基于HRM获得与桃Tssd紧密连锁的SNP标记

鲁振华，牛良，张南南，崔国朝，潘磊，曾文芳，王志强

（中国农业科学院郑州果树研究所/国家桃葡萄品种改良中心/农业部果树育种技术重点实验室，郑州 450009）

基于HRM获得与桃Tssd紧密连锁的SNP标记

鲁振华，牛良，张南南，崔国朝，潘磊，曾文芳，王志强

（中国农业科学院郑州果树研究所/国家桃葡萄品种改良中心/农业部果树育种技术重点实验室，郑州 450009）

【目的】植物中，SNP标记具有分布广泛、分辨率高、共显性和多态性高等特点，是遗传研究的常用分子标记。桃全基因组测序完成，获得了大量SNP位点。利用现有的SNP数据进行简单、快速的SNP基因分型是基因定位、品种鉴定和图谱构建等后续研究的基础。文章拟建立采用高分辨率熔解曲线进行不同类型SNP的基因分型方法，以获得与桃温度敏感半矮生型基因紧密连锁的分子标记。【方法】以普通生长型（ST）单株97-32-46为母本，温度敏感半矮生型单株03-94-2（Tssd）为父本进行人工杂交，利用其分离群体96个后代单株为研究材料。在定位目标基因的区间内开发连锁和不同类型的SNP标记的基础上，采用高分辨率熔解曲线进行SNP的基因分型并获得与目标性状紧密连锁的SNP标记。【结果】明确了DNA模板和Mg2+是影响基因分型的关键因子，并确立了反应体系最佳浓度区间。在15 µL反应体系中模板DNA的量低于5.0 ng时或Mg2+浓度低于1.6 µmol·L-1时则不能完成PCR扩增和基因分型；根据亲本基因型和表型一致的SNP位点设计引物，扩增片段长度在140 bp左右。高分辨率熔解曲线分析可对由单个核苷酸变异引起的4种不同类型的SNP（A/T、A/G、A/C和C/G）进行基因分型，并正确区分了温度敏感半矮生型和普通生长型，与进行Sanger测序鉴定的结果一致。采用96孔板对温度敏感半矮生型和普通生长型各48个分离后代单株进行了PCR扩增和基因分型，确定了遗传距离。分型结果表明高分辨率熔解曲线分析技术可以将96个样杂交后代单株分为温度敏感半矮生型和普通生长型2种，正确地区分了A/A基因型和A/T基因型。在96个样品中仅1个没有成功扩增，在温度敏感半矮生型和普通生长型中各存在1个重组单株。获得与温度敏感半矮生型基因紧密连锁的SNP标记，遗传距离为2.11 cM。【结论】建立了基于高分辨率熔解曲线分析的SNP基因分型。尽管高分辨率熔解曲线分析技术无法区分两种不同纯合类型的SNP变异，但仍不失为区分已知变异SNP的有效方法。在已经获得桃大量SNP的基础上，该体系可用于桃的基因定位、遗传多样性和品种鉴定等研究。

桃；高分辨率熔解曲线；SNP基因分型；温度敏感半矮生型

0 引言

【研究意义】单核苷酸多态性（single nucleotide polymorphism，SNP）是指在基因组水平上单核苷酸的变异，这种变异发生在不同个体的种、品种以及染色相对应的序列间[1]。植物中，SNP分布广，具有分辨率高和共显性等特点[2]，建立基于SNP标记的基因分型体系是进行目标性状快速定位的基础。【前人研究进展】近年来，随着二代测序技术的不断进步和完善，SNP分析被广泛应用于植物的遗传多样性[3]、系统进化分析[4]、全基因组关联分析[5-6]、遗传图谱构建等研究中[7-8]，丰富了分子标记类型，是迄今为止多态性最高的分子标记。用于SNP位点鉴定有Sanger测序[9]、Tilling技术[10]、单链构象多态性（SSCP）[11]、SNP芯片[12]、二代测序[13]等一系列的方法。而高分辨率熔解曲线（high resolution melting，HRM）分析技术是基于研究高温度下双链DNA的分离，进而确定PCR扩增子的遗传变异[14-15]，被广泛应用于小麦、水稻、苹果和梨等作物的SNP、Indel和SSR基因分型研究[16-19]。在桃上，许多性状基因的精细定位均采用了SNP标记技术。如桃分枝角度基因[20]和桃矮化基因[21]。自发现一个变异单株SD9238以来[22]，开展了相关的研究工作，明确了该半矮生性状受显性单基因控制。后续研究发现该温度调控该类型桃节间长度并决定植株高度，命名该类型桃为温度敏感半矮生桃（Temperature-sensitive semi-dwarf for Prunus Persica，PpTssd）[23]。【本研究切入点】尽管果树上已经完成了苹果[24]、草莓[25]、梨[26]以及桃[27]等多个物种全基因序列测定，获得了大量的SNP信息，但是仍缺少快速、低成本、准确的SNP分型技术。同时，获得与桃Tssd紧密连锁的SNP标记是进行目标性状分子鉴定的前提。【拟解决的关键问题】本研究拟从影响HRM基因分型的主要因子Mg2+浓度和DNA模板入手，确定基因分型合适的浓度区间，同时采用杂交群体后代在4种不同类型SNP中进行基因分型验证，建立不同 SNP基因分型的技术体系，可为后续利用HRM技术进行基因定位、品种鉴定以及遗传多样性评价等提供技术支撑。同时，基于此技术，本研究获得了与桃目标性状紧密连锁的SNP，为建立目标性状的分子鉴定体系奠定基础。

1 材料与方法

1.1 研究材料

选取普通生长型单株97-32-46为母本，温度敏感半矮生型单株03-94-2（Tssd）为父本进行杂交，其中，03-94-2来源于本研究小组发现的变异单株‘SD9238’[22]，普通生长型97-32-46（standard type，ST），亲本为丰白和中油桃5号。桃核破壳后，包衣进行层积处理。杂交F1代获得幼苗后，以此分离群体96个单株为HRM基因分型的DNA模板。于4—5月和6月初分别对3年生杂交后代单株进行性状鉴定。在已经定位目标基因的区间内[23]，开发 4种不同类型SNP，以确定HRM基因分型的准确性和重复性。分别以温度敏感半矮型和普通生长型杂交后代分离群体（97-32-46×03-94-2）的6个个体用于基因分型以检验基因型的准确和重复性。任意选取96个杂交后代单株计算SNP标记与目标性状的遗传距离。

1.2 基因组DNA的提取与表型鉴定

取新鲜的桃叶片，硅胶干燥后-20℃冷藏备用。基因组DNA的提取采用CTAB法[28]，略作修改。采用NanoDrop 1000（Thermo Scientific）进行浓度测定，稀释后备用。在温室采用双温度法确定了节间表型，具体表现为较低温度下（22℃左右）节间极短，30℃以上时节间长度接近普通生长型，排除了矮化型的影响（图1）。同时根据植株生长节奏，在4月—5月和6月初进行了表型评价，肉眼观察节间表型并记录结果。半矮生型节间表型表现为4—5月节间长度极短，接近矮化状态；而在6月初后节间长度逐渐接近普通生长型。幼苗和成苗鉴定结果一致。

图1 温度处理植株表型鉴定（左1为ST，左2—4为Tssd）Fig. 1 Phenotype identification based on temperature treatment (Left 1: ST, Right 2 to 4: Tssd)

1.3 目标区域不同基因型SNP开发

根据已经定位的区域并结合亲本基因型和表型一致的SNP设计引物。采用Sanger测序（Invitrogen）在亲本间开发4种不同类型的SNP标记，在SNP位点两翼设计HRM基因分型引物，以用于不同SNP的基因分型。其中，用于对96个杂交后代单株进行连锁分析的引物序列为5′-ATATGTCCCTGGTGGCTTG G-3′和5′-GAGGGCGACTACAGACAGAC-3′，扩增片段长度为90 bp。

1.4 引物的设计与SNP基因分型

引物的设计采用Primer 3.0软件（http://primer3. ut.ee/）进行正反引物设计，退火温度60℃左右。采用HRM master mix（Roche）进行PCR扩增，反应总体积为15 μL。其中含DNA模板、Mg2+、0.17 μmol·L-1正/反引物和7.5 μL HRM master荧光染料（Roche）。利用LightCycler 480II定量PCR仪（Roche）进行PCR扩增和HRM分析。

PCR扩增程序为95℃ 3 min；94℃ 20 s，60℃ 10 s，72℃ 15 s，45个循环；72℃ 5 min，40℃冷却5 min。HRM分析程序为95℃ 1 min，40℃ 1 min，65—95℃读取熔解曲线，温度分辨率0.02℃。高分辨率熔解曲线分析采用Gene Scanning软件（1.5 version）。

2 结果

2.1 模板和Mg2+对基因分型的影响

为研究DNA模板和Mg2+对PCR扩增和基因分型的影响，分别设立了二者的浓度梯度，在一种其他组成不变的情况下研究单因素对基因分型的影响。HRM基因分型反应总体系为15 µL，将DNA模板依次设为2.5、5、7.5、10、12.5和15 µg等6个梯度。Mg2+设为0.4、0.8、1.2、1.6、2.0和2.4 µmol·L-1等6种浓度梯度。当Mg2+浓度小于1.6 µmol·L-1或DNA模板含量小于5 ng时，PCR扩增均不成功，不能完成基于HRM的SNP的基因分型。在设定梯度范围内，高浓度模板对基因分型影响不大。

2.2 SNP基因分型

在已经定位目标性状区间内，开发不同类型的SNP标记。根据碱基配对组合和双亲基因型，选取包括A/T、A/G、A/C和C/G 4种不同类型的SNP标记进行基因分型（图2-B、图3-B、图4-B和图5-B）。同时，参考桃基因序列信息设计4对引物进行SNP基因分型（表1）。基于HRM分析的特点将引物扩增片段长度设计在 150 bp左右，以进行准确分型。基于DNA测序，选取母本表现为aa，父本表现为Aa基因型且与目标性状连锁的SNP标记以检验基于HRM的SNP基因分型。根据测序结果普通生长型DNA序列SNP位点为T/T，温度敏感半矮生型SNP位点A/T，扩增片段长度为124 bp。高分辨率熔解曲线将后代12个分离群体单株分成2种不同类型熔解曲线，红色（6个）为温度敏感半矮生型植株；蓝色（6个）为普通生长型植株，该分型结果与目标性状基因型一致（图SNP基因分型的准确性，采用96孔板对样品进行基因分型。结果表明，96个样品中仅1个样品扩增不成功，其余样品完成了扩增。采用高分辨率熔解曲线可将温度敏感半矮生型和普通生长型区分开来，样品基因分型准确（图 7）。其中，在温度敏感半矮生型中存在一个交换单株A1，普通生长型中存在1个交换单株H11，交换单株后经测序验证，分型结果正确无误。基于以上分型结果，对交换单株进行Sanger测序，验证了 SNP基因分型结果的准确性。获得了与桃 Tssd 2-A），同样地HRM分析结果正确地区分了A/G（图3-A），扩增片段长度为132 bp；A/C（图4-A），扩增片段长度为146 bp和C/G等基因型（图5-A），扩增片段长度为137 bp。HRM分析可区分低至一个碱基的差异。为了检验HRM分析，将目标片段进行了电泳检测，结果表明，PCR扩增成功，且片段大小与目标片段一致（图6-A—图6-D）。

表1 基于HRM分析4种SNP类型引物信息Table 1 Four types of SNP Primer information used for HRM analysis

图2 基于HRM纯合A/A（蓝色曲线）和杂合A/T（红色曲线）位点的SNP鉴定Fig. 2 HRM analysis profile genotyping homozygous A/A (Blue curve) and heterozygous A/T (Red curve)

图3 基于HRM分析纯合G/G（蓝色）和杂合G/A（红色）位点的SNP分型Fig. 3 HRM analysis profile genotyping homozygous G/G (Blue curve) and heterozygous G/A (Red curve)

图4 基于HRM分析纯合C/C（蓝色）和杂合C/A（红色）位点的SNP鉴定Fig. 4 HRM analysis profile genotyping homozygous C/C (Blue curve) and heterozygous C/A (Red curve)

2.3 SNP基因分型的验证与遗传距离分析

图5 基于HRM分析纯合G/G（蓝色）和杂合C/G（红色）位点的SNP鉴定Fig. 5 HRM analysis profile genotyping homozygous G/G (Blue) and heterozygous C/G (Red)

图6 PCR扩增片段琼脂糖凝胶电泳图Fig. 6 PCR amplification fragment profiling of agarose gel electrophoresis

根据已经定位的信息和紧密连锁的SNP标记，选取其中的基因型为AA和AT的SNP验证大量样品的紧密连锁的SNP标记（Scaffold_3 3450405），该标记与目标基因连锁距离为2.11 cM。

3 讨论

基因组的某个位点上单个核苷酸（A、T、C和G）的变化形成一个SNP，SNP检测错误率低、分辨率高、世代间全基因组进化相对稳定[29]，在遗传图谱构建方面，SNP标记优势突出[30-31]。特别是随着基于二代测序技术多个物种基因组测序的完成，SNP标记将逐渐取代SSR、RFLP以及其他分子标记[31]。SNP标记在植物中分布比较广泛。VERDE等[32]通过对56份桃育种材料进行了重测序，共获得了1 022 354个多态性的SNPs，并采用9K的SNP芯片进行了验证。同样，ARANZANA等[33]对47个欧美桃品种23个基因组片段进行测序，发现平均每 598碱基存在一个SNP，每4 189 bp存在一个Indel。大量SNPs的存在可用于基因的定位、遗传多样性评价和特征指纹鉴定提供方便。

图7 基于HRM分析96个样品的SNP基因分型Fig. 7 HRM analysis profile genotyping of 96 samples for homozygous A/A (Blue) and heterozygous A/T (Red)

尽管SNP广泛存在，但由于SNP只有一个碱基的差异，检测相对困难。Sanger测序技术是对SNP进行鉴定的最直接、准确和信息量最完整的方法[9]，但测序成本较高、过程繁杂，同时无法对Poly序列与长重复序列等结构进行鉴定。而采用高分辨率熔解曲线的SNP分型是通过实时监测升温过程中双链DNA荧光染料与PCR扩增产物的结合情况，来判断是否存在SNP[34-35]。由于饱和染料与GC-rich和AT-rich区的亲和无偏好性，HRM不仅可用于分析由SSR、Indel差异引起的多态性[36]，同时可以分析由单个核苷酸差异引起的多态性。

近几年，多年生果树树型研究进展较快，特别是苹果和桃。桃树型研究主要集中在株高和节间长度方面。YAMAMOTO等[37]将控制桃株高矮化基因定位在LG6上。HOLLENDER等[21]获得了矮化性状的基因，明确了矮化性状的遗传和调控机制。VERDE等[38]采用BC1群体将控制节间长度的主效QTL定位于 LG1上。最近，基于二代测序技术，DARDICK等[20]采用了 83个杂交群体单株将控制桃分枝角度的TAC1定位在物理距离2 Mb以内。后采用250个单株，克隆了控制分枝角度的TAC1，并通过转拟南芥验证了该基因的功能，明确了直立型、柱型的遗传和调控机理，这是首个克隆的桃株型基因。

本研究建立了基于HRM的SNP基因分型，并应用于杂交群体，获得了紧密连锁的SNP标记，正确区分了普通生长型（ST）和温度敏感半矮生型（Tssd）。HRM分析能够有效区分不同SNP位点，可用于大规模的分子辅助选中体系中。

4 结论

建立了基于HRM技术对4种不同类型的SNP进行基因分型，确立了基因分型的DNA模板和影响因子Mg2+的浓度区间。尽管HRM技术无法区分双键和三键碱基间（如A/T和C/G）的变异，但仍不失为区分已知变异SNP的有效方法。同时，获得与桃PpTssd基因紧密连锁的SNP标记，遗传距为2.11 cM。

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（责任编辑 李莉）

SNP Marker Tightly Linked to Tssd for Peach Using High Resolution Melting Analysis

LU ZhenHua, NIU Liang, ZHANG NanNan, CUI GuoChao, PAN Lei, ZENG WenFang, WANG ZhiQiang
(Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences/National Peach and Grape Improvement Center/Key Laboratory of Fruit Breeding Technology of Ministry of Agriculture, Zhengzhou 450009)

【Objective】 SNP marker is characterized as highly distribution, high resolution and co-domination in plant organism, which is considered as a promising molecular marker. The achievements of peach whole genome sequencing generated massive amounts of SNPs. How to establish a sensitive and effective method for identifying genotyping of different SNP genotypes is a base of further research on gene mapping, cultivar identification and linkage map construction. 【Method】 The aim of this study was to determine if HRM can detect the genotyping of different SNP genotypes and identify a SNP marker tightly linked to PpTssd gene. The segregation population of semi-dwarf progeny was selected with parents 97-32-46 (ST) × 03-94-2 (Tssd). According togene mapping, different types of SNP markers within mapping region were developed, and the HRM analysis was employed to conduct SNP genotyping and the SNP marker linked to desired traits were generated.【Result】As the key factors for genotyping, the proper concentrations of temperate DNA and Mg2+were established. In the 15 µL PCR reaction system, genotyping could not be complete when template DNA was less than 5 ng and Mg2+was less than 1.6 µmol·L-1. The primers were designed based on the phenotype and genotype, spanning each desired SNP to amplify DNA fragments shorter than 150 bp. HRM analysis could discriminate four types of SNPs (A/T, A/G, A/C, and C/G) occurred via single nucleotide mutation and the result was validated by Sanger sequence. HRM analysis divided temperature-sensitive semi-dwarf and standard type individuals into two groups. A SNP tightly linked to Tssd gene was identified in 96 individuals consisting of 48 Tssd and 48 ST, respectively. The HRM technique distinguished the Tssd and ST into two groups except for one individual with null amplification. Ultimately, the homozygous A/A and heterozygous A/T were identified, and generated a SNP tightly linked to Tssd gene with 2.11cM with two recombinants.【Conclusion】SNP genotyping of different SNPs were established based on HRM analysis. Although HRM could not distinguish two types of homozygote, HRM analysis still can be a effective method for SNP genotyping and can be used for gene mapping, genetic diversity and cultivar identification in peach based on this study.

peach; high resolution melting (HRM); SNP genotyping; temperature-sensitive semi-dwarf type

2016-09-29；接受日期：2016-11-21

国家自然科学基金（31500558、31470679）、中国农业科学院科技创新工程（CAAS-ASTIP-2016-ZFRI）

联系方式：鲁振华，E-mail：luzhenhua@caas.cn。通信作者王志强，E-mail：wangzhiqiang@caas.cn