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遗传多样性评估使用SSR引物的四季豆

博士M Gopinath1Anushree V Katti2K.S.Dayananda1,伊斯梅尔Shareef100,Drishya V.Nair3
  1. 教授,生物技术学系Acharya理工学院,班加罗尔,印度卡纳塔克邦
  2. P。G的学生,生物技术学系Acharya理工学院,班加罗尔,印度卡纳塔克邦
  3. 学生,B。技术在生物技术、Vellore理工学院,Vellore Tamilnadu、印度
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国际创新研究期刊》的研究在科学、工程和技术

文摘

四季豆是其中一个重要的豆科蔬菜生长的嫩豆荚新鲜消费或干种子为“Rajma”。在当前研究中SSR分子标记被用来评估十个品种的遗传多样性四季豆菜豆种质从NBPGR获得登记入册,新德里。各种形态特征和定量特征都被记录下来。在十登记入册,6到达说拥有与所有5个SSR引物的基因。五个SSR引物,两个引物Bmd9和Bmd16乐队在所有十菜豆种质。聚类分析表明,十个基因型被分组在两个集群。SSR分析的目的是促进建设基于SSR遗传连锁图四季豆。

关键字
聚类分析、四季豆、遗传多样性、SSR引物

介绍
四季豆(菜豆L)也被称为„RajmaA¢€Ÿ或扁豆、芸豆或菜豆。四季豆是其中最重要的豆科植物生长的嫩豆荚新鲜消费或加工罐头,冷冻或冷冻干燥的产品。它有抗糖尿病属性有利于膀胱烧伤和心脏问题的自然治愈。雨季作物,它不需要灌溉,甚至当雨量分布在作物周期。然而,拉比作物需要灌溉。灌溉播种后25天是至关重要的。在最佳条件下,2.0 - -2.5吨/公顷的谷物和3.0 - -3.5吨/公顷产草量。植物形态特征的传统方法是一种常见的步骤在植物育种虽然它有一些缺点,因为它是描述性的,容易出错,而且受环境或生理因素的影响。因此,一些分子工具正在采用植物品种鉴定和识别,因为这些技术是可靠的,明确的,且易于采用[19]。分子标记包括生化constituentsA¢€Ÿ即,次生代谢产物在植物和大分子即,蛋白质和脱氧核糖核酸(DNA)。然而,这些植物次生代谢物的分析仅限于产生一个合适的范围的代谢物,可以很容易分析和区分品种。 These metabolites, which are being used as markers, should be neutral to environmental effects or management practices. Hence, amongst the molecular markers used, DNA markers are more suitable and ubiquitous to most of the living organisms [32].DNA markers that are tightly linked to agronomically important traits (called gene „tagging‟) may be used as molecular tools for marker-assisted selection (MAS) in plant breeding [28]. MAS involves using the presence/absence of a marker as a substitute for or to assist in phenotypic selection, in a way which may make it more efficient, effective, reliable and cost- effective compared to the more conventional plant breeding methodology. The use of DNA markers in plant (and animal) breeding has opened a new realm in agriculture called „molecular breeding‟ [27].Simple sequence repeats (SSRs) or microsatellites were first described by Hamada et al. (1982) as short tandemly repeated DNA sequences (2–5 bp in length) widely spread throughout the nuclear genome of eukaryotes [33].Microsatellites are short stretches of DNA which consists of single, di, tri or tetra nucleotide repeats. The ubiquitous nature of such sequence throughout the eukaryotic genome was first highlighted in a number of studies in organisms ranging from yeast to human beings. Microsatellites also represent a rich source of allelic diversity which has been exploited in many types of genetic analysis.SSR markers are locus specific and highly polymorphic. These markers are co-dominant and allow the discrimination of homozygotes and heterozygotes. Fortunately microsatellite primers obtained from one species are very often usable in closely related species of the same genus and sometimes even through genera of the family.Silva et al. (2003) identified RAPD and SSR markers associated with a resistant allele for common bean angular leaf spot (Phaeois ariopsisgriseola) from the line 'ESAL 550', derived from the Andean 'Jalo EEP 558' cultivar, to assist selection of resistant genotypes. One RAPD and one SSR marker were found to be linked in coupling phase to the resistant allele. The SSR marker was amplified by the primer PV-atct001282C, and its distance from the resistant allele was 7.6 cM. This is the most useful marker for indirect selection of resistant plant in segregating populations. A common bean genomic library enriched for microsatellite motifs (ATA), (CA), (CAC) and (GA) was constructed. After screening, 60% of the clones selected from the library enriched for the (ATA) repeat contained microsatellites versus 21% of the clones from the library enriched for (GA) (CA) and (CAC) repeats. Fifteen primer pairs have been developed allowing for the amplification of SSR loci. We have evaluated the genetic diversity of these loci between 45 different bean lines belonging to nine various quality types. A total of 81 alleles was detected at the 15 microsatellite loci with an average of 5.3 alleles per locus. We have investigated the origin of allelic size polymorphism at the locus PvATA20 in which the number of repeats ranges from 24 to 85. We have related these large differences in repeat number to unequal crossing-over between repeated DNA regions. The diversity analysis revealed contrasted levels of variability according to the bean type. The lower level was evidenced for the very fine French bean, showing the effect of breeders intensive selection. 9volune 4 , June 2002).

材料和方法
十五菜豆种质生长在田野和叶样本收集植物基因组DNA的提取。
答:DNA提取
积极收集叶子不受病虫害从每个加入用水冲洗和表面用70%的酒精消毒。树叶被重约0.5克,用于CTAB法提取的DNA。
植物基因组DNA隔离协议:
1)CTAB提取缓冲(100毫米TrisHCl, EDTA 20毫米,1.4毫米生理盐水、2%)(8毫升)和0.8 g的PVP和预热200μl 0.5%β-巯基乙醇。
2)叶组织(2 g)地面与液氮细粉用杵和臼。
3)内容被转移到一个离心管和管再次孵化1小时在600 c水浴。
4)管子都摇动了间歇性每10分钟,冷却到室温,6毫升氯仿:异戊醇(24:1)的混合物被倒相管和混合添加轻轻地直到它形成乳剂。
5)管离心机在13000 rpm 10分钟,水相转移到一个新的离心管,使用切再次提示和氯仿:异戊醇一步是重复
6)明确水相转移到新鲜的离心管和2毫升1.4 m氯化钠和8毫升添加了冰冷的异丙醇,轻轻混合,在-200摄氏度冷藏隔夜。
7)上层的丢弃,颗粒与70%乙醇洗
9)然后DNA丸被离开管暴露在室温下干燥2-3hours直到酒精蒸发。
10)的颗粒在200年resuspendedμl TE缓冲。
孤立的样本存储在-20摄氏度。DNA浓度是由nanotrope和质量验证了agrose凝胶电泳(0.8%)

b DNA扩增
六个SSR引物被用于PCR扩增。的引物列表如下表中所给
图像
所有PCR组件被添加到PCR的管子和插入到井PCR机器。PCR反应混合物准备在10倍20μL卷包含2μL Taq缓冲区,1.5μL核苷酸(2毫米),2μL (F + R) SSR引物(5 pmol /μL), 0.3μL Taq DNA聚合酶(3 U /μL)和2μL提取的DNA (50 ng)。混合物是由20μLμL灭菌蒸馏水的12.2。机器上运行4个小时。删除后PCR产品和凝胶电泳。PCR反应是启动和优化使用热循环程序重复热剖面。PCR程序的设置基于三个步骤。第一步,在95°C是一个初始变性一步3分钟。第二步,运行30周期,每个开始变性为1分钟95°C,其次是退火52 57°C 1分钟和结束在72°C扩展为1分钟,第三步,是最后一个扩展周期为10分钟在72°C。PCR产品是与加载混合染料(0.5%溴酚蓝,0.5%二甲苯苯胺和30%的蔗糖,w / v)在装货前在微型离心机短暂和旋转。PCR产品和100个基点DNA梯用2%琼脂糖凝胶电泳在80伏其次是与溴化乙锭染色分离碎片,可视化的紫外线(UV)透照器。
图像

结果和讨论
根据PCR协议,10个质量好的四季豆到达选择和筛选5 SSR引物。
图像
图像
图像
图像
使用SSR引物PCR产品文章的结果显示,底漆Bmd9和Bmd16节目存在的乐队四季豆登记入册(1 - 10,1:EC540797, 2: IC319825, 3: ARKANOOP, 4: IC319827, 5: EC541908, 6: IC342273, 7: EC500641, 8: EC530923, 9: EC500226, 10: EC115962,(板板2和3),底漆Bmd27显示放大在2,4,5,6,7,8,9 (2:IC319825, 4: IC319827, 5: EC541908, 6: IC342273, 7: EC500641, 8: EC530923, 9: EC500226), Frenchbean登记入册(plate4)。底漆Bmd54显示放大in1-9 (1: EC540797, 2: IC319825, 3: ARKANOOP, 4: IC319827, 5: EC541908, 6: IC342273, 7: EC500641, 8: EC530923, 9: EC500226)四季豆登记入册(plate5)。底漆Bmd154显示放大2 - 9 (2:IC319825, 3: ARKAANOOP, 4: IC319827, 5: EC541908, 6: IC342273, 7: EC500641, 8: EC530923, 9: EC500226)加入四季豆(plate6)。因此,2,4,5,6,7,8 (2:IC319825, 4: IC319827, 5: E541908, 6: IC342273, 7: EC500641, 8: EC530923)四季豆DNA登记入册有特定性状的基因都与上面提到的5个引物。

结论
从目前的研究结果表明,10个基因型属于两大集群。cluster - 1上由variable10 (EC115962),变量9 (EC500226)和变量8 (EC530923),而cluster - 2上的客户包括变量4 (IC319827),变量2 (IC319825)和变量1 (EC540797)。剩下的被安置在之间。在集群1中,变量1 (EC540797)与变量8 (EC530923) 1.7的距离而变9 (EC500226)没有与变量10 (EC115962)和变量8 (EC530923)但这是放置在之间。在集群2中,变量4 (IC319827)与变量2 (IC319825)的距离1。这个链接再次与变量1 (EC540797)的距离1.7。这两个集群是3.8的距离再次联系。但变量3 (ARKAANOOP),变量5 (EC541908),变量6 (IC342273),变量7 (EC500641)没有联系集群1或集群2或链接到集群,它们在1和2之间的集群。这意味着,集群1基因不同的变量(i, e他们有不同的形态特征,如形状、大小、花的颜色,种子颜色,等. .)从变量在cluster - 2上的客户,因此他们可以用于育种工作。

引用
  1. Adesoye我。*和Ojobo。o . A .遗传多样性评估的菜豆l .长白猪NigeriaA¢A€Ÿ年代mid-altitude农业生态的区域国际生物多样性和J ournal Consevation第1辑(13)页。453 - 460年,2012年10月DOI: 10.5897 / IJBC11.216
  2. MavromatisA。G 1, ArvanitoyannisI。2,。Korkovelos1A。E, Giakountis3A Chatzitheodorou1V。答:和。Goulas1C。K *,遗传多样性在菜豆(菜豆l .)希腊长白猪和商业品种:营养成分、RAPD和形态学标记
  3. 一步V V。,Paredes, M. C., Rojo, C. M., Diaz, L. M. and Blair, M. W., Microsatellite marker characterization of Chilean common bean(Phaseolus vulgaris L.) germplasm.Crop Sci., 50:1932–1941,2010.
  4. 毕比,S。,Rengifo, J., Gaitan, E., Duque, M. C. and Tohme, J., Diversity and origin of Andean landraces of common bean.Crop Sci.41: 854-862,2001.
  5. Biswas硕士,Hassan, J. and Hossain, M.M., Assessment of genetic diversity in French bean (Phaseolus vulgaris L) based on RAPD marker. African J. Biotech., 9(23): 5073-5077,2010.
  6. 布莱尔,m . W。,Giraldo, M. C.,Buendia, H. F., Tovar, E., Duque, M. C., Beebe, S. E., , Microsatellite marker diversity in common bean(Phaseolus vulgaris L.). Theor. Appl. Genet., 113: 100–109,2006.
  7. Boutin, s R。,Young, N. D., Olson, T. C., Yu, Z. H., Vallejos, C. E. and Shoemaker, R. C., Genome conservation among three legume genera detected with DNAmarkers. Genome,38(5):928-937.
  8. Burle, m . L。,Fonesca, J. R., Kami, J. A. and Gepts, P., Microsatellite diversity and genetic structure among common bean (Phaseolus vulgaris L.) landraces in Brazil, a secondary center of diversity. Theor. Appl. Genet.,121: 801–813,2010.
  9. Debouck, D.G.,Systematics and morphology. In “common bean: research for crop improvement. Commonwealth Agricultura Bureaux International Wallingford, United Kingdom, 55-118,1991.
  10. 迪亚兹,l . m .和布莱尔·m·W。,Race structure within theMesoamerican gene pool of common bean (Phaseolus vulgarisL.) as determined by microsatellite markers. Theor. AppI. Genet.,114: 143-154,2006.
  11. 杜克,正当,Hand Book of Energy Crops, 1983.
  12. Dursun,。,Haliloglu, K. and Ekinci, M., Characterization of breeding lines of common bean as revealed by RAPD and relationship with morphological traits. Pak. J. Bot., 42(6): 3839-3845,2010.
  13. 步态- N -溶胶,E。Duque, m . C。,Edwards, K. J. and Tohme, J., Microsatellite repeats in common bean (Phaseolus vulgaris): isolation, characterization and cross- Species amplification inPhaseolus ssp. Crop Sci, 42:2128–2136,2002.
  14. Garoff, h . Ansorge, W。,Improvement of DNA sequencing gels. Ana. Biochem., 115: 450– 457,1981.
  15. 罗格·P。,Nodari, R., Tsai, S.M., Koinange, E.M.K., Llaca, V., Gilbertson, R. and Guzman, P., Linkage map in common bean. Annu. Rept. Bean Improv. Coop., 93: 24-25,1993.
  16. 关,R。,Chang, R., Li, Y., Wang, L., Liu, Z., Qiu, L., Genetic diversity comparison between Chinese and Japanese soybeans (Glycine max (L.) Merr.) is revealed by nuclear SSRs, Genet. Resour. Crop Evol., 57:229–242,2010.
  17. 石漠,H。,Petrino, M.G. and Kakunaga, T., A novel repeated element with Z-DNA-forming potential is widely found in evolutionarily diverse eukaryotic genomes. Proc. Natl. Acad. Sci. USA., 79: 6465–6469,1982.
  18. 氧化钾,r·K。Rai, M, K。、氧化钾年代。,Singh, R., Dhawan, A.K., Microsatellite markers: an overview of the recent progressin plants. Euphytica,177:309–334,2011.
  19. 卡普,A。,Krosorich, S., Bhat, K.V., Ayod, I.V.G. and Hodgin, T., Molecular tools in plant genetics resources conservation: A guide to technologies. Tech. bull., 2: 13-29,1997.
  20. 凯利,J.D.英检,P。,Miklas,期票and Coyne, D.P., , Tagging and mapping of genes and QTL and molecular marker-assisted selection for traits of economic importance in bean and cowpea. Field Crop Res., 82: 135-154,2003.
  21. 马丁,A.L.A.哥,核磁共振,Sartorato, A., Rava, C.A., Barros, E.G.D. and Moreira, M.A., Use of markers as a tools to investigate the presence of disease resistance genes in common bean cultivars. Crop Breed. and Appl. Biotech., 2: 125-133,2001,.
  22. 马西,P。,Logozzo, G., Donini, P. and Zeuli, P. S., Analysis of Genetic Structure in Widely Distributed Common Bean Landraces with DifferentPlant Growth Habits Using SSR and AFLP Markers.Crop Sci. 49:187–199,2009.
  23. Miklas,期票,凯利,J.D.毕比,S。E和布莱尔,分子量,b, Common bean breeding for resistance against biotic and abiotic stresses: from classical to MAS breeding. Euphytica, 147: 105–131,2006.
  24. M.W. Blair3 M.C.M. Grisi1, 2, p . Gepts4 c . Brondani2 P.A.A. Pereira2和R.P.V. Brondani2Genetic映射的微卫星标记的一组新的参考菜豆(菜豆)人口BAT93 x Jalo EEP558。ISSN 1676 - 5680
  25. Monaj T,辛格NK, Meenal R, Kumar N。RAPD标记在菜豆种质遗传多样性的分析从喜马拉雅山脉中部。52:315 - 3242005。
  26. (奥利,博和亨氏,B。,The south American wild bean (Phaseolus aborigineus Burk.) as ancestor of the common bean. Econ. Bot., 30: 257-272,1976.
  27. Rafalaski, j .和Tingey年代。,,Genetic diagnostics in Plant breed: RAPDs, microsatellites and machines. Trends Gen., 9: 275-280,1993.
  28. Ribaut J.M. Hoisington, D。,Marker- assisted selection: New tools and strategies. Trends Plant Sci., 3: 236-239,1998.
  29. 席尔瓦G.F.D.,Santos, J.B.D. and Ramalho, M.A.P., Identification of SSR and RAPD markers linked to a resistance allele for angular leaf spot in the common bean (Phaseolus vulgaris) line ESAL 55. Genet. Mol. Biol., 126(4): 459-463,2003.
  30. Smaranika, M。,Manish, K.S., Mohar, S. and Yadav, S.K., Genetic diversity of French bean (Bush type) genotypes in north western Himalayas. Indian J. Plant Genet. Resou., 23(3): 285-287,2010.
  31. 苏萨,m和德尔珈朵。,Mexican Leguminosae: Phytogeography,endemism, and origins. In: Ramamoorthy TP, Bye R, Lot A, Fa J, eds. Biology Diversity of Mexico: Origins and Distribution. New York, USA. Oxford University Press, 459-511,1993.
  32. 斯瓦特,P.J.,Prabhakar, K.R. and Vidya S.G., Molecular markers in plant genome analysis. Curr. Sci., 77: 230–240,1999.
  33. 陶,d,还建议,M。,Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res., 12: 4127–4138,1984.
  34. 女子,M。,Singh, N.K., Rathore, M. and Kumar, N., RAPD markers in the analysis of genetic diversity among common bean germplasm from Central Himalaya. Genet. Resource and Crop Evol., 52: 315–324,2003.
  35. Vallijos,刚建成时,Sakiyama, N.S. and Chase, C.D., A molecular marker based linkage map of Phaseolus vulgaries L. Genetics., 131: 733-740,1992.
  36. 一些K。,shailendra, S., Amit, K.S., Shiveta, S. and Venkatarmana, K.B., Comparative analysis diversity based on morpho-agronomic trait and microsatellite marker in common bean. Euphytica, 170: 249–262,2009.
  37. Waltson, M。,Molecular markers: which one to use? Seed World, 131: 22-29,1993.
  38. 威尔士,j .和Mc摘自M。,Fingerprinting genomes using PCR with arbitrary primers. Nucl. Acids Res., 18: 7213–7218,1990.
  39. 网游,W。,Isolation of plant tissues containing polyphenols and polysaccharides, MRC technical Bulletin No. 7,1999.
  40. 威廉姆斯,j。,Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V., DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acids Res., 18: 6531-6535,1990.
  41. 威廉姆斯,J.G.K.Hanafey,抗议;,Rafalski, J.A. and Tingey, S.V., Genetic analysis using random amplified polymorphic DNA markers. Methods Enzymol., 218: 704-741,1993.
  42. 年轻,R.A.和。凯利,J.D.,RAPD marker flanking the Are gene for anthracnose resistance in common bean. J. Amer. Soc. Hort. Sci., 121: 37-41,1996.
  43. 年轻,R.A.,Melotto, M., Nodari, R.O. and Kelly, J.D., Marker assisted dissection of the oligogenic anthracnose resistance in common bean cultivar, G 2333. Theor. Appl. Genet., 96: 87-94,1998.