摘要：ssr检测新方法介绍
新技术方法的产生发展从来都是来势汹汹、锐不可当的，ssr的检测亦是如此！自从生命科学迈入到atcg时代以来，传统的仅仅靠“跑电泳，比大小”的定性分析，已经越来越无法满足科研工作者对“准确、高效、定量”的要求了。二代高通量测序技术的发展，让这一目标成为现实，天昊生物创新技术—ssrseq，把ssr的“序列信息”及“比例信息”一网打尽！下面就跟随小编看看ssr检测的“前世今生”。
什么是ssr？
ssr (simple sequence repeats，简单序列重复)，或称str（short tandem repeat，短片段串联重复）或者microsatellites（微卫星），广泛的存在于真核生物基因组中。大多数ssrs是非编码序列，可以影响基因表达、剪接、蛋白序列及基因组结构等（1-5）。ssr长度突变频率在每一世代每个位点大概是10-7到10-3之间（6），这远远高于单个碱基10-9左右的突变频率（7-8），从而在基因组中产生了更具多样性的ssrs。尽管ssr序列自身具有高度的变异性，但是它侧翼区域的序列却在物种内具有很高保守性，有时这种保守型甚至在物种间存在（9-12）。ssr相较与其他遗传变异具有几方面特点，包括共显性、高度可重复性和dna检测需要量少等（13-16）。更重要的是，这种ssr序列的多样性和它侧翼序列保守性的结合，使它成为一种理想的遗传分子标记。的确，ssrs已经在包括dna指纹图谱分析、基因作图、亲缘关系鉴定、分子辅助育种、遗传多样性分析、种子纯度及品系鉴定中发挥着重要的作用（16-20）。
ssr多样性的产生原因及传统检测的不足
ssr多样性产生的最主要原因是在ssr复制过程中dna聚合酶固有的“滑移”现象（slippage）造成的(21-27)，这种滑移现象同样可以发生在体外，导致错误的ssr等位基因并增加了ssr准确分型的难度。而且，基于琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳、毛细管电泳这些目前常用的ssr检测方法，普遍存在着分辨率不高、不够准确、效率及通量不高等问题。例如，目前在冬菇、黄麻和木豆中进行的dna指纹图谱分析仅仅用到25、28和48个ssr位点（28-30）。这些有限的ssrs不足以构建高质量ssr指纹图谱用于区分亲缘性高物种间的关系。全基因组重测序虽然一次可以检测大量ssr位点（25, 31-32），但是ssr序列仅仅占整个基因组的很小部分，例如人类基因组中的ssr只占3%左右（33），因此全基因组重测序会获得很多我们关心的ssr以外的冗余序列，这就稀释了所测有用数据的比例，使得ssr位点的测序深度很难超过10-100x（25），这样在合理的测序价格内，利用全基因组重测序的方法就难以得到准确性高的ssr分型。另外，用全基因组重测序进行ssr分型还会导致某些ssr位点的扩增偏好性以及ssr重复序列较高难度的数据分析等问题（34-36）。
天昊生物自主研发的基于二代测序技术的ssr分型新方法--ssrseq，这种方法几乎克服了现存所有检测方法的不足，尤其适合对多ssr位点、超高深度的分型，准确度高，并且分辨率达到单碱基的水平。因此适合所有二倍体动植物及真核微生物的ssr位点分型。另外，我们还成功对六倍体植物—油茶进行了ssr分型。对于多倍体物种来说，我们的ssrseq可以提供不同等位基因的比例数据，从而提高了多倍体物种遗传多样性分析的准确度，获得更加清晰的遗传结构图。
参考文献：
1. li,y.-c., korol,a.b., fahima,t. and nevo,e. (2004) microsatelliteswithin genes: structure, function, and evolution. mol. biol. evol., 21,991–1007.
2. iglesias,a.r., kindlund,e., tammi,m. and wadelius,c. (2004) somemicrosatellites may act as novel polymorphic cis-regulatory elementsthrough transcription factor binding. gene, 341, 149–165.
3. martin,p., makepeace,k., hill,s.a., hood,d.w. and moxon,e.r.(2005) microsatellite instability regulates transcription factor bindingand gene expression. proc. natl. acad. sci. u.s.a., 102, 3800–3804.
4. krishnan,j. and mishra,r.k. (2015) code in the non-coding. proc.indian natl. sci. acad., 81, 609–628.
5. gymrek,m., willems,t., guilmatre,a., zeng,h., markus,b.,georgiev,s., daly,m.j., price,a.l., pritchard,j.k., sharp,a.j. et al.(2016) abundant contribution of short tandem repeats to geneexpression variation in humans. nat. genet., 48, 22–29.
6. buschiazzo,e. and gemmell,n.j. (2006) the rise, fall and renaissanceof microsatellites in eukaryotic genomes. bioessays, 28, 1040–1050.
7. yang,s., wang,l., huang,j., zhang,x., yuan,y., chen,j.-q.,hurst,l.d. and tian,d. (2015) parent-progeny sequencing indicateshigher mutation rates in heterozygotes. nature, 523, 463–467.
8. ossowski,s., schneeberger,k., lucas-lledo,j.i., warthmann,n.,clark,r.m., shaw,r.g., weigel,d. and lynch,m. (2010) the rateand molecular spectrum of spontaneous mutations in arabidopsisthaliana. science, 327, 92–94.
9. moore,s., sargeant,l., king,t., mattick,j., georges,m. andhetzel,d. (1991) the conservation of dinucleotide microsatellitesamong mammalian genomes allows the use of heterologous pcrprimer pairs in closely related species. genomics, 10, 654–660.
10. moodley,y., baumgarten,i. and harley,e. (2006) horsemicrosatellites and their amenability to comparative equid genetics.anim. genet., 37, 258–261.
11. dawson,d.a., horsburgh,g.j., ku¨ pper,c., stewart,i.r.,ball,a.d., durrant,k.l., hansson,b., bacon,i., bird,s. and klein,a.(2010) new methods to identify conserved microsatellite loci anddevelop primer sets of high cross-species utility–as demonstrated forbirds. mol. ecol. resour., 10, 475–494.
12. moodley,y., masello,j.f., cole,t.l., calderon,l.,munimanda,g.k.,thali,m.r., alderman,r., cuthbert,r.j., marin,m., massaro,m.et al. (2015) evolutionary factors affecting the cross-species utility ofnewly developed microsatellite markers in seabirds. mol. ecol.resour., 15, 1046–1058.
13. selkoe,k.a. and toonen,r.j. (2006) microsatellites for ecologists: apractical guide to using and evaluating microsatellite markers. ecol.lett., 9, 615–629.
14. guichoux,e., lagache,l., wagner,s., chaumeil,p., leger,p.,lepais,o., lepoittevin,c., malausa,t., revardel,e., salin,f. et al.(2011) current trends in microsatellite genotyping. mol. ecol.resour., 11, 591–611.
15. schl¨ otterer,c. (2000) evolutionary dynamics of microsatellite dna.chromosoma, 109, 365–371.
16. kaur,s., panesar,p.s., bera,m.b. and kaur,v. (2015) simple sequencerepeat markers in genetic divergence and marker-assisted selection ofrice cultivars: a review. crit. rev. food sci. nutr., 55, 41–49.
17. jarne,p. and lagoda,p.j. (1996) microsatellites, from molecules topopulations and back. trends ecol. evol., 11, 424–429.
18. kim,k.s. and sappington,t.w. (2013) microsatellite data analysis forpopulation genetics. methods mol. biol., 1006, 271–295.
19. chambers,g.k., curtis,c., millar,c.d., huynen,l. andlambert,d.m. (2014) dna fingerprinting in zoology: past, present,future. nvestig.genet., 5, 1–11.
20. borsting,c. and morling,n. (2015) next generation sequencing andits applications in forensic genetics. forensic sci. int.genet., 18, 78–89.
21. ellegren,h. (2004) microsatellites: simple sequences with complexevolution. nat. rev., 5, 435–445.
22. webster,m.t. and hagberg,j. (2007) is there evidence for convergentevolution around human microsatellites? mol. biol. evol., 24,1097–1100.
23. brandstr¨om,m., bagshaw,a.t., gemmell,n.j. and ellegren,h. (2008)the relationship between microsatellite polymorphism andrecombination hot spots in the human genome. mol. biol. evol., 25,2579–2587.
24. kelkar,y.d., tyekucheva,s., chiaromonte,f. and makova,k.d.(2008) the genome-wide determinants of human and chimpanzeemicrosatellite evolution. genome res., 18, 30–38.
25. fungtammasan,a., ananda,g., hile,s.e., su,m.s., sun,c.,harris,r., medvedev,p., eckert,k. and makova,k.d. (2015)accurate typing of short tandem repeats from genome-widesequencing data and its applications. genome res., 25, 736–749.
26. abdulovic,a.l., hile,s.e., kunkel,t.a. and eckert,k.a. (2011) thein vitro fidelity of yeast dna polymerase and polymerase εholoenzymes during dinucleotide microsatellite dna synthesis. dnarepair, 10, 497–505.
27. baptiste,b.a. and eckert,k.a. (2012) dna polymerase kappamicrosatellite synthesis: two distinct mechanisms ofslippage-mediated errors. environ. mol. mutagen., 53, 787–796.
28. zhang,l., cai,r., yuan,m., tao,a., xu,j., lin,l., fang,p. and qi,j.(2015) genetic diversity and dna fingerprinting in jute (corchorusspp.) based on ssr markers. crop j., 3, 416–422.
29. liu,x.b., feng,b., li,j., yan,c. and yang,z.l. (2016) geneticdiversity and breeding history of winter mushroom (flammulinavelutipes) in china uncovered by genomic ssr markers. gene, 591,227–235.
30. njung’e,v., deshpande,s., siambi,m., jones,r., silim,s. and devilliers,s. (2016) ssr genetic diversity assessment of popularpigeonpea varieties in malawi reveals unique fingerprints. electron. j.biotechnol., 21, 65–71.
31. kim,k.-s., noh,c.h., moon,s.-j., han,s.-h. andbang,i.-c. (2016)development of novel tetra-and trinucleotide microsatellite markersfor giant grouper epinepheluslanceolatus using 454 pyrosequencing.mol. biol. rep., 43, 541–548.
32. bozzi,j.a., liepelt,s., ohneiser,s., gallo,l.a., marchelli,p., leyer,i.,ziegenhagen,b. and mengel,c. (2015) characterization of 23polymorphic ssr markers in salix humboldtiana (salicaceae) usingnext-generation sequencing and cross-amplification from relatedspecies. appl. plant sci., 3, 1400120.
33. lander,e.s., linton,l.m., birren,b., nusbaum,c., zody,m.c.,baldwin,j., devon,k., dewar,k., doyle,m., fitzhugh,w. et al.(2001) initial sequencing and analysis of the human genome. nature,409, 860–921.
34. star,b., hansen,m.h., skage,m., bradbury,i.r., godiksen,j.a.,kjesbu,o.s. and jentoft,s. (2016) preferential amplification ofrepetitive dna during whole genome sequencing library creationfrom historic samples. star, 2, 36–45.
35. treangen,t.j. and salzberg,s.l. (2012) repetitive dna andnext-generation sequencing: computational challenges and solutions.nat. rev. genet., 13, 36–46.
36. gymrek,m., golan,d., rosset,s. and erlich,y. (2012) lobstr: ashort tandem repeat profiler for personal genomes. genome res., 22,1154–1162.
部分内容来源：doi: 10.1093/nar/gkx093
往期精选文章：
选择天昊，选择专业！2017年上半年，天昊生物尊龙在线登录的合作伙伴共发表sci文章55篇。截至目前公司尊龙在线登录的合作伙伴发表sci文章总篇数300 ，累计影响因子1000 。我们期待成为您ssr基因分型的优质服务提供商！
苏州工业园区星湖街218号生物纳米园c13楼501单元
86(0)512-6295 9990
86(0)512-6295 9995
