魯振華，牛良，張南南，崔國(guó)朝，潘磊，曾文芳，王志強(qiáng)

（中國(guó)農(nóng)業(yè)科學(xué)院鄭州果樹(shù)研究所/國(guó)家桃葡萄品種改良中心/農(nóng)業(yè)部果樹(shù)育種技術(shù)重點(diǎn)實(shí)驗(yàn)室，鄭州 450009）

基于HRM獲得與桃Tssd緊密連鎖的SNP標(biāo)記

魯振華，牛良，張南南，崔國(guó)朝，潘磊，曾文芳，王志強(qiáng)

（中國(guó)農(nóng)業(yè)科學(xué)院鄭州果樹(shù)研究所/國(guó)家桃葡萄品種改良中心/農(nóng)業(yè)部果樹(shù)育種技術(shù)重點(diǎn)實(shí)驗(yàn)室，鄭州 450009）

【目的】植物中，SNP標(biāo)記具有分布廣泛、分辨率高、共顯性和多態(tài)性高等特點(diǎn)，是遺傳研究的常用分子標(biāo)記。桃全基因組測(cè)序完成，獲得了大量SNP位點(diǎn)。利用現(xiàn)有的SNP數(shù)據(jù)進(jìn)行簡(jiǎn)單、快速的SNP基因分型是基因定位、品種鑒定和圖譜構(gòu)建等后續(xù)研究的基礎(chǔ)。文章擬建立采用高分辨率熔解曲線進(jìn)行不同類型SNP的基因分型方法，以獲得與桃溫度敏感半矮生型基因緊密連鎖的分子標(biāo)記?！痉椒ā恳云胀ㄉL(zhǎng)型（ST）單株97-32-46為母本，溫度敏感半矮生型單株03-94-2（Tssd）為父本進(jìn)行人工雜交，利用其分離群體96個(gè)后代單株為研究材料。在定位目標(biāo)基因的區(qū)間內(nèi)開(kāi)發(fā)連鎖和不同類型的SNP標(biāo)記的基礎(chǔ)上，采用高分辨率熔解曲線進(jìn)行SNP的基因分型并獲得與目標(biāo)性狀緊密連鎖的SNP標(biāo)記?！窘Y(jié)果】明確了DNA模板和Mg2+是影響基因分型的關(guān)鍵因子，并確立了反應(yīng)體系最佳濃度區(qū)間。在15 μL反應(yīng)體系中模板DNA的量低于5.0 ng時(shí)或Mg2+濃度低于1.6 μmol·L-1時(shí)則不能完成PCR擴(kuò)增和基因分型；根據(jù)親本基因型和表型一致的SNP位點(diǎn)設(shè)計(jì)引物，擴(kuò)增片段長(zhǎng)度在140 bp左右。高分辨率熔解曲線分析可對(duì)由單個(gè)核苷酸變異引起的4種不同類型的SNP（A/T、A/G、A/C和C/G）進(jìn)行基因分型，并正確區(qū)分了溫度敏感半矮生型和普通生長(zhǎng)型，與進(jìn)行Sanger測(cè)序鑒定的結(jié)果一致。采用96孔板對(duì)溫度敏感半矮生型和普通生長(zhǎng)型各48個(gè)分離后代單株進(jìn)行了PCR擴(kuò)增和基因分型，確定了遺傳距離。分型結(jié)果表明高分辨率熔解曲線分析技術(shù)可以將96個(gè)樣雜交后代單株分為溫度敏感半矮生型和普通生長(zhǎng)型2種，正確地區(qū)分了A/A基因型和A/T基因型。在96個(gè)樣品中僅1個(gè)沒(méi)有成功擴(kuò)增，在溫度敏感半矮生型和普通生長(zhǎng)型中各存在1個(gè)重組單株。獲得與溫度敏感半矮生型基因緊密連鎖的SNP標(biāo)記，遺傳距離為2.11 cM?！窘Y(jié)論】建立了基于高分辨率熔解曲線分析的SNP基因分型。盡管高分辨率熔解曲線分析技術(shù)無(wú)法區(qū)分兩種不同純合類型的SNP變異，但仍不失為區(qū)分已知變異SNP的有效方法。在已經(jīng)獲得桃大量SNP的基礎(chǔ)上，該體系可用于桃的基因定位、遺傳多樣性和品種鑒定等研究。

桃；高分辨率熔解曲線；SNP基因分型；溫度敏感半矮生型

0 引言

【研究意義】單核苷酸多態(tài)性（single nucleotide polymorphism，SNP）是指在基因組水平上單核苷酸的變異，這種變異發(fā)生在不同個(gè)體的種、品種以及染色相對(duì)應(yīng)的序列間[1]。植物中，SNP分布廣，具有分辨率高和共顯性等特點(diǎn)[2]，建立基于SNP標(biāo)記的基因分型體系是進(jìn)行目標(biāo)性狀快速定位的基礎(chǔ)?！厩叭搜芯窟M(jìn)展】近年來(lái)，隨著二代測(cè)序技術(shù)的不斷進(jìn)步和完善，SNP分析被廣泛應(yīng)用于植物的遺傳多樣性[3]、系統(tǒng)進(jìn)化分析[4]、全基因組關(guān)聯(lián)分析[5-6]、遺傳圖譜構(gòu)建等研究中[7-8]，豐富了分子標(biāo)記類型，是迄今為止多態(tài)性最高的分子標(biāo)記。用于SNP位點(diǎn)鑒定有Sanger測(cè)序[9]、Tilling技術(shù)[10]、單鏈構(gòu)象多態(tài)性（SSCP）[11]、SNP芯片[12]、二代測(cè)序[13]等一系列的方法。而高分辨率熔解曲線（high resolution melting，HRM）分析技術(shù)是基于研究高溫度下雙鏈DNA的分離，進(jìn)而確定PCR擴(kuò)增子的遺傳變異[14-15]，被廣泛應(yīng)用于小麥、水稻、蘋果和梨等作物的SNP、Indel和SSR基因分型研究[16-19]。在桃上，許多性狀基因的精細(xì)定位均采用了SNP標(biāo)記技術(shù)。如桃分枝角度基因[20]和桃矮化基因[21]。自發(fā)現(xiàn)一個(gè)變異單株SD9238以來(lái)[22]，開(kāi)展了相關(guān)的研究工作，明確了該半矮生性狀受顯性單基因控制。后續(xù)研究發(fā)現(xiàn)該溫度調(diào)控該類型桃節(jié)間長(zhǎng)度并決定植株高度，命名該類型桃為溫度敏感半矮生桃（Temperature-sensitive semi-dwarf for Prunus Persica，PpTssd）[23]?！颈狙芯壳腥朦c(diǎn)】盡管果樹(shù)上已經(jīng)完成了蘋果[24]、草莓[25]、梨[26]以及桃[27]等多個(gè)物種全基因序列測(cè)定，獲得了大量的SNP信息，但是仍缺少快速、低成本、準(zhǔn)確的SNP分型技術(shù)。同時(shí)，獲得與桃Tssd緊密連鎖的SNP標(biāo)記是進(jìn)行目標(biāo)性狀分子鑒定的前提。【擬解決的關(guān)鍵問(wèn)題】本研究擬從影響HRM基因分型的主要因子Mg2+濃度和DNA模板入手，確定基因分型合適的濃度區(qū)間，同時(shí)采用雜交群體后代在4種不同類型SNP中進(jìn)行基因分型驗(yàn)證，建立不同 SNP基因分型的技術(shù)體系，可為后續(xù)利用HRM技術(shù)進(jìn)行基因定位、品種鑒定以及遺傳多樣性評(píng)價(jià)等提供技術(shù)支撐。同時(shí)，基于此技術(shù)，本研究獲得了與桃目標(biāo)性狀緊密連鎖的SNP，為建立目標(biāo)性狀的分子鑒定體系奠定基礎(chǔ)。

1 材料與方法

1.1 研究材料

選取普通生長(zhǎng)型單株97-32-46為母本，溫度敏感半矮生型單株03-94-2（Tssd）為父本進(jìn)行雜交，其中，03-94-2來(lái)源于本研究小組發(fā)現(xiàn)的變異單株‘SD9238’[22]，普通生長(zhǎng)型97-32-46（standard type，ST），親本為豐白和中油桃5號(hào)。桃核破殼后，包衣進(jìn)行層積處理。雜交F1代獲得幼苗后，以此分離群體96個(gè)單株為HRM基因分型的DNA模板。于4—5月和6月初分別對(duì)3年生雜交后代單株進(jìn)行性狀鑒定。在已經(jīng)定位目標(biāo)基因的區(qū)間內(nèi)[23]，開(kāi)發(fā) 4種不同類型SNP，以確定HRM基因分型的準(zhǔn)確性和重復(fù)性。分別以溫度敏感半矮型和普通生長(zhǎng)型雜交后代分離群體（97-32-46×03-94-2）的6個(gè)個(gè)體用于基因分型以檢驗(yàn)基因型的準(zhǔn)確和重復(fù)性。任意選取96個(gè)雜交后代單株計(jì)算SNP標(biāo)記與目標(biāo)性狀的遺傳距離。

1.2 基因組DNA的提取與表型鑒定

取新鮮的桃葉片，硅膠干燥后-20℃冷藏備用。基因組DNA的提取采用CTAB法[28]，略作修改。采用NanoDrop 1000（Thermo Scientific）進(jìn)行濃度測(cè)定，稀釋后備用。在溫室采用雙溫度法確定了節(jié)間表型，具體表現(xiàn)為較低溫度下（22℃左右）節(jié)間極短，30℃以上時(shí)節(jié)間長(zhǎng)度接近普通生長(zhǎng)型，排除了矮化型的影響（圖1）。同時(shí)根據(jù)植株生長(zhǎng)節(jié)奏，在4月—5月和6月初進(jìn)行了表型評(píng)價(jià)，肉眼觀察節(jié)間表型并記錄結(jié)果。半矮生型節(jié)間表型表現(xiàn)為4—5月節(jié)間長(zhǎng)度極短，接近矮化狀態(tài)；而在6月初后節(jié)間長(zhǎng)度逐漸接近普通生長(zhǎng)型。幼苗和成苗鑒定結(jié)果一致。

圖1 溫度處理植株表型鑒定（左1為ST，左2—4為Tssd）Fig. 1 Phenotype identification based on temperature treatment (Left 1: ST, Right 2 to 4: Tssd)

1.3 目標(biāo)區(qū)域不同基因型SNP開(kāi)發(fā)

根據(jù)已經(jīng)定位的區(qū)域并結(jié)合親本基因型和表型一致的SNP設(shè)計(jì)引物。采用Sanger測(cè)序（Invitrogen）在親本間開(kāi)發(fā)4種不同類型的SNP標(biāo)記，在SNP位點(diǎn)兩翼設(shè)計(jì)HRM基因分型引物，以用于不同SNP的基因分型。其中，用于對(duì)96個(gè)雜交后代單株進(jìn)行連鎖分析的引物序列為5′-ATATGTCCCTGGTGGCTTG G-3′和5′-GAGGGCGACTACAGACAGAC-3′，擴(kuò)增片段長(zhǎng)度為90 bp。

1.4 引物的設(shè)計(jì)與SNP基因分型

引物的設(shè)計(jì)采用Primer 3.0軟件（http://primer3. ut.ee/）進(jìn)行正反引物設(shè)計(jì)，退火溫度60℃左右。采用HRM master mix（Roche）進(jìn)行PCR擴(kuò)增，反應(yīng)總體積為15 μL。其中含DNA模板、Mg2+、0.17 μmol·L-1正/反引物和7.5 μL HRM master熒光染料（Roche）。利用LightCycler 480II定量PCR儀（Roche）進(jìn)行PCR擴(kuò)增和HRM分析。

PCR擴(kuò)增程序?yàn)?5℃ 3 min；94℃ 20 s，60℃ 10 s，72℃ 15 s，45個(gè)循環(huán)；72℃ 5 min，40℃冷卻5 min。HRM分析程序?yàn)?5℃ 1 min，40℃ 1 min，65—95℃讀取熔解曲線，溫度分辨率0.02℃。高分辨率熔解曲線分析采用Gene Scanning軟件（1.5 version）。

2 結(jié)果

2.1 模板和Mg2+對(duì)基因分型的影響

為研究DNA模板和Mg2+對(duì)PCR擴(kuò)增和基因分型的影響，分別設(shè)立了二者的濃度梯度，在一種其他組成不變的情況下研究單因素對(duì)基因分型的影響。HRM基因分型反應(yīng)總體系為15 μL，將DNA模板依次設(shè)為2.5、5、7.5、10、12.5和15 μg等6個(gè)梯度。Mg2+設(shè)為0.4、0.8、1.2、1.6、2.0和2.4 μmol·L-1等6種濃度梯度。當(dāng)Mg2+濃度小于1.6 μmol·L-1或DNA模板含量小于5 ng時(shí)，PCR擴(kuò)增均不成功，不能完成基于HRM的SNP的基因分型。在設(shè)定梯度范圍內(nèi)，高濃度模板對(duì)基因分型影響不大。

2.2 SNP基因分型

在已經(jīng)定位目標(biāo)性狀區(qū)間內(nèi)，開(kāi)發(fā)不同類型的SNP標(biāo)記。根據(jù)堿基配對(duì)組合和雙親基因型，選取包括A/T、A/G、A/C和C/G 4種不同類型的SNP標(biāo)記進(jìn)行基因分型（圖2-B、圖3-B、圖4-B和圖5-B）。同時(shí)，參考桃基因序列信息設(shè)計(jì)4對(duì)引物進(jìn)行SNP基因分型（表1）?；贖RM分析的特點(diǎn)將引物擴(kuò)增片段長(zhǎng)度設(shè)計(jì)在 150 bp左右，以進(jìn)行準(zhǔn)確分型?；贒NA測(cè)序，選取母本表現(xiàn)為aa，父本表現(xiàn)為Aa基因型且與目標(biāo)性狀連鎖的SNP標(biāo)記以檢驗(yàn)基于HRM的SNP基因分型。根據(jù)測(cè)序結(jié)果普通生長(zhǎng)型DNA序列SNP位點(diǎn)為T/T，溫度敏感半矮生型SNP位點(diǎn)A/T，擴(kuò)增片段長(zhǎng)度為124 bp。高分辨率熔解曲線將后代12個(gè)分離群體單株分成2種不同類型熔解曲線，紅色（6個(gè)）為溫度敏感半矮生型植株；藍(lán)色（6個(gè)）為普通生長(zhǎng)型植株，該分型結(jié)果與目標(biāo)性狀基因型一致（圖SNP基因分型的準(zhǔn)確性，采用96孔板對(duì)樣品進(jìn)行基因分型。結(jié)果表明，96個(gè)樣品中僅1個(gè)樣品擴(kuò)增不成功，其余樣品完成了擴(kuò)增。采用高分辨率熔解曲線可將溫度敏感半矮生型和普通生長(zhǎng)型區(qū)分開(kāi)來(lái)，樣品基因分型準(zhǔn)確（圖 7）。其中，在溫度敏感半矮生型中存在一個(gè)交換單株A1，普通生長(zhǎng)型中存在1個(gè)交換單株H11，交換單株后經(jīng)測(cè)序驗(yàn)證，分型結(jié)果正確無(wú)誤。基于以上分型結(jié)果，對(duì)交換單株進(jìn)行Sanger測(cè)序，驗(yàn)證了 SNP基因分型結(jié)果的準(zhǔn)確性。獲得了與桃 Tssd 2-A），同樣地HRM分析結(jié)果正確地區(qū)分了A/G（圖3-A），擴(kuò)增片段長(zhǎng)度為132 bp；A/C（圖4-A），擴(kuò)增片段長(zhǎng)度為146 bp和C/G等基因型（圖5-A），擴(kuò)增片段長(zhǎng)度為137 bp。HRM分析可區(qū)分低至一個(gè)堿基的差異。為了檢驗(yàn)HRM分析，將目標(biāo)片段進(jìn)行了電泳檢測(cè)，結(jié)果表明，PCR擴(kuò)增成功，且片段大小與目標(biāo)片段一致（圖6-A—圖6-D）。

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

圖2 基于HRM純合A/A（藍(lán)色曲線）和雜合A/T（紅色曲線）位點(diǎn)的SNP鑒定Fig. 2 HRM analysis profile genotyping homozygous A/A (Blue curve) and heterozygous A/T (Red curve)

圖3 基于HRM分析純合G/G（藍(lán)色）和雜合G/A（紅色）位點(diǎn)的SNP分型Fig. 3 HRM analysis profile genotyping homozygous G/G (Blue curve) and heterozygous G/A (Red curve)

圖4 基于HRM分析純合C/C（藍(lán)色）和雜合C/A（紅色）位點(diǎn)的SNP鑒定Fig. 4 HRM analysis profile genotyping homozygous C/C (Blue curve) and heterozygous C/A (Red curve)

2.3 SNP基因分型的驗(yàn)證與遺傳距離分析

圖5 基于HRM分析純合G/G（藍(lán)色）和雜合C/G（紅色）位點(diǎn)的SNP鑒定Fig. 5 HRM analysis profile genotyping homozygous G/G (Blue) and heterozygous C/G (Red)

圖6 PCR擴(kuò)增片段瓊脂糖凝膠電泳圖Fig. 6 PCR amplification fragment profiling of agarose gel electrophoresis

根據(jù)已經(jīng)定位的信息和緊密連鎖的SNP標(biāo)記，選取其中的基因型為AA和AT的SNP驗(yàn)證大量樣品的緊密連鎖的SNP標(biāo)記（Scaffold_3 3450405），該標(biāo)記與目標(biāo)基因連鎖距離為2.11 cM。

3 討論

基因組的某個(gè)位點(diǎn)上單個(gè)核苷酸（A、T、C和G）的變化形成一個(gè)SNP，SNP檢測(cè)錯(cuò)誤率低、分辨率高、世代間全基因組進(jìn)化相對(duì)穩(wěn)定[29]，在遺傳圖譜構(gòu)建方面，SNP標(biāo)記優(yōu)勢(shì)突出[30-31]。特別是隨著基于二代測(cè)序技術(shù)多個(gè)物種基因組測(cè)序的完成，SNP標(biāo)記將逐漸取代SSR、RFLP以及其他分子標(biāo)記[31]。SNP標(biāo)記在植物中分布比較廣泛。VERDE等[32]通過(guò)對(duì)56份桃育種材料進(jìn)行了重測(cè)序，共獲得了1 022 354個(gè)多態(tài)性的SNPs，并采用9K的SNP芯片進(jìn)行了驗(yàn)證。同樣，ARANZANA等[33]對(duì)47個(gè)歐美桃品種23個(gè)基因組片段進(jìn)行測(cè)序，發(fā)現(xiàn)平均每 598堿基存在一個(gè)SNP，每4 189 bp存在一個(gè)Indel。大量SNPs的存在可用于基因的定位、遺傳多樣性評(píng)價(jià)和特征指紋鑒定提供方便。

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

盡管SNP廣泛存在，但由于SNP只有一個(gè)堿基的差異，檢測(cè)相對(duì)困難。Sanger測(cè)序技術(shù)是對(duì)SNP進(jìn)行鑒定的最直接、準(zhǔn)確和信息量最完整的方法[9]，但測(cè)序成本較高、過(guò)程繁雜，同時(shí)無(wú)法對(duì)Poly序列與長(zhǎng)重復(fù)序列等結(jié)構(gòu)進(jìn)行鑒定。而采用高分辨率熔解曲線的SNP分型是通過(guò)實(shí)時(shí)監(jiān)測(cè)升溫過(guò)程中雙鏈DNA熒光染料與PCR擴(kuò)增產(chǎn)物的結(jié)合情況，來(lái)判斷是否存在SNP[34-35]。由于飽和染料與GC-rich和AT-rich區(qū)的親和無(wú)偏好性，HRM不僅可用于分析由SSR、Indel差異引起的多態(tài)性[36]，同時(shí)可以分析由單個(gè)核苷酸差異引起的多態(tài)性。

近幾年，多年生果樹(shù)樹(shù)型研究進(jìn)展較快，特別是蘋果和桃。桃樹(shù)型研究主要集中在株高和節(jié)間長(zhǎng)度方面。YAMAMOTO等[37]將控制桃株高矮化基因定位在LG6上。HOLLENDER等[21]獲得了矮化性狀的基因，明確了矮化性狀的遺傳和調(diào)控機(jī)制。VERDE等[38]采用BC1群體將控制節(jié)間長(zhǎng)度的主效QTL定位于 LG1上。最近，基于二代測(cè)序技術(shù)，DARDICK等[20]采用了 83個(gè)雜交群體單株將控制桃分枝角度的TAC1定位在物理距離2 Mb以內(nèi)。后采用250個(gè)單株，克隆了控制分枝角度的TAC1，并通過(guò)轉(zhuǎn)擬南芥驗(yàn)證了該基因的功能，明確了直立型、柱型的遺傳和調(diào)控機(jī)理，這是首個(gè)克隆的桃株型基因。

本研究建立了基于HRM的SNP基因分型，并應(yīng)用于雜交群體，獲得了緊密連鎖的SNP標(biāo)記，正確區(qū)分了普通生長(zhǎng)型（ST）和溫度敏感半矮生型（Tssd）。HRM分析能夠有效區(qū)分不同SNP位點(diǎn)，可用于大規(guī)模的分子輔助選中體系中。

4 結(jié)論

建立了基于HRM技術(shù)對(duì)4種不同類型的SNP進(jìn)行基因分型，確立了基因分型的DNA模板和影響因子Mg2+的濃度區(qū)間。盡管HRM技術(shù)無(wú)法區(qū)分雙鍵和三鍵堿基間（如A/T和C/G）的變異，但仍不失為區(qū)分已知變異SNP的有效方法。同時(shí)，獲得與桃PpTssd基因緊密連鎖的SNP標(biāo)記，遺傳距為2.11 cM。

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（責(zé)任編輯 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

國(guó)家自然科學(xué)基金（31500558、31470679）、中國(guó)農(nóng)業(yè)科學(xué)院科技創(chuàng)新工程（CAAS-ASTIP-2016-ZFRI）

聯(lián)系方式：魯振華，E-mail：luzhenhua@caas.cn。通信作者王志強(qiáng)，E-mail：wangzhiqiang@caas.cn