3.本文利用POPGENE version 1.32软件对各位点的观测的等位基因数目(Na)、有效等位基因数目(Ne)、Nei基因多样性(H)和香农信息指数(I)进行了统计分析。观测的最高等位基因数目为1.5833(E-AAG/ M-CTA),最低为1.3241(E-AAG/ M-CAT),所有位点平均的等位基因数目为1.4178;有效等位基因数目最高为1.2618(E-AAG/ M-CTA),最低为1.1610(E-AAG/ M-CAA),平均有效等位基因数目为1.1874;各位点的Nei基因多样性为0.0727(E-AAG/ M-CAT)-0.1603(E-AAG/ M-CTA),平均为0.1133;香农信息指数为0.1166(E-AAG/ M-CAT)-0.2486(E-AAG/ M-CTA),平均为0.1752,方差分析显示H和I在多数位点具有显著性差异,少数位点具有极显著差异。说明山东石榴遗传多样性较为丰富。
4.基于Lei& Li相似系数利用UPGMA法进行聚类分析,在相似系数0.786处将25个石榴品种划分为四大类,进一步用AFLP技术证明山东石榴品种遗传多样性丰富。
本研究根据AFLP结果探讨了山东石榴的亲缘关系,并对进一步的品种选育进行了展望。
Punica granatum L., alternate name‘An Shiliu’,‘Ruo Liu’,‘Tian Jiang’which belongs to Punicaceae, Punica L., occurred originally Central Asia. It is a deciduous fruit tree in temperate zone. Punica granatum L.of all over the world belongs to this species. Owing to the differences and selectivities to the adaptability of different ecological environments and domesticating cultivation and artificial selection, cultivation groups of different geological environments were coming into being. The genetic diversities of pomegranate cultivars in Shandong were analyzed using molecular systematical theories and methods by AFLP. This paper provided the basis for the selection and breeding of Shandong pomegranate cultivars.The main results were as follows:
1. The morphological characteristics of pomegranate was analyzed from 25 cultivars including‘Damayatian’,‘Qingpiruanzi’,‘Sanbaitian’,‘Dahongpisuan’,‘Chongbanmanao’,‘Mudanshiliu’,‘Baipisuan’,‘Bingtandong’,‘Chongbanbaishiliu’,‘Xiaohongpitian’,‘Qingpixiehuatian’,‘Qingpigangliu’,‘Moshiliu’,‘Yuejishiliu’,‘Hongpimayatian’,‘Zaozhuangduanzhihong’,‘Juzimi’,‘Luyusuan’,‘Xiaoqingpitian’,‘Daqingpitian’,‘Dahongpitian’,‘Daqingpisuan’,‘Taishansanbaishiliu’,‘Taishanhong’,‘Dahongpao’. The results showed that the differences between peel colour and petal colour were great and there were no significant correlations. The main flavour of fruits was sweetness. The simple flower hold the most portion of the flowers and then double-petalled blossoms.
2. The AFLP maker technique was carried out with eight pairs primers from 64 pairs. The total 1728 bands were amplified in 25 pomegranate and the polymorphic loci were 722 which had a proportion of 41.78%. The number and the percentage of polymorphic loci amplified by differert primers combination were obviously differernt. The polymorphic loci amplified by 8 pairs primers ranged from 70(E-AAG/M- CAT)to 126(E-AAG/M- CTA), and the main polymorphic loci were 90.25. The percentage of polymorphic loci amplified by E-AAG/M- CTA combination were the highest (P=58.33%) while E-AAG/M- CAT the lowest (P=32.41%). The main percentage of polymorphic loci was 41.789%. The results indicated that distributions of the restriction sites at the DNA level of these cultivars had wide variations.
3. The Na, Ne, H and I of observed locis were statistical analyzed using POPGENE version 1.32 software. The highest number of observed alleles were 1.5833(E-AAG/ M-CTA)and the lowest were 1.3241(E-AAG/ M-CAT). The average number of alleles were 1.4178. The highest effective number of alleles were 1.2618(E-AAG/ M-CTA)and the lowest were 1.1610(E-AAG/ M-CAA). The average effective number of alleles were 1.1874. The gene diversities of all allels were 0.0727(E-AAG/ M-CAT)-0.1603(E-AAG/ M-CTA)while the main was 0.1133. The I was 0.1166(E-AAG/ M-CAT)-0.2486(E-AAG/ M-CTA)while the main was 0.1752. Variance analysis showed that H and I had differences at the most locis while significant differences at few locis. The results showed that pomegranate of Shandong had rich genetic diversities.
4. The cluster analysis of the Lei & Li was carried out by UPGMA. 25 cultivars were divided into four groups at 0.786 level of similarity coefficient. The genetic diversities of Shandong pomegranate were further proved by AFLP.
The genetic relationships of pomegranate in Shandong were discussed according to the AFLP results. The prospects of selection and breeding of Punica granatum L. cultivars were also put forward in this paper.
引文
1陈德均.我国石榴的种质资源及地理分布[J].河南科技,1989(10)
2陈瑞阳.中国农学会遗传资源学会.1994.中国作物遗传资源.北京:中国农业出版社,1982
3陈新露,赵祥云等. 1995.应用RAPD技术评价丁香品种间遗传关系.园艺学报,2:171~175
4陈延惠,张四普,胡青霞等.不同方法对石榴叶片DNA提取效果的影响[J] .河南农业大学学报,2005,39(2):182~186
5程亚东、侯乐峰.石榴高产技术[M].山东科学技术出版社.济南:1999,1
6戴思兰,陈俊愉,李文斌.菊花起源的RAPD分析.植物学报,1998.11:1053~1059
7丁之恩,徐迎碧,刘韶,周先锋,陈红梅. 6个石榴品种的光合和蒸腾特性.经济林研究, 2008,26(1):15~19
8冯玉增,梅亭,康宇静等.中国石榴的生产科研现状及产业开发建议[J].落叶果树, 2006(1):11~15
9冯玉增,宋梅亭,赵艳丽等.河南省石榴优良品种资源[J].中国种业, 2001(2): 28~29
10冯玉增,宋梅亭.我国石榴生产现状与发展建议[J].林业科技开发,2003,14(5):7~9
11冯玉增、宋梅亭、宋长治.河南省石榴种质资源的研究.中国果树[J]1998,15(4):370～375
12冯玉增.河南省的石榴品种资源评价与利用[J].果树科学,1998(4)
13冯夏莲,何承忠,张志毅等.植物遗传多样性研究方法概述[J].南林学院学报, 2006( 26): 69~79.
14符政德,邓开清,唐志澄.芷江石榴品种资源及糖石榴繁育技术研究[J].湖南林业科技, 1998,25(3): 35~38
15巩雪梅,张明水,宋丰顺等.中国石榴品种资源经济性状研究[J].植物遗传资源学报, 2004, 5(1):17~21
16巩雪梅.石榴品种资源遗传变异分子标记研究[D].合肥:安徽农业大学.2004.
17古丽米热,董海丽,居来提.新疆石榴产业现状与未来发展[J].西北园艺, 2003(6): 7~8
18桂枝,袁庆华,蔺海明. RAPD标记技术及其在牧草研究中的应用.草业科学,2001,12:50~54
19侯乐峰,耿道鹏,苏成.峄城石榴种质资源研究[J].落叶果树,2006(5): 19~21
20何天明,陈学森,吴燕.从蔷薇科果树硅胶干燥叶片中制备DNA.石河子大学学报,2004,22 (4):316~319
21贾继增.分子标记种质资源鉴定与分子标记育种[J].中国农业科学,1996,29(4):1~4
22蒋世高,王继龙,杨应龙.会泽盐水石榴[J].果树科学, 1995, 12(2):126~128
23李秀娟,徐鹏,刘金勇.枣庄市石榴资源与产业化开发[J].中国林福特产, 2005(1): 54~55
24李宗圈.豫石榴一号、二号、三号新品种介绍[J].林业科技通讯, 1996(1): 38
25李淑娴,陈志银,沈永宝等.松属树种种苗鉴定中AFLP指纹技术的研究.南京林业大学学报(自然科学版), Vol.27,No.3 May.2003:1~6.
26梁玉英.突尼斯软籽石榴引种栽培试验初报[J].山西果树, 2001(3): 8~9
27陆丽娟,巩雪梅,朱立武.中国石榴品种资源种子硬度性状研究[J].安徽农业大学学报, 2006, 33(3):356~359.
28卢龙斗,刘素霞,邓传良等. RAPD技术在石榴品种分类上的应用.果树学报2007, 24(5): 634～639.
29刘素霞,洪达,高武军等.石榴RAPD反应体系优化及亲缘关系研究.安徽农业科学,2007(35)36:11775~11777
30马克平.试论生物多样性的概念[J].生物多样性, 1993, 1( 1): 20-22.
31马艳青,刘志敏,邹学校.辣椒种质资源的RAPD分析.湖南农业大学学报,2003,29:120~123
32庞朝友,杜雄明,马峄英.棉属种间杂交基因渐渗系SSR标记及其表型性状的聚类分析.作物学报, 2006. 32(9):1371~1378.
33钱迎倩,马克平.生物多样性研究的原理与方法[M].北京,1994. 13~36.
34邱英雄,傅承新,孔航辉.杨梅不同品种的ISSR分析.农业生物技术学报,2002,10(4):343~346
35沈浩,刘登义.遗传多样性概述[J].生物学杂志, 2001, 18( 3): 5~9.
36沈进,朱立武,张水明,巩雪梅,陆丽娟.中国石榴核心种质的构建.中国农学通报, 2008, 24(5): 265~271
37宋红竹.杨属AFLP遗传多样性研究和杨树品种分子指纹图谱构建[D]中国林业科学研究院, 2006.
38孙广宇,张荣,彭友良. 2003.随机扩增多态性DNA的重复性和可靠性问题.植物保护,4:44~46
39孙玉刚,张承安,史传铎等.优质石榴新品种‘泰山大红’[J].园艺学报, 2004, 31(2): 278
40谭兴荣.四川会理石榴产业化开发的构想.林业科技开发,2001(15): 24~25
41 WEA,中国科学院生物多样性委员会译.全球生物多样性策略[M].北京:中国标准出版社,1992.
42陶文静,刘大钧. AFLP技术及其在植物基因组研究中的应用.世界农业,1998.236:16~19
43田家祥.特大籽石榴新品种-巨籽蜜[J].烟台果树, 2003(4): 56
44汪小飞,向其柏,尤传楷等.石榴品种分类研究[J].南京林业大学学报. 2006, 30(4): 81~84
45汪小全,邹喻苹,张大明等. RAPD应用于遗传多样性和系统学研究汇总的问题[J].植物学报,1996.38(12):954~962
46王斌,翁曼丽. AFLP的原理及其应用.杂交水稻.1996.5:27~30
47王伏雄.中国植物花粉形态[M].北京:科学出版社,1995:328
48王关林,方宏筠.植物基因工程(第二版).北京:科学出版社,742~744
49宣继萍,章镇.适合于苹果的ISSR反应体系的建立.植物生理学通讯,2002,38(4):549~550
50徐凯,钟家煌,杨军.安徽省石榴优良品种资源[J].作物品种资源, 1997(3): 48~50
51徐迎碧,丁之恩,姚玉敏等. 4个石榴品种的染色体核型分析.经济林研究2008,26(1):47~52
52续九如,赵秉伦,王生民.临潼石榴遗传资源研究[J].经济林研究, 1993, 11(1):13~17.
53夏铭.遗传多样性研究进展[J].生态学杂志, 1999, 18( 3): 92~96.
54杨官品,Saghaimaroof M,张启发等.水稻-多拷贝微卫星多态性遗传分析1998. 2:27~30
55杨荣萍,龙雯虹,张宏等.云南25份石榴资源的RAPD分析[J].果树学报.2007, 24(2):226~229
56易干军,霍合强,陈大成等.荔枝品种亲缘关系的AFLP分析[J].园艺学报,2003,30(4):399~403 .
57苑兆和,尹燕雷,李自峰等.石榴果实香气物质的研究[J].林业科学,2008. 44(1):65~69
58苑兆和,尹燕雷,曲健禄等.中国石榴栽培群体遗传多样性的荧光AFLP分析[J].遗传学报, 2007, 34(12): 1061~1071
59苑兆和,尹燕雷,朱丽琴等.山东石榴品种遗传多样性的荧光AFLP分析[J].园艺学报, 2008. 35(1):107~122
60苑兆和,朱丽琴,尹燕雷等.山东石榴果实鞣质含量的差异[J].山东林业科技, 2007.6:7-9
61苑兆和,尹燕雷,朱丽琴等.石榴保健功能的研究进展[J].山东林业科学,2008.1:91-93
62张虹,郭亚力,姚立华等.蒙自地区石榴种植现状及发展建议.广西热带农业,2003(2): 46~48
63张军.临选系列软籽石榴[J].现代种业, 2003(5): 42
64张军.陕西临潼石榴优良品种[J].中国果树, 2004(3): 29~31
65张明水,朱立武,青平乐等.安徽石榴品种资源经济性状模糊综合评判[J].安徽农业大学学报, 2002, 29(3):297~300
66张润光.石榴贮期生理变化及保险技术研究.陕西师范大学硕士学位论文, 2006
67张秀英,葛春民,贾继增.山羊草属五个基本基因组RAPD分析.植物学报,1999.4:393~397
68赵国荣,朱立武,张明水等.软籽石榴新品种"红玛瑙籽"[J].园艺学报,2007,34(1): 260
69赵淑清,武维华. DNA分子标记和基因定位.生物技术通报,2000.6:1~3
70赵先贵,肖玲.中国石榴科花粉形态的研究[J].西北植物学报,1996, 16(1):52~55
71周光洁,袁永勇,曾凡哲,先开泽,陈正国.中国石榴生产的现状及发展前景.西南农业学报, 1995.8(1): 111~116.
72周兴文,杨秋生,李永华.牡丹基因组AFLP银染反应体系的建立和优化[J].河南农业大学学报, 2006, (06):602~607
73周翔陆.石榴丰产栽培图说[M].北京:中国林业出版社, 1995.1~2
74朱诚,刘之燕. RAPD技术与桂花品种分类和鉴定.广西植物,1999.2:190~192
75朱立武,张水明,巩雪梅等.软籽石榴新品种'红玉石籽'[J].园艺学报, 2005, 32(5): 965
76朱红霞.牡丹AFLP荧光反应体系的建立和优化.北方园艺2007(10):164~167
77朱立武,贾兵,张水明等.优质大果石榴新品种-白玉石籽[J].中国南方果树. 2004,33(5):69~70
78朱振东,贾继增.小麦SSR标记的发展及应用.遗传,2003,25:355~360
79邹喻苹,葛颂,王晓东.系统与进化植物学中的分子标记.北京:科学出版社.2001.
80邹云昌.云南蒲缥10个优良石榴品种[J].中国果树, 2005(3):26~27
81 Anesini C, Perez C. Screening of plants used in Argentine folk medicine for antimicrobial activity. J Ethnopharmacol , 1993. 39:119~128
82 Artik N, Cemeroglu B, Murakami H, Mori T. Determination of phenolic compounds in pomegranate juice by HPLC. Fruit Process, 1998.8: 492~499.
83 Becker J, Henu M. Mapping of digested and undigested random amplified microsatellite polymorphisms in barley. Genome, 1995. 39:991~998
84 Beckmann J, Soller M, Restriction fragment length polymorphisms in genetic improvement: method ologies mapping and costs. Theor Appl Genet, 1983. 67:34~43
85 Ben Nasr N, Ayed M. Quantitative determination of the polyphenolic content of pomegranate peel. Z Lebensm Unters Forsch, 1996.203: 374~378.
86 Botstein D, White R, Skolnick M et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J HumGenet, 1980. 32:314~331
87 Diffenbach C, et al. PCR技术实验指南.北京:科学出版社. 1998.
88 Ephrain Philip Lansky, Gregory Harrison, Paul Froom, Wen G. Jiang. Pomegranate (Punica granatum) pure chemicals show possible synergistic inhibition of human PC-3 prostate cancer cell invasion MatrigelTM. Inverstigational New Drugs, 2005. 23:121~122.
89 Mavlyanov SM, Islambekov SY, Karimdzhanov AK, Ismailov AI. Polyphenols of pomegranate peels show marked antitumor and anti-viral action. Khim Prir Soedin, 1997.33: 124~126.
90 Messaoud Mars, Mohamed Marrakchi. Diversity of pomegrante (Punica granatum L.) germplasm in Tunisia. Genetic Resources and Crop Evolution, 1999.46:461~467.
91 Nam Deuk Kim, Rajendra Mehta, Weiping Yu et al.. Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer[J]. Breast Cancer Research and Treatment, 2002, 71 :203~217
92 Paran I, Michelmore RW. Development of reliable PCR -based marker linked to downy mildew resistance genes in lettuce. Theor Appl Genet, 1993.85:985~993
93 Ponce-Macotela M, Navarro-Alegria I, Martinez-Gordillo MN, Alvarez-Chacon R. In vitro effect against Giardia of 14 plant extracts.Rev Invest Clin, 1994. 46: 343~347.
94 Roder M, Korzum V, Wendehake K. et al. Amicrosatellite map of wheat. Genetics, 1998.149:2007~2023
95 Vierling R. Nguyen. Use of RAPD marker to determine the genetic diversity of dipoid, wheat genotypes. Theor Appl Genet, 1993. 7-8:835~838
96 Wang D G, Fan J B, Siao C J, et al. Large-scale identification, mapping, and genotying of single-nucleotide polymorphisms in the human genome. Science, 1998.280:1077~1082
97 Welsh J, Honeycutt R, Mcdelland M. Parentage determination in maize hybrids using the arbitrarily primed polymerase chain reaction. Theor Appl Genet, 1991.4:573~576
98 Zabeau M, Vos P .S. 1993 . Elective retriction fragment amplification: a general method for DNA fingerprinting. European Patent Application Number:92402629. 7.Publication Number:0534858
99 Zhang J, Zhan B, Yao X, Gao Y, Shong J. Antiviral activity of tannin from the pericarp of Punica granatum L against Herpes virus in vitro. Chung Kuo Chung Yao Tsa Chih 1995. 20:556~558.
