茄子品种DUS测试指南的研制

[目的] 植物品种特异性(distinctness)、一致性(uniformity)和稳定性(stability) 测试(简称：DUS测试)是品种保护、品种审定、品种登记的必要条件。研制茄子(Solanum melongena L.)品种DUS测试指南旨在为测试和品种权实质审查工作提供科学依据。[方法]以《植物新品种特异性、一致性和稳定性测试指南 总则》(GB/T19557.1)为总体原则,以国际植物新品种保护联盟颁布的《植物新品种特异性、一致性和稳定性测试指南 茄子》(TG/117/4)为参考,通过对243份茄子品种的田间试验和多次同行专家修订,完成茄子的DUS测试指南(国家标准报批稿)。[结果]指南确定了42个测试性状及描述方法,筛选出20个标准品种,明确了茄子特判定、一致性和稳定性判定的原则。[结论]指南为我国茄子DUS测试的品种描述和特异性、一致性、稳定性判定提供了技术标准。笔者重点介绍了指南的编制原则和过程、测试性状的选择和标准品种的选用,以及特异性、一致性、稳定性的判定标准。     

Discriminating maize inbred lines using molecular and DUS data

Growing numbers of candidate varieties, decrease of their variability for morphological traits, and internationalization of the national list all contribute to excessive increase of the trial costs, thus creating the need for the improvement of current variety evaluation procedures, especially regards their distinctness, Uniformity, and Stability (DUS) component. Due to rapid advancement in molecular techniques, the use of molecular markers in DUS testing as a complement to, or replacement of, morphological observations became the subject of great interest in scientific studies, and consequently topic for discussion within International Union for the Protection of New Varieties of Plants (UPOV). In order to explore the potential of simple sequence repeat (SSR) markers for distinctness tests, present study involved set of 41 maize inbred lines that were scored for 32 DUS characters prescribed by UPOV and genotyped at 28 SSR loci. Results were largely in favor of the use of molecular markers, revealing or confirming their already known advantages over morphological markers like better consistency with the pedigree, and relatively higher discriminative power. However, their integration into DUS testing protocols still depends upon resolving of several important issues.     

龙眼新品种DUS测试指南的研制

植物新品种特异性(distinctness)、一致性(uniformity)和稳定性(stability)（简称DUS）测试指南是审查机关开展植物新品种实质审查工作的基础和技术手册。国际植物新品种保护联盟(UPOV)技术文件《TGP/7 植物新品种测试指南的研制》是各国研制植物新品种测试指南的依据与标准。龙眼DUS指南在严格遵循TGP/7文件的基础上,通过借鉴现有的龙眼种质资源描述方法并结合育种实际,最终确定了45个性状、26个标准品种和DUS判定标准。笔者主要叙述了龙眼DUS测试指南的研制原则、测试性状的选择与确定、性状分级标准、DUS判定标准以及标准品种的选择,并指出将分子标记技术与形态测试技术相结合是今后DUS测试指南研制的发展方向。     

Suitability of mapped sequence tagged microsatellite site markers for establishing distinctness, uniformity and stability in aromatic rice

At present, testing for distinctness, uniformity and stability (DUS) of crop varieties relies on a set of morphological characters. These characters suffer fromthe limitations of number, interaction with the environment in which the variety grows and subjectivity in decision-making. The potential of DNA-based markers such as sequence tagged microsatellite site (STMS), for establishing DUS merits investigation. In the present study, a set of 55 mapped STMS markers, selected from 12 linkage groups of rice genome, was used to examine distinctness of 23 aromatic rice genotypes including the commercially important Basmati varieties. Forty-one of these markers (74.5%) showed polymorphism between the varieties. The number of alleles per locus ranged from 2–4 with an average of 2.3. The polymorphism information content (PIC) of the markers varied from 0.083 to 0.665 with an average of 0.338. All the varieties could be differentiated from each other at a low probability (0.07×10^-13) of identical match by chance. The marker-based clustering of the varieties corresponded with the known phenotypic classification, thereby providing confidence in the distinctness established by the mapped STMS markers. The utility of these markers to study uniformity and stability was analysed using a commercially important crossbred Basmati rice variety Pusa Basmati 1(IET-10364) that contributes about 40–50% of Basmati rice export from India. Genotyping of twenty individual plants, grown from the nucleus, breeder, foundation, certified and farmer's saved seed samples using all the 55 markers revealed no variation among the plants. These observations suggested that the set of mapped markers employed in this study could be further used for establishing distinctness of aromatic rice varieties and for studying DUS of the important commercial variety Pusa Basmati 1. This revised version was published online in July 2006 with corrections to the Cover Date.     

基于DUS测试的标准品种形态性状稳定性和重要性分析

为了进一步提高水稻品种特异性(Distinctness)、一致性(Uniformity)和稳定性(Stability)(简称DUS)测试的准确性,以水稻DUS测试指南中列出的49份水稻标准品种为材料,应用AMMI模型对水稻品种50个DUS测试性状进行了稳定性分析,并采用随机森林算法对测试性状的重要性进行了评价。结果表明,不同性状的稳定性差异较大,以质量性状和假质量性状为主的目测性状稳定性高,而以数量性状为主的量测性状稳定性较低。不同性状的重要性参数差异甚大,总体上,以数量形式表示的量测性状的重要性参数较大,而以分级代码表示的目测性状重要性参数相对较小。对于茎节包露、穗类型、穗立形状、外颖茸毛和叶茸毛等稳定性较低、重要性参数不高的性状,可从水稻DUS测试指南中去除;对成熟期、抽穗期等稳定性较低但在生产实践中很重要的农艺性状,宜安排在申请者所在地参试点测试,使其表达状态真实表现;或者采用标准品种进行校正,从而提高测试的准确性。     

海南不同季节对水稻新品种DUS测试性状表达的效应分析

为了探究海南不同季节对水稻新品种特异性、一致性和稳定性（简称DUS）测试性状表达的影响效应,为优化海南水稻DUS测试的操作规程提供参考,为海南水稻新品种DUS测试技术的完善提供实践经验与理论支撑,根据水稻植物新品种DUS测试指南,按照相关原理和技术规则,通过对4份水稻材料进行4个独立的生长周期的测试,研究其相关性状的表达与季节及年度间的效应,结果表明：除穗芒外,不同季节的4个独立测试周期中,质量性状观测结果稳定一致;14个假质量性状的表达结果不影响对申请品种的DUS判定;10个量测性数量性状在方差分析中出现显著性差异,但是申请品种与近似品种的变化和差异保持相对不变,不影响其DUS的判定结论;17个目测数量性状的表达无显著差异。因此,在海南进行不同季节的水稻测试对其DUS的判定无显著影响,可考虑多点多季节测试,加快水稻实质审查的进程。     

蝴蝶兰新品种DUS测试指南的研制

植物新品种特异性、一致性和稳定性(DUS)测试是新品种保护的基础和依据,为适应中国蝴蝶兰的发展以及与国际接轨的需要,研制了蝴蝶兰DUS测试指南。蝴蝶兰DUS测试指南共78个性状,22个标准品种,笔者就测试指南研制的原则、性状表的构建、标准品种的选择以及DUS判定标准进行阐述。     

Assessment of the uniformity of wheat and tomato varieties at DNA microsatellite loci

By analysing a number (20–38) of individuals from selected varieties of wheat and tomato, we have been able to assess intra-varietal uniformity at certain micro satellite (simple sequence repeat, SSR) loci. In total, 45 varieties of wheat were analysed at between 7–9 different SSR loci, and 10 varieties of tomato were analysed at six loci. The results showed that there was variation both between varieties and between microsatellites in the degree of non-uniformity observed, and it was possible to identify a number of different probable sources of non-uniformity. Twenty-four of the wheat varieties and nine of the tomato varieties were sufficiently uniform to meet the standards currently applied for distinctness, uniformity and stability (DUS) testing using phenotypic characteristics. The implications for the potential future use of SSRs in DUS testing are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

巴西橡胶树DUS测试指南研制初探

为加强橡胶树新品种保护工作,在国际新品种保护联盟（UPOV）对DUS测试的基本要求和品种的生物学特性的基础上,制定巴西橡胶树DUS测试指南,阐述了其研制过程,品种测试的特殊性,性状的筛选原则和发展方向等。建议橡胶树DUS测试标准不宜定太高,测试性状要能体现中国育种水平和方向,并遵循UPOV原则。并认为分子标记技术与表型测试技术相结合将逐步成为橡胶树DUS测试的主要手段。     

Prospects for applications of genomic tools in registration testing and seed certification of ryegrass varieties

The ryegrass (Lolium) species, perennial ryegrass (Lolium perenne L.) and Italian ryegrass (Lolium multiflorum Lam.), are the two most important pasture grass species for global temperate regions and are also useful for amenity purposes. Due to an outbreeding reproductive habit, high levels of genetic heterogeneity are present within cultivated varieties. A continual increase in delivery of new cultivars to the marketplace, along with limited capacity to discriminate on the basis of morphological characteristics in a grow‐out test, has caused difficulties for distinctness, uniformity and stability (DUS) testing in the current plant variety protection (PVP) system. A range of genomic tools and resources have been generated for ryegrasses, which provide new opportunities and challenges to the current PVP system. In this review, the currently available genomic tools and resources are described, along with prospects for applications to the PVP system and testing for seed certification and purity accreditation.     

红花新品种DUS测试指南的制订

植物新品种特异性(distinctness)、一致性(uniformity)和稳定性(Stability)(简称DUS)测试是新品种保护的技术基础和授权的科学依据。DUS测试指南既是指导测试机构开展DUS测试工作的技术手册,同时还是审批机关审查新品种DUS的技术标准。红花（Carthamus tinctorius L.）俗称草红花或杜红花,是一种油、药及工业多用途作物,其经济价值较高。本文详细地论述了红花DUS测试指南的适用对象、测试性状的选择与确定、性状分级与标准品种的选用、红花DUS判定标准的确定。红花DUS测试指南的制订对增强红花新品种保护,促进我国植物新品种保护事业的发展具有积极意义。     

栽培环境对番茄部分数量性状DUS测试结果的影响

为了探索不同栽培环境对番茄部分数量性状DUS测试结果的影响,选取对30个番茄DUS测试标准品种和2个测试品种及其选定的2个近似品种,进行塑料大棚和露地2种不同的栽培环境对番茄10个数量性状的DUS测试结果影响的研究。结果表明：2种栽培环境对除第一花序着生节位和心室数以外的8个数量性状均存在显著或极显著的影响,导致在部分数量性状代码判别和特异性的判定上存在着差异。通过标准品种数量性状分级标准的校正,可以有效消除因栽培环境不同而造成的影响。     

Construction of an integrated microsatellite and key morphological characteristic database of potato varieties on the EU common catalogue

The European Union Common Catalogue (EUCC) for potato contains over 1000 varieties. Each year member states add varieties to the list after they have undergone Distinctness, Uniformity and Stability (DUS) testing according to international guidelines. A rapid and robust method for variety identification to aid the management and maintenance of existing variety collections and for the screening of new candidate varieties would therefore be a highly useful tool for DUS testing stations. A database containing key morphological characteristics and microsatellite data was constructed for varieties on the 2006 list of the EUCC for potato. Rules for scoring SSR markers in different laboratories were established to allow a harmonized scoring of markers. Almost all varieties (99.5%) were shown to have unique molecular profiles and in pair wise comparisons 99.99% of all variety pairs could be distinguished. This clearly shows the versatility of the markers and database for identifying potato samples.     

Applicability of molecular markers in the context of protection of new varieties of cucumber

The growing number of candidate varieties presented every year at each national Plant Variety Protection Office and their decreasing genetic variability forces strategies to be adopted that will reduce costs without losing rigour when deciding about the acceptance or rejection of a candidate variety. Molecular markers have been envisaged as a reliable tool to establish differences, but can molecular markers be used for assessing distinctness? A comparison between a molecular and a morphological characterization of 41 seed samples belonging to 36 cucumber varieties and including several external controls has been carried out to investigate the applicability of molecular markers in the context of distinctness, uniformity and stability (DUS) tests and the protection of new varieties. Both types of character indicate the same relationships among main groups, Dutch and Beth‐Alpha types, and Gerking and Slice types, the correlation between distance matrices being only 0.6489. Varieties considered uniform for morphological traits proved not to be so for molecular markers. Therefore, molecular characterization is not offering the same estimates of uniformity and relatedness between varieties as does morphological characterization. External controls were used to establish distinction limits with morphological and molecular dendrograms to focus on those varieties, which were suspiciously similar. The results with cucumber show that molecular identity could be used to assess any lack of distinctness and so corroborate the morphological assessment of candidate varieties. The morphological trait ‘type’ provides distinct characterization of varieties and the molecular characterization of candidate varieties could be used to design better field experiments to assess distinction within each group of morphological cucumber type.     

Marker‐assisted backcross breeding for improvement of drought tolerance in bread wheat (Triticum aestivum L. em Thell)

Drought stress is presently a major productivity limiting factor in wheat. This study developed five wheat lines with inbuilt tolerance to drought stress using marker‐assisted backcross breeding (MABB) approach employing three linked quantitative trait loci (QTLs) in an initial population of 516 BC₁F₁ plants. The high‐yielding wheat cultivar ‘HD2733’ grown over last few years extensively in the eastern plains of India is largely sensitive to drought and is used as the recurrent parent. ‘HI1500’ released for water‐limiting conditions and carrying drought‐tolerant QTLs was used as donor parent. MABB lines were advanced using foreground and background selection, coupled with stringent phenotyping. We identified 29 lines that were homozygous for targeted QTLs in different combinations with background recovery range of 89.2%–95.4%. Further evaluation of selected lines for physiological traits and distinctness, uniformity and stability (DUS) characters under rainfed condition identified five potential varieties for national varietal evaluation programme in the zone. The report is first of its kind in implementing known QTLs for the development of drought‐tolerant wheat lines through MABB approach.     

玉米新品种DUS测试中数量性状的适宜样本容量研究

DUS（Distinctness, Uniformity and Stability）测试是对申请品种权的植物新品种的特异性、一致性和稳定性进行测试。DUS测试结果直接影响到植物新品种是否能被授予保护权。在DUS测试中,数量性状是完成品种描述和进行特异性判定的重要部分,而数量性状的调查花费人工最多、工作量最大,怎样能缩小工作量又能反映数量性状调查的准确性,这是本试验研究的目的。目前,玉米测试指南已经成为国家指南,指南要求每个数量性状测量40个数据。本试验利用10个测试品种、全株10个数量性状,对每个性状的40个已测样本进行统计分析,根据抽样分布理论和区间估计的方法确定每个性状的最小取样容量。     

小麦区试品系DUS测试的分子标记

为了确定测试小麦(Triticum aestivum L.)区试品系特异性、一致性、稳定性(DUS)的分子标记,采用156个来自我国不同麦区的品种对SSR、EST-SSR和AFLP-SCAR标记的1334对引物进行筛选,根据在染色体上分布均匀、多态性信息指数较高、带型清晰、不同等位变异的带型易于区分及PCR产物稳定的原则,筛选出105对小麦品种DUS测试的分子标记引物,包括63对SSR引物、21对EST-SSR引物和21对AFLP-SCAR引物,可以检测122个位点的754个等位变异,平均每条染色体上被检测位点5.8个,平均每个位点包含7.2个等位变异。根据DUS测试的需求、引物的染色体分布、PIC值大小和带型特点,将105对引物分为21对核心引物、29对一级备用引物和55对二级备用引物。核心引物分辨力较高,可以完成约80%品系的特异性检测,约95%品系的种子纯度检测和约60%品系的一致性、稳定性检测;备用引物用于确定品系DNA位点纯合率和相似品种(品系)之间的遗传相似系数,以判断DNA指纹相同或相似的品种(品系)之间的相似性和特异性,评价核心标记中具有非纯位点的品系的DNA位点纯合度,同时完成核心引物未能完成的少数品系的种子纯度检测。通过在2006-2007、2007-2008、2008-2009年度对464个冬小麦区试品系DUS测试中的应用,证明105对引物具有很好的代表性和实用性,可以完成90%以上参试品系的DUS检测。