利用ISSR标记及rDNA-ITS序列分析河北省野生铁线莲的亲缘关系

doi:10.16420/j.issn.0513-353x.2020-0953

摘要/Abstract

摘要：

通过ISSR和rDNA-ITS 两种分子标记方法对河北省17个类群的野生铁线莲种间亲缘关系进行了鉴定。利用UPGMA法对ISSR扩增的条带结果进行聚类分析,并用PCR法扩增ITS,通过Clustal X,MEGA等软件比较和分析ITS的序列特征,最后对两种分子标记的结果综合分析。结果表明,利用筛选出的ISSR引物扩增出多态性条带104条,多态性百分比为100%,等位基因数（N_a）为2.00,有效等位基因数（N_e）为1.3069,Nei’s基因多样性（H）为0.2196,Shannon’s多样性信息指数（I）为0.3675。聚类结果显示,在遗传距离为0.66处,17种野生铁线莲被分为5类;铁线莲的17个铁线莲样本ITS序列长度为543 ~ 561 bp,含变异位点87个,信息位点37个,特异性鉴别位点50个。其中,长瓣铁线莲与半钟铁线莲、太行铁线莲与狭裂太行铁线莲遗传距离为0,槭叶铁线莲与其他种的遗传距离较大,亲缘关系较远。

关键词: 铁线莲, ISSR, ITS, 种质资源, 亲缘关系

Abstract:

The ISSR and rDNA-ITS sequence analysis were used to identify the genetic relationship of 17 wild species of Clematis L. in Hebei Province. The ISSR amplified result was conducted cluster analysis by UPGMA method. After PCR amplification,the ITS sequence characteristics were compared and analyzed by Clustal X,MEGA and other software. Finally,the results of these two molecular markers method were comprehensively analyzed. The results showed that primer combinations can amplify 104 polymorphism bands with the percentage of polymorphic bands（PPB）of 100.0%. The average observed number of alleles（N_a）was 2.00;the number of effective alleles（N_e）was 1.3069;the Nei’s gene diversity（H）was 0.2196;the Shannon’s information index（I）was 0.3675. According to the clustering results,17 species were divided into five groups at a genetic distance of 0.66. Moreover,the length of ITS sequences varied from 543 to 561 bp,including 87 variable sites,37 information sites and 50 singleton specific discrimination sites in the 17 Clematis L. species. The genetic distance between C. macropetala and C. ochotensis,C. kirilowii and C. kirilowii var. chaneti was 0,and the genetic distance between C. acerfolia and others was large and the genetic relationship was far.

Key words: Clematis, ISSR, ITS, germplasm resource, genetic relationship

中图分类号:

S682
S687.3

王鑫, 李明阳, 田琳, 刘冬云. 利用ISSR标记及rDNA-ITS序列分析河北省野生铁线莲的亲缘关系[J]. 园艺学报, 2021, 48(9): 1755-1767.

WANG Xin, LI Mingyang, TIAN Lin, LIU Dongyun. ISSR and rDNA-ITS Sequence Analysis of the Genetic Relationship of Clematis in Hebei Province[J]. Acta Horticulturae Sinica, 2021, 48(9): 1755-1767.

图/表 10

参考文献 21

[1]	Jiang Ming, Zhou Ying-qiao, Li Rong-rong. 2011. Sequence analysis of ITS of 8 medicinal plants of Clematis. Chinese Herbal Medicine, 42(9):1802-1806. (in Chinese)
	蒋明, 周英巧, 李嵘嵘. 2011. 铁线莲属8种药用植物ITS序列分析. 中草药, 42(9):1802-1806.
[2]	Li Xiao, Wang Pei, Zhao Long, Ju Ai-hua. 2012. rDNA-ITS sequence analysis of Clematis mongolicum. Journal of Inner Mongolia Medical College, 34(4):325-330. (in Chinese)
	李骁, 王佩, 赵龙, 鞠爱华. 2012. 铁线莲属蒙药材的rDNA-ITS序列分析. 内蒙古医学院学报, 34(4):325-330.
[3]	Liu Jing-jing, Gao Yi-ke. 2013. Investigation on germplasm resources of clematis in Beijing area. Heilongjiang Agricultural Science,(4):65-69. (in Chinese)
	刘晶晶, 高亦珂. 2013. 北京地区野生铁线莲属植物种质资源调查研究. 黑龙江农业科学,(4):65-69.
[4]	Liu Qing-chao, Wang Kui-ling, Lu Wan-pei, Liu Qing-hua. 2014. Reasearch progress of Clematis in China. Journal of Plant Genetic Resource, 15(3):483-490,497. (in Chinese)
	刘庆超, 王奎玲, 卢婉佩, 刘庆华. 2014. 我国铁线莲属植物资源研究进展. 植物遗传资源学报, 15(3):483-490,497.
[5]	Mu Lin, Xie Lei. 2011. Systematic location of Clematis acerfolia—analysis of ITS and chloroplast DNA sequences. Journal of Beijing Forestry University, 33(5):49-55. (in Chinese)
	穆琳, 谢磊. 2011. 槭叶铁线莲的系统位置初探——来自ITS和叶绿体DNA序列片段的分析. 北京林业大学学报, 33(5):49-55.
[6]	Qian Ren-juan, Zheng Jian, Hu Qing-di, Ma Xiao-hua, Zhang Xu-Le. 2017. Research progress on ornamental germplasm resources of Clematis in China. Chinese Agricultural Science Bulletin, 33(21):75-81. (in Chinese)
	钱仁卷, 郑坚, 胡青荻, 马晓华, 张旭乐. 2017. 中国铁线莲属观赏种质资源研究进展. 中国农学通报, 33(21):75-81.
[7]	Salem S A, Muhammad A K, Hussein M M, Ehab H E, Muhammad A, Muhammad F. 2019. Biochemical and molecular characterization of cowpea landraces using seed storage protein and SRAP marker patterns. Saudi Journal of Biological Sciences, 26(1):74-82. doi: 10.1016/j.sjbs.2018.09.004 URL
[8]	Sheng Lu. 2015. Karyotype analysis and molecular systematics of Clematis[Ph. D. Dissertation]. Nanjing:Nanjing Forestry University.
[9]	盛璐. 2015. 铁线莲属植物的核型分析及分子系统学研究[博士论文]. 南京:南京林业大学.
[10]	Shi Ling-yan, Liu Bao-wei, Gu Xin-ying. 2019. Application of biochemical and ISSR molecular markers in identification of Auricularia auricular. Northern Horticulture, 43(9):136-141. (in Chinese)
	史灵燕, 刘保卫, 顾新颖. 2019. 生化和ISSR分子标记在黑木耳品种鉴定中的应用, 北方园艺, 43(9):136-141.
[11]	Sun Zheng-hai, Wang Jin, Li Shi-feng, Lin Kai-wen, Hu Yi-chen, Wang Shi-jin. 2012. Establishment and optimization of ISSR-PCR reaction system for Rattan. Journal of Yunnan Agricultural University(Natural Science), 27(5):746-750. (in Chinese)
	孙正海, 王锦, 李世峰, 林开文, 胡祎晨, 王仕锦. 2012. 滑叶藤ISSR-PCR反应体系建立及优化. 云南农业大学学报(自然科学), 27(5):746-750.
[12]	Wang Jiang-yong, Qiao Qian, Zhang Jie, Hu Feng-rong. 2020. Research progress of Clematis and application in garden. Journal of Shandong Agricultural University(Natural Science), 51(2):217-221. (in Chinese)
	王江勇, 乔谦, 张杰, 胡凤荣. 2020. 铁线莲属植物研究进展与园林应用. 山东农业大学学报(自然科学版), 51(2):217-221.
[13]	Wang Nan, Wang Jin, Li Zong-yan, Yang Kun-mei, He Hui, Xiao Zhen-dong. 2016. ISSR-PCR reaction system optimization and primer screening of Clematis horticulture varieties. Northern Horticulture,(1):80-83. (in Chinese)
	王楠, 王锦, 李宗艳, 杨坤梅, 何辉, 肖振东. 2016. 铁线莲园艺品种ISSR-PCR反应体系优化与引物筛选. 北方园艺,(1):80-83.
[14]	Wang W T, Xie L. 2007. A revision of Clematis sect. Tubulosae(Ranunculaceae). Acta Phytotaxonomica Suiica, 45:425-457.
[15]	Wang Wen-cai, Li Liang-qian. 2005. A new classification system of Clematis. Acta Botanica Taxa,(5):431-488. (in Chinese)
	王文采, 李良千. 2005. 铁线莲属一新分类系统. 植物分类学报,(5):431-488.
[16]	Xie L, Shi J H, Li L Q. 2005. Identity of Clematis tatarinowii and C. pinnata var. ternatifolia(Ranunculaceae). Annales Botanici Fennici, 42:305-308.
[17]	Xie L, Wen J, Li L Q. 2011. Phylogenetic analyses of Clematis(Ranunculaceae)based on sequences of nuclear ribosomal ITS and three plastid regions. Systematic Botany, 36:907-921. doi: 10.1600/036364411X604921 URL
[18]	Yang Jiu-yan, Yang Jie, Yang Ming-bo, Ju Aihua, Qu bi. 2006. ISSR analysis of medicinal plants of the genus Caragana in Ordos Plateau. Chinese Herbal Medicine, 37(10):1562-1566. (in Chinese)
	杨九艳, 杨劼, 杨明博, 鞠爱华, 渠弼. 2006. 鄂尔多斯高原锦鸡儿属药用植物的ISSR分析. 中草药, 37(10):1562-1566.
[19]	Zhao Huan, Wu Wei, Zheng You-liang, Pan Hong-mei, Zhai Juan-yuan. 2009. Application of ribosomal DNA ITS sequence analysis in the study of medicinal plants. Shi Zhen Chinese Medicine, 20(4):959-962. (in Chinese)
	赵欢, 吴卫, 郑有良, 潘红梅, 翟娟园. 2009. 核糖体DNA ITS序列分析在药用植物研究中的应用. 时珍国医国药, 20(4):959-962.
[20]	Zhu Hong, Zhu Shu-xia, Li Yong-fu, Yi Xian-gui, Duan Yi-fan, Wang Xian-rong. 2018. Leaf phenotypic variation in natural populations of Cerasus dielsiana. Chinese Journal of Plant Ecology, 42(12):1168-1178. (in Chinese) doi: 10.17521/cjpe.2018.0196 URL
	朱弘, 朱淑霞, 李涌福, 伊贤贵, 段一凡, 王贤荣. 2018. 尾叶樱桃天然种群叶表型性状变异研究. 植物生态学报, 42(12):1168-1178.
[21]	Zhu Yuan-di, Cao Min-ge, Xu Zheng, Wang Kun, Zhang Wen. 2014. Phylogenetic relationship between Xinjiang wild apple(Malus sieversii Roem.) and Chinese apple(Malus × domestica subsp. chinesnsis)based on ITS and matK sequences. Acta Horticulturae Sinica, 41(2):227-239. (in Chinese)
	朱元娣, 曹敏格, 许正, 王昆, 张文. 2014. 基于ITS和matK序列探讨新疆野苹果与中国苹果的系统演化关系. 园艺学报, 41(2):227-239.

编号 No.	种 Species	植株类型 Plant type
编号 No.	种 Species	生活型 Life form	叶型 Leaf form	花萼 Calyx	花色 Sepals color	花型 Flower form	花期（月份） Blooming period	果期（月份） Fruiting period	采集地 Collection place
1	黄花铁线莲 Clematis intricata	木质藤本 Woody vines	一至二回羽状复叶 One to two pinnately compound leaves	4	黄色 Yellow	钟状 Bell shape	6 ~ 7	8 ~ 9	张家口市蔚县 Yu County,Zhangjiakou City
2	芹叶铁线莲 C. aethusifolia	草质藤本 Herbaceous vines	一至二回羽状复叶 One to two pinnately compound leaves	4	淡黄色 Canary yellow	钟状 Bell shape	7 ~ 8	9	邯郸市武安市 Wu’an City,Handan City
3	褐毛铁线莲 C. fusca	草质藤本 Herbaceous vines	一回羽状复叶 One pinnately compound leaves	4	紫色 Purple	钟状 Bell shape	6 ~ 7	8 ~ 9	秦皇岛市青龙县 Qinglong County, Qinhuangdao City
4	钝萼铁线莲 C. peterae	木质藤本 Woody vines	一回羽状复叶 One pinnately compound leaves	4 ~ 5	白色 White	轮状 Wheel shape	5 ~ 6	7 ~ 8	邯郸市磁县 Ci County,Handan City
5	大叶铁线莲 C. heracleifolia	灌木 Shrub	三出复叶 Ternately compound leaves	4	蓝紫色 Bluish violet	筒状 Tubular	7 ~ 8	9 ~ 10	保定市易县 Yi County,Baoding City
6	毛果扬子铁线莲 C. puberula	草质藤本 Herbaceous vines	一回羽状或二回三出复叶One pinnately or two ternately compound leaves	4	白色 White	十字型 Cross	7 ~ 9	9 ~ 10	邯郸市磁县 Ci County,Handan City
7	狭裂太行铁线莲 C. kirilowii var. chaneti	木质藤本 Woody vines	一回羽状复叶 One pinnately compound leaves	4或 5 ~ 6	白色 White	轮状 Wheel shape	6 ~ 8	8 ~ 9	保定市满城区 Mancheng District, Baoding City
8	太行铁线莲 C. kirilowii	木质藤本 Woody vines	一回羽状复叶 One pinnately compound leaves	4或 5 ~ 6	白色 White	轮状 Wheel shape	6 ~ 8	8 ~ 9	保定市易县 Yi County,Baoding City
9	短尾铁线莲 C. brevicaudata	木质藤本 Woody vines	二回三出复叶 Bipinnately and ternately compound leaves	4	白色 White	轮状 Wheel shape	7 ~ 8	9 ~ 10	保定市阜平县 Fuping County,Baoding City
10	卷萼铁线莲 C. tubulosa	灌木 Shrub	三出复叶 Ternately compound leaves	4	蓝紫色 Bluish violet	筒状 Tubular	7 ~ 8	10	承德市兴隆县 Xinglong County, Chengde City
11	粗齿铁线莲 C. argentilucida	木质藤本 Woody vines	一回羽状复叶 One pinnately compound leaves	4	白色 White	轮状 Wheel shape	5 ~ 7	7 ~ 10	石家庄市赞皇县 Zanhuang County, Shijiazhuang City
12	灌木铁线莲 C. fruticosa	灌木 Shrub	一回羽状复叶 One pinnately compound leaves	4	黄色 Yellow	钟状 Bell shape	7 ~ 8	10	张家口市涿鹿县 Zhuolu County, Zhangjiakou City
13	棉团铁线莲 C. hexapetala	木质藤本 Woody vines	单叶 Simple leaf	4 ~ 8	白色 White	轮状 Wheel shape	6 ~ 7	8 ~ 9	承德市兴隆县 Xinglong County, Chengde City
14	羽叶铁线莲 C. pinnata	木质藤本 Woody vines	一回羽状复叶 One pinnately compound leaves	4	白色 White	筒状 Tubular	9 ~ 10	11 ~ 12	秦皇岛市青龙县 Qinglong County, Qinhuangdao City
15	长瓣铁线莲 C. macropetala	木质藤本 Woody vines	二回三出复叶 Bipinnately and ternately compound leaves	4	蓝色或淡紫色 Blue or lavender	钟状 Bell shape	7	8	保定市易县 Yi County,Baoding City
16	槭叶铁线莲 C. acerfolia	灌木 Shrub	单叶 Simple leaf	5 ~ 8	白色或粉红色 White or pink	高脚碟状 Salver- form	3月中旬 ~ 4月中旬	5 ~ 6	石家庄市赞皇县 Zanhuang County, Shijiazhuang City
17	半钟铁线莲 C. ochotensis	木质藤本 Woody vines	二回三出复叶 Bipinnately and ternately compound leaves	4	淡蓝色 Light blue	钟状 Bell shape	5 ~ 6	7 ~ 8	保定市易县 Yi County,Baoding City

编号 No.	种 Species	植株类型 Plant type
编号 No.	种 Species	生活型 Life form	叶型 Leaf form	花萼 Calyx	花色 Sepals color	花型 Flower form	花期（月份） Blooming period	果期（月份） Fruiting period	采集地 Collection place
1	黄花铁线莲 Clematis intricata	木质藤本 Woody vines	一至二回羽状复叶 One to two pinnately compound leaves	4	黄色 Yellow	钟状 Bell shape	6 ~ 7	8 ~ 9	张家口市蔚县 Yu County,Zhangjiakou City
2	芹叶铁线莲 C. aethusifolia	草质藤本 Herbaceous vines	一至二回羽状复叶 One to two pinnately compound leaves	4	淡黄色 Canary yellow	钟状 Bell shape	7 ~ 8	9	邯郸市武安市 Wu’an City,Handan City
3	褐毛铁线莲 C. fusca	草质藤本 Herbaceous vines	一回羽状复叶 One pinnately compound leaves	4	紫色 Purple	钟状 Bell shape	6 ~ 7	8 ~ 9	秦皇岛市青龙县 Qinglong County, Qinhuangdao City
4	钝萼铁线莲 C. peterae	木质藤本 Woody vines	一回羽状复叶 One pinnately compound leaves	4 ~ 5	白色 White	轮状 Wheel shape	5 ~ 6	7 ~ 8	邯郸市磁县 Ci County,Handan City
5	大叶铁线莲 C. heracleifolia	灌木 Shrub	三出复叶 Ternately compound leaves	4	蓝紫色 Bluish violet	筒状 Tubular	7 ~ 8	9 ~ 10	保定市易县 Yi County,Baoding City
6	毛果扬子铁线莲 C. puberula	草质藤本 Herbaceous vines	一回羽状或二回三出复叶One pinnately or two ternately compound leaves	4	白色 White	十字型 Cross	7 ~ 9	9 ~ 10	邯郸市磁县 Ci County,Handan City
7	狭裂太行铁线莲 C. kirilowii var. chaneti	木质藤本 Woody vines	一回羽状复叶 One pinnately compound leaves	4或 5 ~ 6	白色 White	轮状 Wheel shape	6 ~ 8	8 ~ 9	保定市满城区 Mancheng District, Baoding City
8	太行铁线莲 C. kirilowii	木质藤本 Woody vines	一回羽状复叶 One pinnately compound leaves	4或 5 ~ 6	白色 White	轮状 Wheel shape	6 ~ 8	8 ~ 9	保定市易县 Yi County,Baoding City
9	短尾铁线莲 C. brevicaudata	木质藤本 Woody vines	二回三出复叶 Bipinnately and ternately compound leaves	4	白色 White	轮状 Wheel shape	7 ~ 8	9 ~ 10	保定市阜平县 Fuping County,Baoding City
10	卷萼铁线莲 C. tubulosa	灌木 Shrub	三出复叶 Ternately compound leaves	4	蓝紫色 Bluish violet	筒状 Tubular	7 ~ 8	10	承德市兴隆县 Xinglong County, Chengde City
11	粗齿铁线莲 C. argentilucida	木质藤本 Woody vines	一回羽状复叶 One pinnately compound leaves	4	白色 White	轮状 Wheel shape	5 ~ 7	7 ~ 10	石家庄市赞皇县 Zanhuang County, Shijiazhuang City
12	灌木铁线莲 C. fruticosa	灌木 Shrub	一回羽状复叶 One pinnately compound leaves	4	黄色 Yellow	钟状 Bell shape	7 ~ 8	10	张家口市涿鹿县 Zhuolu County, Zhangjiakou City
13	棉团铁线莲 C. hexapetala	木质藤本 Woody vines	单叶 Simple leaf	4 ~ 8	白色 White	轮状 Wheel shape	6 ~ 7	8 ~ 9	承德市兴隆县 Xinglong County, Chengde City
14	羽叶铁线莲 C. pinnata	木质藤本 Woody vines	一回羽状复叶 One pinnately compound leaves	4	白色 White	筒状 Tubular	9 ~ 10	11 ~ 12	秦皇岛市青龙县 Qinglong County, Qinhuangdao City
15	长瓣铁线莲 C. macropetala	木质藤本 Woody vines	二回三出复叶 Bipinnately and ternately compound leaves	4	蓝色或淡紫色 Blue or lavender	钟状 Bell shape	7	8	保定市易县 Yi County,Baoding City
16	槭叶铁线莲 C. acerfolia	灌木 Shrub	单叶 Simple leaf	5 ~ 8	白色或粉红色 White or pink	高脚碟状 Salver- form	3月中旬 ~ 4月中旬	5 ~ 6	石家庄市赞皇县 Zanhuang County, Shijiazhuang City
17	半钟铁线莲 C. ochotensis	木质藤本 Woody vines	二回三出复叶 Bipinnately and ternately compound leaves	4	淡蓝色 Light blue	钟状 Bell shape	5 ~ 6	7 ~ 8	保定市易县 Yi County,Baoding City

引物名称 Primer name	引物序列 Primer sequence	退火温度/℃ Annealing temperature	位点数 Number of loci	多态性位点数 Number of polymorphic loci	多态性位点百分率/% Percentage of polymorphic loci
U815	CTCTCTCTCTCTCTCTG	54.0	11	11	100
U824	TCTCTCTCTCTCTCTCG	55.4	12	12	100
U834	AGAGAGAGAGAGAGAGYT	57.5	7	7	100
U835	AGAGAGAGAGAGAGAGYC	50.4	9	9	100
U836	AGAGAGAGAGAGAGAGYA	55.4	6	6	100
U840	GAGAGAGAGAGAGAGAYT	55.4	7	7	100
U841	GAGAGAGAGAGAGAGAYC	55.4	10	10	100
U843	CTCTCTCTCTCTCTCTRA	54.0	8	8	100
U844	CTCTCTCTCTCTCTCTRC	55.4	6	6	100
U845	CTCTCTCTCTCTCTCTRG	55.4	10	10	100
U866	CTC CTC CTC CTC CTC CTC	55.4	7	7	100
U899	CATGGTGTTGGTCATTGTTCCA	51.8	11	11	100

引物名称 Primer name	引物序列 Primer sequence	退火温度/℃ Annealing temperature	位点数 Number of loci	多态性位点数 Number of polymorphic loci	多态性位点百分率/% Percentage of polymorphic loci
U815	CTCTCTCTCTCTCTCTG	54.0	11	11	100
U824	TCTCTCTCTCTCTCTCG	55.4	12	12	100
U834	AGAGAGAGAGAGAGAGYT	57.5	7	7	100
U835	AGAGAGAGAGAGAGAGYC	50.4	9	9	100
U836	AGAGAGAGAGAGAGAGYA	55.4	6	6	100
U840	GAGAGAGAGAGAGAGAYT	55.4	7	7	100
U841	GAGAGAGAGAGAGAGAYC	55.4	10	10	100
U843	CTCTCTCTCTCTCTCTRA	54.0	8	8	100
U844	CTCTCTCTCTCTCTCTRC	55.4	6	6	100
U845	CTCTCTCTCTCTCTCTRG	55.4	10	10	100
U866	CTC CTC CTC CTC CTC CTC	55.4	7	7	100
U899	CATGGTGTTGGTCATTGTTCCA	51.8	11	11	100

编号 No.	样本 Sample	ITS		ITS1		ITS2
编号 No.	样本 Sample	长度/bp Size	G + C/%	长度/bp Size	G + C/%	长度/bp Size	G + C/%
1	黄花铁线莲 C. intricata	550	60.00	164	59.15	227	65.60
2	芹叶铁线莲 C. aethusifolia	549	60.88	171	60.36	219	67.10
3	褐毛铁线莲 C. fusca	561	61.50	180	61.67	223	66.80
4	钝萼铁线莲 C. peterae	550	61.27	172	60.47	219	67.60
5	大叶铁线莲 C. heracleifolia	550	61.64	172	61.05	219	68.00
6	毛果扬子铁线莲 C. puberula	543	60.89	164	58.90	220	67.70
7	狭裂太行铁线莲 C. kirilowii var. chaneti	544	61.76	165	59.39	220	70.00
8	太行铁线莲 C. kirilowii	545	61.76	165	59.76	221	69.70
9	短尾铁线莲 C. brevicaudata	550	61.38	172	60.47	219	67.90
10	卷萼铁线莲 C. tubulosa	550	61.57	172	61.05	219	67.90
11	粗齿铁线莲 C. argentilucida	550	61.68	172	60.59	219	68.50
12	灌木铁线莲 C. fruticosa	543	60.04	165	56.97	219	67.10
13	棉团铁线莲 C. hexapetala	549	61.38	169	59.17	221	69.20
14	羽叶铁线莲 C. pinnata	550	61.79	172	61.05	219	68.50
15	长瓣铁线莲 C. macropetala	548	60.95	171	59.65	218	67.40
16	槭叶铁线莲 C. acerfolia	559	62.79	180	63.89	220	68.60
17	半钟铁线莲 C. ochotensis	557	61.40	179	60.89	219	67.60