分子标记在可可基因库管理方面的应用
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摘要
可可(Theobroma cacao L.)(2n=2x=20)是梧桐科热带常绿植物,它是世界上重要的经济作物之一,其种子(可可豆)是生产巧克力的主要原料,全球年产量约400万吨。当以原始植物材料作为科研领域研究对象时,通常称为"Cacao",而作为栽培植物或是生产原料时,通常称为‘'Cocoa"或"Chocolate"。可可原产于南美洲亚马逊河上游的热带雨林,早在3000多年前就被玛雅人驯化栽培。目前,可可在非洲、东南亚和拉丁美洲等热带地区广泛种植,主要生产国有加纳、巴西、尼日利亚、厄瓜多尔、洪都拉斯、菲律宾和马来西亚等。
一直以来,可可被划分为三大种群,即Criollo、Forastero和Trinitario。其中,Criollo种群原产于中美洲和加勒比地区,是众所周知的“细味(Fine Flavor)"可可豆,是风味最好的巧克力生产原料。Forastero种群则包括多态性较高的野生可可以及最初在南美洲驯化的栽培种,目前主要在非洲西部的加纳和南美洲的巴西广泛栽培,该种群约占世界可可豆总产量的90%左右。而Trinitario种群被认为是由Criollo和Forastero两个种群杂交而来,是在18世纪由于大规模“扫帚病”肆虐而保留下来的具有抗病性状的杂交后代。
目前,全球范围内有两个国际公共领域的可可资源中心,一个是由西印度大学(University of West Indies)的可可研究组主持运作的特立尼达国际可可基因库(International Cacao Genebank, Trinidad)(ICG, T),该可可基因库是在世界上最大的可可资源保护区,收集保存有大约2500株可可树,其中87个株系是收集自南美洲和中美洲的种子繁殖或无性繁殖的种群,许多株系树木是由原苗圃直接目前迁移至此地。因此,这个可可基因库可以提供已分布在世界各地的许多重要的参考基因型。另一个是哥斯达黎加“热带农业研究与高等教育中心”(Centro Agronomico Tropical de Investigaciony Ensenanza, CATIE,英文名称：Tropical Agriculture Research and Higher Education Center)负责维护并管理的“CATIE国际可可资源圃”(International CATIE Cacao Collection)(Turnbulle等,2004)。目前,已经有3500多株可可树被收纳入这两大国际资源中心,并由英国雷丁大学(University of Reading)的国际检疫中心(International Quarantine Center, ICQC)负责病虫害的检验检疫。在2003年,由英国雷丁大学主办,国际可可资源库和可可检疫中心承办,共同建立了国际可可种质资源数据库(Cacao Germplasm Database, ICGD),由法国国际农业发展机构CIRAD承担该数据库的维护和管理工作。
在自然状态下保存的可可种子,其生活力较低,若在低温条件下长期保存,其种子生活力会完全丧失。因此,可可资源的异地保存主要采取扦插、分蘖等无性繁殖的方式。这种保存方式为可可异地资源库的建立和管理带来了很多新的问题。其中,最主要的问题就是容易出现标签挂错或标签丢失的现象,从而造成在可可资源的农艺性状的评价、鉴定以及育种应用等工作上的混乱与失误,为资源学研究造成财力、物力和人力等方面的浪费。据不完全估计,目前国际上几个主要可可异地保存资源库的标签挂错率平均在30%左右,个别库甚至高达40%。因此,亟需对目前可可基因库中的资源进行种质鉴定和遗传多样性分析,从而指导可可资源的保存与管理工作。
针对以上问题,本论文的研究内容包括如下三个相对独立的试验,共同证明如何利用分子标记手段有效解决可可资源保存与管理中的实际问题,为其他物种基因库管理提供重要的范例。
(1)利用SSR标记进行Trinitario种群的遗传多样性分析及遗传信息鉴定
为了更深入的探讨Trinitario种群的来源与遗传关系,本研究通过SSR(Single Sequence Repeat,简单重复序列)分子标记手段,在核苷酸水平上分析农家栽培驯化Trinitario可可种群的遗传多样性和种群结构,推测特定栽培群体中各个体之间的亲缘关系,鉴定并验证父母本植株与杂交子代植株,核实个体标签与植株是否对应,剔除重复标签并对错挂的标签进行更正,以保证所挂标签真实可靠,对于可可种质资源基因库的维护并管理具有重要的意义。
本研究以特立尼达群岛国际可可基因库中保存的Trinitario杂交种群中的59个特立尼达皇家学院选择系(Imperial College Selection) ICS系列品种为研究对象,利用美国农业部农业研究中心多年生植物试验室前期筛选出的15个稳定高效的SSR标记分析该种群的遗传多样性。试验采摘新鲜健康的叶片,经简单硅胶干燥处理,作为提取DNA的试验样品,使用DNeasy Plant试剂盒(Qiagen Inc)提取高纯度基因组DNA。15对SSR引物由科罗拉多州Proligo生物公司合成,5’-正向引物由加利福尼亚州Beckman公司使用WellRED荧光染料进行标记。引物使用Invitrogen公司SuperMix配制热启动PCR supermixes进行PCR扩增。利用Beckman Coulter公司生产的CEQTM8800遗传分析系统,将扩增的PCR产物,利用毛细管电泳分离。根据试验仪器参数设置建议进行数据的准备处理,使用CEQTM8800片段分析软件7.0.55版本进行数据分析,该分析系统将自动处理SSR片段的大小计算到小数点后两位,并采用该系统分级导向软件进行等位基因的分级区划。运用GIMLET软件进行基因型匹配的重复鉴定,并通过贝叶斯分配测试。使用Structure V2.0进行聚类分析,该程序采用了全贝叶斯方法,运用程序Geneclass进行经典基因频率和贝叶斯分配的测试。
研究结果表明,在供试材料中,ICS2、ICS83和ICS95三个株系的个体基因型在15个等位基因位点完全匹配,通过检查取样地点记录比对树木的相对位置,判断认为这三个株系编号的株系应当更正为同一标签编号,是存在标签贴错的工作失误,从而纠正并完善该可可树木基因库的管理与记录。而且,仅由mTcCIR211、mTcCIR15、mTcCIR40、mTcCIR37、mTcCIR24、mTcCIR33和mTcCIR12等7个位点就可以基本分辨是否为同一株系的重复。PowerMarker绘制聚类树状图种群结构的分析结果显示,供试样品中ICS1、ICS10、ICS35、 ICS57、ICS77、ICS80和ICS100等8个株系可能是由于在异地保存中贴错标签而混入ICS种群中的个体。
由于SSR标记位点的限制性,对于确定种群内的遗传多样性并不能明确鉴定,因而本论文对新型的SNP标记在可可种群鉴定及亲缘关系研究中应用进行了探讨。
(2)利用SNP标记鉴定农家可可品种的遗传多样性和亲缘关系
SNP标记检测可以不需要分离DNA的大小,直接实现高通量的自动化检测模式。而且等位基因的单核苷酸多态性鉴定的失误率极低。虽然SNP标记已广泛应用于植物品种识别许多其它作物,但在可可基因型鉴定和多样性评估等方而尚无报道。本研究旨在筛选最小数量的的SNP标记组合从而实现大规模基因分型,并将成为可可基因库的管理维护的重要手段于有力的科学依据。
本论文的第二部分内容即探讨了采用SNP标记对中南美洲洪都拉斯和尼加拉瓜地区的可可资源进行种群多样性鉴定以及种群内株系之间是否存在杂交父母本与子代亲缘关系的判定与研究。供试样品包含来自洪都拉斯与尼加拉瓜的84个农家品系,另有来自国际可可基因库的31个已知明确遗传身份的参考系克隆共同参与多态性分析,参考系拥有已知的SSR分子标记结果与DNA指纹图谱,可以作为本研究中参考系种质资源分组的依据。这些种质主要收集自洪都拉斯和尼加拉瓜地区的一些非官方的农业研究机构,以及生产精品巧克力的私营公司的专属可可种植规划区,在巧克力生产行业领域内被认为是风味优良的种群,其中大部分的供试株系是由多家巧克力工厂拥有该资源种群的相关背景。样品的收集处理与DNA提取方法基本与前章试验流程相同。在覆盖可可树整个基因组的10条染色体的1560个候选单核苷酸多态性位点中,本研究选取其中100个多态性位点,使用MALDI-TOF质谱分析仪(Sequenom)在华盛顿大学人类遗传学部门核心实验室进行SNP基因分型的试验。
研究结果显示,在这100个SNP标记位点中,有4个SNP标记均在115株可可样品单株中呈现单态性存在,另有26个SNP标记在的基因序列数据存在15%以上的缺失数据,可能是DNA质量偏低的原因造成不能呈现完整的标记结果。将剩余的70个SNP标记提供了有效的数据结果进行分析表明,84个农家品系与31个参考系克隆的结果相比,平均观测杂合度以及平均期望杂合度呈现小幅度偏低,近缘杂交系数略高,但整体的遗传多样性水平基本相当。通过对样品进行配对检测以及同胞可能性检验(probability of identity, PID),其中有16个样品存在重复,并可以据检测结果将这16个材料分为6个同义克隆小组中,有3个小组与参考克隆Criollo13、Criollo22以及Amelonado15完全匹配,为同义重复样品。其中,农家采集的可可品种的总体数据显示,存在11.9%以上的重复概率。
本研究采用主座标轴分析确定遗传距离,并通过聚类分析探讨亲缘关系,且两种方法都可以将供试的115个克隆清晰的分成五个组。第一组是完全由古老的Criollo构成,但同时小组之间也存在有微小的多态性差异；第二组是来自尼加拉瓜的农家杂交Trinitario,存在明显的遗传多样性；第三组仅包括Lower Amazon Forastero克隆,与参考对照系中的Amelonado克隆(Amelonado15, Amelonado22, SIAL325)具有相当高的遗传相似性；第四组主要由来自洪都拉斯的Trinitario杂交品种,并且与第三组中的来自尼加拉瓜的Trinitario种群存在较大遗传差距,反而它们更接近参考系克隆中的Upper Amazon Forastero以及厄瓜多尔的Ecuadorian Nacional杂交系(CLM78和JA10/33),同时也表明这一组的成员不属于传统经典意义上的Trinitario杂交系,而且很大的可能性是由于Upper Amazon Forastero的基因渗入的结果；第五组的成员包括所有供试参考系,包括Upper Amazon Forastero克隆以及Ecuadorian Nacional杂交系克隆。
在本研究采用的供试样品中有53个Trinitario型农家栽培品种,其中28个样品得到了确认的亲本身份,其置信水平为80%。此外,待定亲本与五个参考系克隆的亲缘关系分析结果显示,Amelonado与23个农家栽培品种和一个参考系克隆TRD86具有亲缘关系,是它们的共同亲本。而古老的Criollo并未作为杂交亲本参与农家栽培品种的遗传学贡献。但是Criollo与来自尼加拉瓜的Matagalpa34具有直接亲缘关系,作为遗传亲本的置信水平达80%。与此同时,Criollo也被确定对于参考系Trinitario克隆OC77、ICS95和ICS39的亲本身份,置信水平为95%,同时被鉴定作为RIM113的杂交亲本,置信水平80%。另外,四个Upper Amazon Forastero克隆IMC27、IMC47、IMC63以及JA10/33被确定为农家栽培品种以及参考系克隆ICS97的可能亲本。聚类分析的结果作为亲本与子代的遗传关系鉴定的主要依据,在聚类树状图中,栽培品种作为杂交子代遗传关系较近,并且具有相同的亲本来源。以SNP基因分型数据的聚类树状分析结果显示,28株样品树被重新命名标记为Criollo,其中,22株极可能来源于古老Criollo的基因型,其余三分之二的样品与参考系Trinitario参考系克隆聚类在同一组,认为是具有尼加拉瓜Trinitario的基因型。
(3)利用SNP标记鉴定加纳可可种群的遗传多样性
本论文最后一部分的研究内容主要是致力于探求以较少数目的SNP多态性位点标记,准确地识别个体的基因型差异,这对于种质资源保护和种子传播等工作的高效进行以及节约科研成本都是有重要的意义。SNP标记的高通量基因分型以及绘制指纹图谱的功能特点,可以应用于大规模可可的遗传信息识别与基因分型,是高效快速提供可可基因身份证明的强大准确的工具。
根据第二部分的研究结果,筛选54个SNP位点作为用于本部分研究的标记手段,验证最少位点数目在可可品种鉴定工作中的可行性与可靠性。
本部分研究试验材料收集自位于非洲的加纳可可研究中心所保存的种质资源(Cocoa Research Institute of Ghana, CRIG),材料来自8个种植园的160株可可树,选取新鲜嫩叶作为供试材料。另外选择已知遗传身份的36个材料作为参考系克隆,并且加入来自特立尼达国际可可基因库的29个参考系克隆,共同参与该部分的检测与分析研究。提取DNA样品并运用NanoDrop分光光度仪测定浓度,方法流程与第一、二部分试验基本一致。基于前一章的SNP检测结果,在覆盖整个可可基因组10个染色体水平的多态性及其分布的1560个SNP位点中选取54个标记作为本研究的候选SNPs,在华盛顿大学圣路易斯分校人类遗传学部试验室,利用MALDI-TOF质谱(Sequenom)仪器进行基因分型检测。
结果显示,通过多位点匹配统计检测,确定了NA79、PA150和IMC76等三个株系的标签不正确,同时AMAZ-2和PA303克隆呈现高程度匹配,推测是同一克隆而挂错了标签。被用作参考系克隆中,拥有相同的名称但在SNP标记结果中的多个位点呈现不同的结果。本研究中认定,存在5个以上位点不相互匹配,即应当挂上不同的标签,在名称或编号上有所区分。通过将所有的54个位点的检测分析,同时存在多个有差异位点的概率是10-6。总体而言,多位点匹配的统计检测发现在供试的160株样品中,有149株可以与参考系克隆的基因型相匹配,植株所对应的标签真实准确。同时,将单独取样的39个样本的SNP多态性结果进行种群结构和亲缘族谱关系分析,软件STRUCTURE统计结果表示,该种群最有可能的遗传基因型数目为K=5。来自加纳的39个可可样品,以及29个参考系克隆共同被分作Amelonado、IMC、SCA/Ucayali、Morona、Nanay和Parinari等6个群体,这与前人研究的结果相互印证。但是SCA/Ucayali和Morona种质类群在本研究中得到分离。包含NA32和NA34在内的PA和IMC父母本明显对于T60、T63、T65和T79等株系有遗传学贡献,更加进一步验证T85/799的父母本分别为IMC60与NA34。另外IMC和Amelonado与T16/613家族相关.证实前人研究IMC24为亲本之一,同时本研究结果表明,亲本另一方是来自于Amelonado种质类群。通过亲缘关系鉴定与分析,在95%的置信水平上基础上,确定了6对杂交父母亲本组合,在80%的置信水平上确定了一对杂交父母亲本组合。STRUCTURE软件对父母本子代关系进行聚类分析,同样支持了这一结果。实验表明,本研究所采用的54个SNP位点是稳定而可靠的,其误差率不高于10%,同时,通过这54个SNP标记结果的描述性统计分析,在44个供试样品中具有独特SNP基因型的平均期望杂合度为0.336,实际检测杂合度为0.269。在实地观察工作中得到的平均亲近系数是0.218。通过亲缘鉴定和模型分配,以参考对照系的已知遗传身份为依据,验证杂交记载亲本的身份并验证杂交亲本的遗传背景。研究表明,仅采用一小组多态性SNP标记对可可基因型鉴定,就可以提供足够的遗传信息进行基因分型。这个检测体系可以适用于大规模进行可可基因分型。
综上所述,本研究采用SSR分子标记技术对于特立尼达群岛的国际可可资源库的Trinatario杂交可可ICS种群进行标签缺失或重复鉴定,确定并更正该种群的部分株系在异地保存过程中标签挂错。本研究首次采用SNP单核苷酸多态性标记,对可可种质资源进型多态性鉴定,在单核苷酸水平上的多态性证明该技术将是国际可可资源基因库的管理维护的重要手段,可以实现在短时间内高通量筛选大量单核甘酸位点进行农家栽培品种及野生可可资源的遗传学鉴定。首次采用SNP标记,以单核苷酸多态性标记作为可靠依据,鉴定不同标签编号但是是同一品种,或是相同标签编号但实质是不同品种等历史上资源异地保存的遗留失误问题等,为分布于世界各地的可可种质资源进行归类整理提供有效的科技手段。首次采用SNP标记技术,更加明确的阐述可可作为巧克力产业的原材料,部份可可栽培品种的杂交父母本来源一直以来存在的争议问题。采用单核苷酸多态性分辨鉴定父母本与杂交子代的亲缘关系,为可可国际基因库的维护与完善提供可靠而高效的鉴定手段。
Cacao (Theobroma cacao L.)(2n=2x=20), is an important Sterculiaceae tropical evergreen plant origin from Central and South America, as early as3,000years ago was domesticated and cultivated by the Mayans. Now it is widely planted in Africa, Southeast Asia and Latin America. Cacao is one of the world's major crops, cocoa beans, are the main resource of chocolate industry, with the global annual production about4million tons. The world's major cocoa growing areas cultivated mainly in Latin America, South America, West Africa, Southern Asia and other tropical along the equator, but this region is also important for biodiversity conservation areas.
The conservation of cacao germplasm requires maintaining living trees in genebanks within tropical regions. There are two international public cacao collection, one is the International Cacao Genebank, Trinidad (ICG, T) and the other is International CATIE Cacao Collection (IC3). ICG, T is managed by the Cocoa Research Unit (CRU) of the University of West Indies and IC3by the Centro Agronomico Tropical de Investigacion y Ensenanza (CATIE) of Costa Rica (Beakele and Beakele,1996). Germplasm held in these two international collections is available for breeders via the international quarantine center located at the University of Reading in UK. These genebanks are supported by both public and industrial funding. Information of cacao germplasm held in these international collections is currently keeping in the International Cacao Germplasm Database (ICGD) which is hosted by the University of Reading in UK, as well as Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement (CIRAD) in France.
Since cocoa seed can not be stay alive for a long time, either to be stored under low temperature conditions. Therefore, only in the field of cocoa germplasm vivo preservation. This kind of offsite mode management has brought many problems. One major problem is mislabeling or label lost will cause to the confusion in evaluation, identification and breeding process of cocoa. The major rate of mislabeling within current international cocoa offsite repository is around30%to40%by average. The most urgent task of cocoa Repository Management is to correct the mislabeling in cacao collection fileds. That will be great help to assist documented pedigree and evaluation of genetic variaties.
It is commonly accepted that there are three groups of cocoa germplasm. The first group is Criollo, which was originally distributed in Central America and the Caribbean, and is well known for its "fine-flavor" beans. The second group is Forastero, which includes diverse wild and more recently cultivated populations originally from South America. Today Forastero is widely grown in West Africa and Brazil, and accounts for approximately90%of the cocoa beans produced in the world. Finally, there is the Trinitario group, which is believed to be the result of hybridizations between Criollo and Forastero types in the18th century. The original hybrid zone is the island of Trinidad.
The SSR (Simple Sequence Repeat) polymorphic markers and SNP (Single Nucleotide Polymorphism) markers are used to explore diverse populations of the best molecular identification means, and provide accurate and reliable molecular basis for the correction of mislabeling germplasm for theoretical guidance and support.
Experiment I. Assessing genetic identity and population structure of Trinitario cocoa germplasm using microsatellite markers
In this experiment, SSR markers were used to characterize a Trinitario cacao collection in the International Genebank in Trinidad. Trinitario cocoa is indigenous to the island of Trinidad and is believed to have had a very small number of founding parents in the18th century. However, rarely known about the individual identity and population structure in this group of cocoa germplasm.
This study was aimed at assessing the population structure and the extent of mislabeling in an indigenous Trinitario collection, and tested the hypothesis that the Trinitario were founded by a few progenitors. Multilocus microsatellite data were generated using15pairs of SSR primers. We used a capillary electrophoresis as previously described (Saunders et al,2004) using a CEQTM8000genetic analysis system (Beckman Coulter Inc.). Data analysis was performed using the CEQTM8000Fragment Analysis software version7.0.55according to manufacturers" recommendations (Beckman Coulter Inc). SSR fragment sizes were automatically calculated to two decimal places by the CEQTM8000Genetic Analysis System. Allele calling was performed using the CEQTM8000bining wizard software (CEQTM8000software version7.0.55, Beckman Coulter Inc.).
Synonymous mislabeling was identified by matching multilocus genotypes among individuals. Population structure was assessed by a Bayes method of sample partition. Heterozygosity deficiency was estimated as indication of recent population bottleneck. All microsatellite loci were polymorphic, with the number of alleles per locus ranging from2to10(mean=5.8). The mean expected heterozygosity ranged from0.307to0.656, with an average of0.552. The multi-locus fingerprints unambiguously identified all59individuals and three of them were found to be synonymous. Probability of identity predicted that the top7loci were necessary, yet sufficient, to distinguish with greater than99%certainty between the Trinitario individuals. Wilcoxon and Sign tests showed no heterozygosity excess. The distance-based cluster analysis, multi-dimensional scaling based on genetic relatedness, and Bayesian cluster method all showed that there is population admixture in the ICS Trinirario germplasm.
Analysis showed, ICS2, ICS83and ICS95three clones in15alleles are exactly match, by checking the relative position and compared to trees, it is determined that this three should be correct as the same lable. Moreover, only by the seven loci mTcCIRll, mTcCIR15, mTcCIR40, mTcCIR37, mTcCIR24, mTcCIR33and mTcCIR12can tell whether the same basic lines repetition. Population structure analysis was results showed by PowerMarker that the samples ICS1, ICS10, ICS35, ICS46, ICS57, ICS77, ICS80and ICS100other eight clones may be due to off-site preservation mislabeled and mixed ICS groups of individuals.
It shows that SSR fingerprinting is highly efficient to identify cacao accessions. We demonstrated that the Trinitario cocoa is comprised of more than one source population thus should not be treated as a single uniform genetic group in conservation and breeding.
Experiment II. Genetic diversity and parentage in farmer varieties of cacao from Honduras and Nicaragua as revealed by single nucleotide polymorphism (SNP) markers
Although SSR markers are efficient tool for cacao germplasm characterization, this system has several drawbacks for large scale DNA fingerprinting. Compared to SSR markers, SNP assays can be done without separating DNA by size, and therefore can be automated in high throughput assay format. The di-allelic nature of SNPs offers a much lower error rate in allele calling. Moreover, there is a higher consistency in allele calling across laboratories. While SNP markers have been widely used in plant varietal identification in many other crops, the efficacy of using SNP markers for cacao genotype identification and diversity assessment remain to be investigated. Large scale genotyping using a small set of SNP markers is still in great demand by the cacao community for a broad range of research and field applications.
Experiment Ⅲ. Verification of genetic identity of introduced cacao germplasm in Ghana using single nucleotide polymorphism (SNP) markers
Cocoa is an important global commodity with an annual production that exceeded4million tons in2010, of which75%was produced in West Africa. Ghana alone produced850,000tons of cacao, accounting for21%of the world's total output in2010(FAOSTAT, http://faostat3.fao.org/home/index.html).
In the present study, high-throughput genotyping with SNP markers was used to fingerprint160cacao trees in the CRIG germplasm collection. These accessions had been originally introduced from international germplasm collections. The study also included100international clonesas references. DNA concentration was determined with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). Based on the level of polymorphism and on their distribution across the ten chromosomes in cacao,54SNP markers were selected from1560candidate SNPs that had been developed using cDNA sequences from a wide range of cacao organs (Argout et al.2008). SNP genotyping was performed at the Human Genetics Division Genotyping Core facility, Washington University, St. Louis, using MALDI-TOF mass spectrometry (Sequenom, Inc., San Diego, CA).
Key descriptive statistics for measuring the infonnativeness of the SNP markers were calculated, including observed heterozygosity, expected heterozygosity, minor allele frequency, Inbreeding coefficient and probability of identity (Evett and Weir1998; Waits et al.2001). The program GenAlEx6.2(Peakall and Smouse2006;2012) was used for computation.
For the identification of mislabeling (off-type), SNP profile of100reference trees maintained in the International Cacao Genebank, Trinidad (ICG,T) were used in the analysis. The genetic identity of the100reference trees has been characterized by both SNP (Zhang, personal communication and SSR fingerprinting (Zhang et al.2009b; Motilal et al.2010; Johnson et al.2009). Pairwise multilocus matching was applied among each pair of individual trees, including the reference trees from the International germplasm collections, using the same program. Accessions with same names as the reference trees, but not matching them, were declared off-types. For the multilocus matching, the option to ignore missing data was used. Discriminating power of the SNP loci was computed using the probability of identity (PID, Waits et al.2001) option implemented in the same computer program.
The multilocus SNP profiles, generated by the Sequenom Mass Spectrometry platform, were compared with the SNP profiles of reference trees maintained in the international cacao collections. Comparison of the multilocus SNP profiles with the reference accessions identified seven intraclonal mislabelings in accessions NA79, PA150and IMC76. The multilocus matching also found that AMAZ-2and PA303were mislabeled. With all53loci considered, the combined probability of identity was in the order of10-9. Overall, the procedure of multilocus matching with known reference trees led to the identification of149true-to-type trees out of160tested samples. Based on the value of delta K, the model-based approach of STRUCTURE indicated K=5as the most probable number of genetic clusters. The tested cacao accessions from the Ghana cacao collection, as well as the100reference accessions were stratified as germplasm groups of Amelonado, IMC, SCA/Ucayali, Morona, Nanay and Parinari respectively. The assigned memberships for all the tested trees from Ghana were compatible with their known parentage germplasm groups. The assignment test of the T clones confirmed their recorded parental germplasm groups. The parental group of PA, IMC (which includes parental clones NA32and NA34) were clearly reflected in the admixed ancestry profiles of T60, T63, T65and T79. A full genetic background of IMC was revealed for accession T85/799, supporting its recorded parentage of IMC60and NA34(a member of the IMC germplasm group; Motamayor et al.2008). In addition, admixed ancestry of IMC and Amelonado was revealed for T16/613family, which not only supported the recorded parentage of IMC24, but also detected that the other parent came from the Amelonado group.
Of the eight candidate parent-offspring relationships, the results of parentage inference confirmed six pairs at the95%confidence level and one pair (NA34-T80/799) at the80%confidence level (Table4). For offspring T16/613, only one parent (Amelonado22) was identified at the>80%confidence level because the reference genotype of maternal parent IMC24was not available. The result of parent-offspring assignment supported the outcome of model-based clustering analysis by the STRUCTURE program.
The comparison unambiguously identified mislabeled trees. For materials introduced as hybrid seeds without an available reference genotype, parentage analysis and model-based assignment were applied to verify their recorded parentage and genetic background. Our study shows that a small set of polymorphic SNP markers can provide a robust and accurate result for cacao genotype identification. This protocol can be applied for large-scale genotyping of cacao as well as for many other crops.
In summary, it shows that SSR fingerprinting is highly efficient to identify cacao accessions Trinatario from International Cocoa Cocoa ICS collection through this experiment. We demonstrated that the Trinitario cocoa is comprised of more than one source population thus should not be treated as a single uniform genetic group in conservation and breeding duplicate identification. The first study using single nucleotide polymorphism SNP markers for cocoa germplasm into polymorphism identified in single nucleotide polymorphisms prove that the level of technology will be the International Cocoa Genebank resource management and maintenance an important tool that can be achieved in a short time for a large number of high-throughput screening of single nucleotide loci cultivars, as well as wild cocoa farm genetic resources identified. For the first time using SNP markers, as a reliable basis for identification of mislabling and other historical legacy of offsite resources issues. Identification of single nucleotide polymorphisms to distinguish parental genetic relationship with hybrids and provide reliable and efficient identification for cacao world collections.
一直以来,可可被划分为三大种群,即Criollo、Forastero和Trinitario。其中,Criollo种群原产于中美洲和加勒比地区,是众所周知的“细味(Fine Flavor)"可可豆,是风味最好的巧克力生产原料。Forastero种群则包括多态性较高的野生可可以及最初在南美洲驯化的栽培种,目前主要在非洲西部的加纳和南美洲的巴西广泛栽培,该种群约占世界可可豆总产量的90%左右。而Trinitario种群被认为是由Criollo和Forastero两个种群杂交而来,是在18世纪由于大规模“扫帚病”肆虐而保留下来的具有抗病性状的杂交后代。
目前,全球范围内有两个国际公共领域的可可资源中心,一个是由西印度大学(University of West Indies)的可可研究组主持运作的特立尼达国际可可基因库(International Cacao Genebank, Trinidad)(ICG, T),该可可基因库是在世界上最大的可可资源保护区,收集保存有大约2500株可可树,其中87个株系是收集自南美洲和中美洲的种子繁殖或无性繁殖的种群,许多株系树木是由原苗圃直接目前迁移至此地。因此,这个可可基因库可以提供已分布在世界各地的许多重要的参考基因型。另一个是哥斯达黎加“热带农业研究与高等教育中心”(Centro Agronomico Tropical de Investigaciony Ensenanza, CATIE,英文名称：Tropical Agriculture Research and Higher Education Center)负责维护并管理的“CATIE国际可可资源圃”(International CATIE Cacao Collection)(Turnbulle等,2004)。目前,已经有3500多株可可树被收纳入这两大国际资源中心,并由英国雷丁大学(University of Reading)的国际检疫中心(International Quarantine Center, ICQC)负责病虫害的检验检疫。在2003年,由英国雷丁大学主办,国际可可资源库和可可检疫中心承办,共同建立了国际可可种质资源数据库(Cacao Germplasm Database, ICGD),由法国国际农业发展机构CIRAD承担该数据库的维护和管理工作。
在自然状态下保存的可可种子,其生活力较低,若在低温条件下长期保存,其种子生活力会完全丧失。因此,可可资源的异地保存主要采取扦插、分蘖等无性繁殖的方式。这种保存方式为可可异地资源库的建立和管理带来了很多新的问题。其中,最主要的问题就是容易出现标签挂错或标签丢失的现象,从而造成在可可资源的农艺性状的评价、鉴定以及育种应用等工作上的混乱与失误,为资源学研究造成财力、物力和人力等方面的浪费。据不完全估计,目前国际上几个主要可可异地保存资源库的标签挂错率平均在30%左右,个别库甚至高达40%。因此,亟需对目前可可基因库中的资源进行种质鉴定和遗传多样性分析,从而指导可可资源的保存与管理工作。
针对以上问题,本论文的研究内容包括如下三个相对独立的试验,共同证明如何利用分子标记手段有效解决可可资源保存与管理中的实际问题,为其他物种基因库管理提供重要的范例。
(1)利用SSR标记进行Trinitario种群的遗传多样性分析及遗传信息鉴定
为了更深入的探讨Trinitario种群的来源与遗传关系,本研究通过SSR(Single Sequence Repeat,简单重复序列)分子标记手段,在核苷酸水平上分析农家栽培驯化Trinitario可可种群的遗传多样性和种群结构,推测特定栽培群体中各个体之间的亲缘关系,鉴定并验证父母本植株与杂交子代植株,核实个体标签与植株是否对应,剔除重复标签并对错挂的标签进行更正,以保证所挂标签真实可靠,对于可可种质资源基因库的维护并管理具有重要的意义。
本研究以特立尼达群岛国际可可基因库中保存的Trinitario杂交种群中的59个特立尼达皇家学院选择系(Imperial College Selection) ICS系列品种为研究对象,利用美国农业部农业研究中心多年生植物试验室前期筛选出的15个稳定高效的SSR标记分析该种群的遗传多样性。试验采摘新鲜健康的叶片,经简单硅胶干燥处理,作为提取DNA的试验样品,使用DNeasy Plant试剂盒(Qiagen Inc)提取高纯度基因组DNA。15对SSR引物由科罗拉多州Proligo生物公司合成,5’-正向引物由加利福尼亚州Beckman公司使用WellRED荧光染料进行标记。引物使用Invitrogen公司SuperMix配制热启动PCR supermixes进行PCR扩增。利用Beckman Coulter公司生产的CEQTM8800遗传分析系统,将扩增的PCR产物,利用毛细管电泳分离。根据试验仪器参数设置建议进行数据的准备处理,使用CEQTM8800片段分析软件7.0.55版本进行数据分析,该分析系统将自动处理SSR片段的大小计算到小数点后两位,并采用该系统分级导向软件进行等位基因的分级区划。运用GIMLET软件进行基因型匹配的重复鉴定,并通过贝叶斯分配测试。使用Structure V2.0进行聚类分析,该程序采用了全贝叶斯方法,运用程序Geneclass进行经典基因频率和贝叶斯分配的测试。
研究结果表明,在供试材料中,ICS2、ICS83和ICS95三个株系的个体基因型在15个等位基因位点完全匹配,通过检查取样地点记录比对树木的相对位置,判断认为这三个株系编号的株系应当更正为同一标签编号,是存在标签贴错的工作失误,从而纠正并完善该可可树木基因库的管理与记录。而且,仅由mTcCIR211、mTcCIR15、mTcCIR40、mTcCIR37、mTcCIR24、mTcCIR33和mTcCIR12等7个位点就可以基本分辨是否为同一株系的重复。PowerMarker绘制聚类树状图种群结构的分析结果显示,供试样品中ICS1、ICS10、ICS35、 ICS57、ICS77、ICS80和ICS100等8个株系可能是由于在异地保存中贴错标签而混入ICS种群中的个体。
由于SSR标记位点的限制性,对于确定种群内的遗传多样性并不能明确鉴定,因而本论文对新型的SNP标记在可可种群鉴定及亲缘关系研究中应用进行了探讨。
(2)利用SNP标记鉴定农家可可品种的遗传多样性和亲缘关系
SNP标记检测可以不需要分离DNA的大小,直接实现高通量的自动化检测模式。而且等位基因的单核苷酸多态性鉴定的失误率极低。虽然SNP标记已广泛应用于植物品种识别许多其它作物,但在可可基因型鉴定和多样性评估等方而尚无报道。本研究旨在筛选最小数量的的SNP标记组合从而实现大规模基因分型,并将成为可可基因库的管理维护的重要手段于有力的科学依据。
本论文的第二部分内容即探讨了采用SNP标记对中南美洲洪都拉斯和尼加拉瓜地区的可可资源进行种群多样性鉴定以及种群内株系之间是否存在杂交父母本与子代亲缘关系的判定与研究。供试样品包含来自洪都拉斯与尼加拉瓜的84个农家品系,另有来自国际可可基因库的31个已知明确遗传身份的参考系克隆共同参与多态性分析,参考系拥有已知的SSR分子标记结果与DNA指纹图谱,可以作为本研究中参考系种质资源分组的依据。这些种质主要收集自洪都拉斯和尼加拉瓜地区的一些非官方的农业研究机构,以及生产精品巧克力的私营公司的专属可可种植规划区,在巧克力生产行业领域内被认为是风味优良的种群,其中大部分的供试株系是由多家巧克力工厂拥有该资源种群的相关背景。样品的收集处理与DNA提取方法基本与前章试验流程相同。在覆盖可可树整个基因组的10条染色体的1560个候选单核苷酸多态性位点中,本研究选取其中100个多态性位点,使用MALDI-TOF质谱分析仪(Sequenom)在华盛顿大学人类遗传学部门核心实验室进行SNP基因分型的试验。
研究结果显示,在这100个SNP标记位点中,有4个SNP标记均在115株可可样品单株中呈现单态性存在,另有26个SNP标记在的基因序列数据存在15%以上的缺失数据,可能是DNA质量偏低的原因造成不能呈现完整的标记结果。将剩余的70个SNP标记提供了有效的数据结果进行分析表明,84个农家品系与31个参考系克隆的结果相比,平均观测杂合度以及平均期望杂合度呈现小幅度偏低,近缘杂交系数略高,但整体的遗传多样性水平基本相当。通过对样品进行配对检测以及同胞可能性检验(probability of identity, PID),其中有16个样品存在重复,并可以据检测结果将这16个材料分为6个同义克隆小组中,有3个小组与参考克隆Criollo13、Criollo22以及Amelonado15完全匹配,为同义重复样品。其中,农家采集的可可品种的总体数据显示,存在11.9%以上的重复概率。
本研究采用主座标轴分析确定遗传距离,并通过聚类分析探讨亲缘关系,且两种方法都可以将供试的115个克隆清晰的分成五个组。第一组是完全由古老的Criollo构成,但同时小组之间也存在有微小的多态性差异；第二组是来自尼加拉瓜的农家杂交Trinitario,存在明显的遗传多样性；第三组仅包括Lower Amazon Forastero克隆,与参考对照系中的Amelonado克隆(Amelonado15, Amelonado22, SIAL325)具有相当高的遗传相似性；第四组主要由来自洪都拉斯的Trinitario杂交品种,并且与第三组中的来自尼加拉瓜的Trinitario种群存在较大遗传差距,反而它们更接近参考系克隆中的Upper Amazon Forastero以及厄瓜多尔的Ecuadorian Nacional杂交系(CLM78和JA10/33),同时也表明这一组的成员不属于传统经典意义上的Trinitario杂交系,而且很大的可能性是由于Upper Amazon Forastero的基因渗入的结果；第五组的成员包括所有供试参考系,包括Upper Amazon Forastero克隆以及Ecuadorian Nacional杂交系克隆。
在本研究采用的供试样品中有53个Trinitario型农家栽培品种,其中28个样品得到了确认的亲本身份,其置信水平为80%。此外,待定亲本与五个参考系克隆的亲缘关系分析结果显示,Amelonado与23个农家栽培品种和一个参考系克隆TRD86具有亲缘关系,是它们的共同亲本。而古老的Criollo并未作为杂交亲本参与农家栽培品种的遗传学贡献。但是Criollo与来自尼加拉瓜的Matagalpa34具有直接亲缘关系,作为遗传亲本的置信水平达80%。与此同时,Criollo也被确定对于参考系Trinitario克隆OC77、ICS95和ICS39的亲本身份,置信水平为95%,同时被鉴定作为RIM113的杂交亲本,置信水平80%。另外,四个Upper Amazon Forastero克隆IMC27、IMC47、IMC63以及JA10/33被确定为农家栽培品种以及参考系克隆ICS97的可能亲本。聚类分析的结果作为亲本与子代的遗传关系鉴定的主要依据,在聚类树状图中,栽培品种作为杂交子代遗传关系较近,并且具有相同的亲本来源。以SNP基因分型数据的聚类树状分析结果显示,28株样品树被重新命名标记为Criollo,其中,22株极可能来源于古老Criollo的基因型,其余三分之二的样品与参考系Trinitario参考系克隆聚类在同一组,认为是具有尼加拉瓜Trinitario的基因型。
(3)利用SNP标记鉴定加纳可可种群的遗传多样性
本论文最后一部分的研究内容主要是致力于探求以较少数目的SNP多态性位点标记,准确地识别个体的基因型差异,这对于种质资源保护和种子传播等工作的高效进行以及节约科研成本都是有重要的意义。SNP标记的高通量基因分型以及绘制指纹图谱的功能特点,可以应用于大规模可可的遗传信息识别与基因分型,是高效快速提供可可基因身份证明的强大准确的工具。
根据第二部分的研究结果,筛选54个SNP位点作为用于本部分研究的标记手段,验证最少位点数目在可可品种鉴定工作中的可行性与可靠性。
本部分研究试验材料收集自位于非洲的加纳可可研究中心所保存的种质资源(Cocoa Research Institute of Ghana, CRIG),材料来自8个种植园的160株可可树,选取新鲜嫩叶作为供试材料。另外选择已知遗传身份的36个材料作为参考系克隆,并且加入来自特立尼达国际可可基因库的29个参考系克隆,共同参与该部分的检测与分析研究。提取DNA样品并运用NanoDrop分光光度仪测定浓度,方法流程与第一、二部分试验基本一致。基于前一章的SNP检测结果,在覆盖整个可可基因组10个染色体水平的多态性及其分布的1560个SNP位点中选取54个标记作为本研究的候选SNPs,在华盛顿大学圣路易斯分校人类遗传学部试验室,利用MALDI-TOF质谱(Sequenom)仪器进行基因分型检测。
结果显示,通过多位点匹配统计检测,确定了NA79、PA150和IMC76等三个株系的标签不正确,同时AMAZ-2和PA303克隆呈现高程度匹配,推测是同一克隆而挂错了标签。被用作参考系克隆中,拥有相同的名称但在SNP标记结果中的多个位点呈现不同的结果。本研究中认定,存在5个以上位点不相互匹配,即应当挂上不同的标签,在名称或编号上有所区分。通过将所有的54个位点的检测分析,同时存在多个有差异位点的概率是10-6。总体而言,多位点匹配的统计检测发现在供试的160株样品中,有149株可以与参考系克隆的基因型相匹配,植株所对应的标签真实准确。同时,将单独取样的39个样本的SNP多态性结果进行种群结构和亲缘族谱关系分析,软件STRUCTURE统计结果表示,该种群最有可能的遗传基因型数目为K=5。来自加纳的39个可可样品,以及29个参考系克隆共同被分作Amelonado、IMC、SCA/Ucayali、Morona、Nanay和Parinari等6个群体,这与前人研究的结果相互印证。但是SCA/Ucayali和Morona种质类群在本研究中得到分离。包含NA32和NA34在内的PA和IMC父母本明显对于T60、T63、T65和T79等株系有遗传学贡献,更加进一步验证T85/799的父母本分别为IMC60与NA34。另外IMC和Amelonado与T16/613家族相关.证实前人研究IMC24为亲本之一,同时本研究结果表明,亲本另一方是来自于Amelonado种质类群。通过亲缘关系鉴定与分析,在95%的置信水平上基础上,确定了6对杂交父母亲本组合,在80%的置信水平上确定了一对杂交父母亲本组合。STRUCTURE软件对父母本子代关系进行聚类分析,同样支持了这一结果。实验表明,本研究所采用的54个SNP位点是稳定而可靠的,其误差率不高于10%,同时,通过这54个SNP标记结果的描述性统计分析,在44个供试样品中具有独特SNP基因型的平均期望杂合度为0.336,实际检测杂合度为0.269。在实地观察工作中得到的平均亲近系数是0.218。通过亲缘鉴定和模型分配,以参考对照系的已知遗传身份为依据,验证杂交记载亲本的身份并验证杂交亲本的遗传背景。研究表明,仅采用一小组多态性SNP标记对可可基因型鉴定,就可以提供足够的遗传信息进行基因分型。这个检测体系可以适用于大规模进行可可基因分型。
综上所述,本研究采用SSR分子标记技术对于特立尼达群岛的国际可可资源库的Trinatario杂交可可ICS种群进行标签缺失或重复鉴定,确定并更正该种群的部分株系在异地保存过程中标签挂错。本研究首次采用SNP单核苷酸多态性标记,对可可种质资源进型多态性鉴定,在单核苷酸水平上的多态性证明该技术将是国际可可资源基因库的管理维护的重要手段,可以实现在短时间内高通量筛选大量单核甘酸位点进行农家栽培品种及野生可可资源的遗传学鉴定。首次采用SNP标记,以单核苷酸多态性标记作为可靠依据,鉴定不同标签编号但是是同一品种,或是相同标签编号但实质是不同品种等历史上资源异地保存的遗留失误问题等,为分布于世界各地的可可种质资源进行归类整理提供有效的科技手段。首次采用SNP标记技术,更加明确的阐述可可作为巧克力产业的原材料,部份可可栽培品种的杂交父母本来源一直以来存在的争议问题。采用单核苷酸多态性分辨鉴定父母本与杂交子代的亲缘关系,为可可国际基因库的维护与完善提供可靠而高效的鉴定手段。
Cacao (Theobroma cacao L.)(2n=2x=20), is an important Sterculiaceae tropical evergreen plant origin from Central and South America, as early as3,000years ago was domesticated and cultivated by the Mayans. Now it is widely planted in Africa, Southeast Asia and Latin America. Cacao is one of the world's major crops, cocoa beans, are the main resource of chocolate industry, with the global annual production about4million tons. The world's major cocoa growing areas cultivated mainly in Latin America, South America, West Africa, Southern Asia and other tropical along the equator, but this region is also important for biodiversity conservation areas.
The conservation of cacao germplasm requires maintaining living trees in genebanks within tropical regions. There are two international public cacao collection, one is the International Cacao Genebank, Trinidad (ICG, T) and the other is International CATIE Cacao Collection (IC3). ICG, T is managed by the Cocoa Research Unit (CRU) of the University of West Indies and IC3by the Centro Agronomico Tropical de Investigacion y Ensenanza (CATIE) of Costa Rica (Beakele and Beakele,1996). Germplasm held in these two international collections is available for breeders via the international quarantine center located at the University of Reading in UK. These genebanks are supported by both public and industrial funding. Information of cacao germplasm held in these international collections is currently keeping in the International Cacao Germplasm Database (ICGD) which is hosted by the University of Reading in UK, as well as Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement (CIRAD) in France.
Since cocoa seed can not be stay alive for a long time, either to be stored under low temperature conditions. Therefore, only in the field of cocoa germplasm vivo preservation. This kind of offsite mode management has brought many problems. One major problem is mislabeling or label lost will cause to the confusion in evaluation, identification and breeding process of cocoa. The major rate of mislabeling within current international cocoa offsite repository is around30%to40%by average. The most urgent task of cocoa Repository Management is to correct the mislabeling in cacao collection fileds. That will be great help to assist documented pedigree and evaluation of genetic variaties.
It is commonly accepted that there are three groups of cocoa germplasm. The first group is Criollo, which was originally distributed in Central America and the Caribbean, and is well known for its "fine-flavor" beans. The second group is Forastero, which includes diverse wild and more recently cultivated populations originally from South America. Today Forastero is widely grown in West Africa and Brazil, and accounts for approximately90%of the cocoa beans produced in the world. Finally, there is the Trinitario group, which is believed to be the result of hybridizations between Criollo and Forastero types in the18th century. The original hybrid zone is the island of Trinidad.
The SSR (Simple Sequence Repeat) polymorphic markers and SNP (Single Nucleotide Polymorphism) markers are used to explore diverse populations of the best molecular identification means, and provide accurate and reliable molecular basis for the correction of mislabeling germplasm for theoretical guidance and support.
Experiment I. Assessing genetic identity and population structure of Trinitario cocoa germplasm using microsatellite markers
In this experiment, SSR markers were used to characterize a Trinitario cacao collection in the International Genebank in Trinidad. Trinitario cocoa is indigenous to the island of Trinidad and is believed to have had a very small number of founding parents in the18th century. However, rarely known about the individual identity and population structure in this group of cocoa germplasm.
This study was aimed at assessing the population structure and the extent of mislabeling in an indigenous Trinitario collection, and tested the hypothesis that the Trinitario were founded by a few progenitors. Multilocus microsatellite data were generated using15pairs of SSR primers. We used a capillary electrophoresis as previously described (Saunders et al,2004) using a CEQTM8000genetic analysis system (Beckman Coulter Inc.). Data analysis was performed using the CEQTM8000Fragment Analysis software version7.0.55according to manufacturers" recommendations (Beckman Coulter Inc). SSR fragment sizes were automatically calculated to two decimal places by the CEQTM8000Genetic Analysis System. Allele calling was performed using the CEQTM8000bining wizard software (CEQTM8000software version7.0.55, Beckman Coulter Inc.).
Synonymous mislabeling was identified by matching multilocus genotypes among individuals. Population structure was assessed by a Bayes method of sample partition. Heterozygosity deficiency was estimated as indication of recent population bottleneck. All microsatellite loci were polymorphic, with the number of alleles per locus ranging from2to10(mean=5.8). The mean expected heterozygosity ranged from0.307to0.656, with an average of0.552. The multi-locus fingerprints unambiguously identified all59individuals and three of them were found to be synonymous. Probability of identity predicted that the top7loci were necessary, yet sufficient, to distinguish with greater than99%certainty between the Trinitario individuals. Wilcoxon and Sign tests showed no heterozygosity excess. The distance-based cluster analysis, multi-dimensional scaling based on genetic relatedness, and Bayesian cluster method all showed that there is population admixture in the ICS Trinirario germplasm.
Analysis showed, ICS2, ICS83and ICS95three clones in15alleles are exactly match, by checking the relative position and compared to trees, it is determined that this three should be correct as the same lable. Moreover, only by the seven loci mTcCIRll, mTcCIR15, mTcCIR40, mTcCIR37, mTcCIR24, mTcCIR33and mTcCIR12can tell whether the same basic lines repetition. Population structure analysis was results showed by PowerMarker that the samples ICS1, ICS10, ICS35, ICS46, ICS57, ICS77, ICS80and ICS100other eight clones may be due to off-site preservation mislabeled and mixed ICS groups of individuals.
It shows that SSR fingerprinting is highly efficient to identify cacao accessions. We demonstrated that the Trinitario cocoa is comprised of more than one source population thus should not be treated as a single uniform genetic group in conservation and breeding.
Experiment II. Genetic diversity and parentage in farmer varieties of cacao from Honduras and Nicaragua as revealed by single nucleotide polymorphism (SNP) markers
Although SSR markers are efficient tool for cacao germplasm characterization, this system has several drawbacks for large scale DNA fingerprinting. Compared to SSR markers, SNP assays can be done without separating DNA by size, and therefore can be automated in high throughput assay format. The di-allelic nature of SNPs offers a much lower error rate in allele calling. Moreover, there is a higher consistency in allele calling across laboratories. While SNP markers have been widely used in plant varietal identification in many other crops, the efficacy of using SNP markers for cacao genotype identification and diversity assessment remain to be investigated. Large scale genotyping using a small set of SNP markers is still in great demand by the cacao community for a broad range of research and field applications.
Experiment Ⅲ. Verification of genetic identity of introduced cacao germplasm in Ghana using single nucleotide polymorphism (SNP) markers
Cocoa is an important global commodity with an annual production that exceeded4million tons in2010, of which75%was produced in West Africa. Ghana alone produced850,000tons of cacao, accounting for21%of the world's total output in2010(FAOSTAT, http://faostat3.fao.org/home/index.html).
In the present study, high-throughput genotyping with SNP markers was used to fingerprint160cacao trees in the CRIG germplasm collection. These accessions had been originally introduced from international germplasm collections. The study also included100international clonesas references. DNA concentration was determined with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). Based on the level of polymorphism and on their distribution across the ten chromosomes in cacao,54SNP markers were selected from1560candidate SNPs that had been developed using cDNA sequences from a wide range of cacao organs (Argout et al.2008). SNP genotyping was performed at the Human Genetics Division Genotyping Core facility, Washington University, St. Louis, using MALDI-TOF mass spectrometry (Sequenom, Inc., San Diego, CA).
Key descriptive statistics for measuring the infonnativeness of the SNP markers were calculated, including observed heterozygosity, expected heterozygosity, minor allele frequency, Inbreeding coefficient and probability of identity (Evett and Weir1998; Waits et al.2001). The program GenAlEx6.2(Peakall and Smouse2006;2012) was used for computation.
For the identification of mislabeling (off-type), SNP profile of100reference trees maintained in the International Cacao Genebank, Trinidad (ICG,T) were used in the analysis. The genetic identity of the100reference trees has been characterized by both SNP (Zhang, personal communication and SSR fingerprinting (Zhang et al.2009b; Motilal et al.2010; Johnson et al.2009). Pairwise multilocus matching was applied among each pair of individual trees, including the reference trees from the International germplasm collections, using the same program. Accessions with same names as the reference trees, but not matching them, were declared off-types. For the multilocus matching, the option to ignore missing data was used. Discriminating power of the SNP loci was computed using the probability of identity (PID, Waits et al.2001) option implemented in the same computer program.
The multilocus SNP profiles, generated by the Sequenom Mass Spectrometry platform, were compared with the SNP profiles of reference trees maintained in the international cacao collections. Comparison of the multilocus SNP profiles with the reference accessions identified seven intraclonal mislabelings in accessions NA79, PA150and IMC76. The multilocus matching also found that AMAZ-2and PA303were mislabeled. With all53loci considered, the combined probability of identity was in the order of10-9. Overall, the procedure of multilocus matching with known reference trees led to the identification of149true-to-type trees out of160tested samples. Based on the value of delta K, the model-based approach of STRUCTURE indicated K=5as the most probable number of genetic clusters. The tested cacao accessions from the Ghana cacao collection, as well as the100reference accessions were stratified as germplasm groups of Amelonado, IMC, SCA/Ucayali, Morona, Nanay and Parinari respectively. The assigned memberships for all the tested trees from Ghana were compatible with their known parentage germplasm groups. The assignment test of the T clones confirmed their recorded parental germplasm groups. The parental group of PA, IMC (which includes parental clones NA32and NA34) were clearly reflected in the admixed ancestry profiles of T60, T63, T65and T79. A full genetic background of IMC was revealed for accession T85/799, supporting its recorded parentage of IMC60and NA34(a member of the IMC germplasm group; Motamayor et al.2008). In addition, admixed ancestry of IMC and Amelonado was revealed for T16/613family, which not only supported the recorded parentage of IMC24, but also detected that the other parent came from the Amelonado group.
Of the eight candidate parent-offspring relationships, the results of parentage inference confirmed six pairs at the95%confidence level and one pair (NA34-T80/799) at the80%confidence level (Table4). For offspring T16/613, only one parent (Amelonado22) was identified at the>80%confidence level because the reference genotype of maternal parent IMC24was not available. The result of parent-offspring assignment supported the outcome of model-based clustering analysis by the STRUCTURE program.
The comparison unambiguously identified mislabeled trees. For materials introduced as hybrid seeds without an available reference genotype, parentage analysis and model-based assignment were applied to verify their recorded parentage and genetic background. Our study shows that a small set of polymorphic SNP markers can provide a robust and accurate result for cacao genotype identification. This protocol can be applied for large-scale genotyping of cacao as well as for many other crops.
In summary, it shows that SSR fingerprinting is highly efficient to identify cacao accessions Trinatario from International Cocoa Cocoa ICS collection through this experiment. We demonstrated that the Trinitario cocoa is comprised of more than one source population thus should not be treated as a single uniform genetic group in conservation and breeding duplicate identification. The first study using single nucleotide polymorphism SNP markers for cocoa germplasm into polymorphism identified in single nucleotide polymorphisms prove that the level of technology will be the International Cocoa Genebank resource management and maintenance an important tool that can be achieved in a short time for a large number of high-throughput screening of single nucleotide loci cultivars, as well as wild cocoa farm genetic resources identified. For the first time using SNP markers, as a reliable basis for identification of mislabling and other historical legacy of offsite resources issues. Identification of single nucleotide polymorphisms to distinguish parental genetic relationship with hybrids and provide reliable and efficient identification for cacao world collections.
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