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s00122-020-03609-w","16:\"https%3a%2f%2fdoi.org%2f10.1007%2fs00122-020-03609-w\"|27:\"12\"|31:12"],[20,"）。何中虎研究组的硕士研究生武玉莹同学为该论文的第一作者，夏先春研究员为该论文的通讯作者。该项研究得到国家自然科学基金项目（","27:\"12\"|31:3"],[20,"31461143021","27:\"12\"|31:12"],[20,"）资助。","27:\"12\"|31:3"],[20,"\n","24:\"ai0q\"|linespacing:\"150\""],[20,"参考文献：","27:\"12\"|31:3|8:1"],[20,"\n","24:\"sysq\"|linespacing:\"150\""],[20,"long="" ym,="" chao="" ws,="" ma="" gj,="" xu="" ss="" and="" qi="" ll,="" 2017.="" an="" innovative="" snp="" genotyping="" method="" adapting="" to="" multiple="" platforms="" throughputs.="" theoretical="" applied="" genetics,="" 130:="" 597-607.="" doi:="" s00122-016-2838-4.","27:\"12\"|31:12"],[20,"\n","24:\"vaon\"|linespacing:\"150\""],[20,"rasheed="" a,="" wen="" w’e,="" gao="" fm,="" zhai="" sn,="" jin="" h,="" liu="" jd,="" guo="" q,="" zhang="" yj,="" dreisigacker="" s,="" xia="" xc="" he="" zh,="" 2016.="" development="" validation="" of="" kasp="" assays="" for="" genes="" underpinning="" key="" economic="" traits="" in="" bread="" wheat.="" 129:="" 1843-1860.="" s00122-016-2743-x.="" ","27:\"12\"|31:12"]]'="">开发高通量、低成本的基因特异性标记可显著提高小麦分子标记辅助选择的准确性和效率。目前，KASP（Kompetitive allele-specific PCR）标记广泛应用于小麦分子育种（Rasheed et al. 2016），但是KASP标记有3个缺点：（1）两个正向引物只有3’端一个碱基的差异，部分标记的特异性较差；（2） PCR反应的MasterMix需要从LGC或其他公司购买，成本较高；（3）PCR产物不能用常规PAGE胶检测。
针对KASP标记的不足之处，我们参考Long et al.（2017）的方法，根据已克隆基因或等位基因的序列信息，结合基因测序技术，获得46个基因的特异性SNP或InDel，通过检测体系的优化，建立了准确性高、通量灵活、检测成本低、平台兼容性好的小麦基因特异性STARP（Semi-thermal asymmetric reverse PCR）标记检测体系，开发了30个小麦品质性状的基因特异性STARP标记，7个与小麦生物和非生物胁迫抗性相关的基因特异性STARP标记，19个产量和适应性相关的基因特异性STARP标记，并利用305份小麦品种（系）和3个RIL群体对标记的有效性进行了验证。
STARP是一种创新的SNP和InDel基因分型平台，该技术可以由研究者自行设计引物，操作灵活并具有平台兼容性。在STARP技术中，PEA引物 1和PEA引物 2是由研究人员自行设计并合成的通用引物，两者在结构上被荧光序列标签（FAM和HEX）及猝灭基团（Dabsyl）修饰，在序列上有4 bp的差异（AGAG-3’）。因此，相对于KASP标记，STARP技术显著降低了PCR反应成本；PEA引物 1和PEA引物2的结构特征使得它们在低温下均形成发卡结构，有效地提高了Touchdown过程中PCR反应的效率和特异性；PEA引物2中4 bp的插入产生了等位基因间PCR产物的序列长度差异，因此最终的PCR产物既可以通过无凝胶的荧光信号检测也可以利用基于凝胶的片段大小分离。此外，STARP技术中两个AMAS引物3'端的第3或第4个碱基处引入碱基错配（LONG et al. 2017），使得两个等位基因在进行初始PCR扩增时能够获得更高的特异性。PCR反应体系在STARP技术中也扮演着重要角色。首先，为了保证基因分型结果的准确可靠性，明确AMAS引物、PEA引物和通用反向引物的比例是首要条件。本研究通过多次试验，最终确定了每条AMAS引物、每条PEA引物和通用反向引物的最佳比例为2:5:10。其次，STARP技术中PCR扩增的循环数是影响最终荧光信号强弱的因素之一。本研究发现，在PCR程序中第3步的循环数过高（42-46个）将会导致引物二聚体的扩增进而形成假阳性荧光信号；一般情况下，39个循环足以满足最终PCR荧光信号检测；少数情况下，在39个循环的基础上增加3个循环可以获得理想的分型结果。
图1. Ppo-D1基因STARP标记的验证。a、Ppo-D1位点的STARP标记在40份已知基因型小麦品种中的荧光信号图，b、Ppo-D1位点的STARP标记在305份小麦品种（系）中的基因分型结果2020年5月25日《Theor Appl Genet》杂志在线发表了何中虎研究团队这一研究成果（https://doi.org/10.1007/s00122-020-03609-w）。该研究组的硕士研究生武玉莹同学为该论文的第一作者，夏先春研究员为该论文的通讯作者。该项研究得到国家自然科学基金项目（31461143021）资助。参考文献：
LONG YM, CHAO WS, MA GJ, XU SS and QI LL, 2017. An innovative SNP genotyping method adapting to multiple platforms and throughputs. Theoretical and Applied Genetics, 130: 597-607. DOI: 10.1007/s00122-016-2838-4.RASHEED A, WEN W’E, GAO FM, ZHAI SN, JIN H, LIU JD, GUO Q, ZHANG YJ, DREISIGACKER S, XIA XC and HE ZH, 2016. Development and validation of KASP assays for genes underpinning key economic traits in bread wheat. Theoretical and Applied Genetics, 129: 1843-1860. DOI: 10.1007/s00122-016-2743-x.
