茶树抗旱机理和抗旱育种研究进展

茶树原产于中国西南地区,喜温暖潮湿的气候条件,其中水分对茶树的生长十分重要。笔者对茶树在干旱胁迫条件下的抗旱机理研究以及抗旱育种的现状进行综述,针对当前研究中存在的问题并结合当前分子生物学的发展,提出今后在茶树抗旱研究的研究重点：一是利用现代功能基因组研究技术,发掘更多的抗旱基因,深入研究茶树的抗旱机制;二是加强茶树转基因工程的研究。     

Rapid screening and selection of low-caffeine-containing tea (Camellia sinensis) trees by a colorimetric method

A simple and rapid colorimetric method for the determination of caffeine has been developed. Tea tree lines from the Hadong region in South Korea differ widely in their caffeine contents and this colorimetric method facilitated an efficient screening strategy for the selection of low‐caffeine‐containing lines. A high correlation was observed between the values obtained with colorimetric and high‐performance liquid chromatographic analysis. Among the selected tree lines, the caffeine content for the lowest caffeine‐containing tea tree line (H‐19) was 178.35 μg/g dry weight basis, which was 61‐fold less productive than the highest caffeine‐producing tea tree line (H‐82). Caffeine contents of the selected tea tree lines remained similar during the corresponding collection periods. The colorimetric method is of great practical value in screening low‐caffeine‐containing tea trees.     

土壤水分胁迫下茶树部分渗透调节物质的变化

为了了解不同茶树品种对干旱环境的适应性,分析了土壤水分胁迫对茶树部分渗透调节物质含量变化的影响。以茶树‘铁观音’和‘福鼎大白茶’2年生幼苗为材料,采用盆栽试验的方法,研究了在正常供水(土壤含水量为田间最大持水量的75%)、轻度水分胁迫(55%)、中度水分胁迫(35%)和重度水分胁迫(20%)条件下,茶树部分渗透调节物质的变化情况。结果表明,正常供水条件下,‘铁观音’叶片相对含水量、脯氨酸含量低于‘福鼎大白茶’,可溶性蛋白和可溶性糖含量高于‘福鼎大白茶’;水分胁迫下‘铁观音’、‘福鼎大白茶’叶片相对含水量随胁迫程度的加大而降低,可溶性蛋白质、可溶性糖以及脯氨酸的含量随胁迫程度的加大而增多,但在重度胁迫下,‘福鼎大白茶’的可溶性蛋白质及脯氨酸含量下降。在相同的水分胁迫条件下,‘铁观音’叶片相对含水量的降幅都小于‘福鼎大白茶’,而可溶性蛋白、可溶性糖和脯氨酸含量的增幅都比‘福鼎大白茶’大。表明在土壤水分胁迫下‘铁观音’的渗透调节适应能力高于‘福鼎大白茶’。     

茶树cDNA-AFLP银染技术体系的建立

为了挖掘安白茶白化期相关的差异基因,建立并优化了茶树叶片cDNA-AFLP扩增反应体系,进行cDNA-AFLP分析。在安吉白茶叶片cDNA-AFLP的试验中,分别对20 μL预扩增和选择性扩增反应体系中的4种反应条件影响因子进行不同梯度优化的研究,包括引物、Mg2+、dNTP及Taq DNA聚合酶的用量。PCR预扩增20 μL反应体系中,引物75 ng/20 μL,Mg2+ 2.0 mM,dNTP 0.2 mM,Taq DNA聚合酶1 U时预扩增效果较好;PCR选择性扩增20 μL反应体系中,引物75 ng/20 μL,Mg2+ 2.5 mM,dNTP 0.2 mM,Taq DNA聚合酶1.5 U时选择性扩增效果较好。通过试验,获得较为理想的预扩增和选择性扩增体系。     

茶树金属耐受蛋白基因CsMTP11的克隆及功能分析

金属耐受蛋白MTP(metal tolerance protein)是阳离子转运蛋白(CDF)家族的重要成员,在植物重金属转运过程中发挥重要调控作用。本研究以中茶108茶树为试验材料,通过RT-PCR和RACE方法克隆到茶树重金属耐受蛋白基因CsMTP11(Gen Bank登录号为KX450265),其全长c DNA为1197 bp,编码398个氨基酸残基,其编码蛋白分子量为44.85 k D,等电点为5.34。在线软件分析表明,CsMTP11蛋白具有5个跨膜结构域,且含有CDF家族的其他保守结构域。系统进化树分析结果表明,茶树CsMTP11与葡萄VvMTP11进化同源性最近,其氨基酸序列相似度高达90%。基因表达模式分析表明,CsMTP11基因在茶树老叶中的表达量最高,根中的表达量最低,另外,CsMTP11基因受重金属Mn和Co离子胁迫诱导表达。CsMTP11-YFP融合蛋白在拟南芥原生质体共定位试验表明,CsMTP11-YFP融合蛋白定位于质膜。CsMTP11在酿酒酵母及其突变株中的异源表达可以提高其对重金属Mn和Co离子的耐受性。综上所述,茶树CsMTP11属于Mn-CDF亚家族,可能参与茶树对重金属锰和钴的转运过程。     

Conventional and contemporary approaches for drought tolerance rice breeding: Progress and prospects

Drought is becoming a major threat to rice farming across the globe owing to the depletion of water tables in rice-growing belts. Drought affects rice plants at multiple stages, causing damage at morphological and physio-biochemical levels, leading to severe losses that exceed losses from all other stresses. The amalgamation of conventional breeding methods with modern molecular biology tools and biometrical methods could help accelerate the genetic gain for drought tolerance in rice. Many drought-tolerance traits with genetic determinants have been identified and exploited for tolerance rice variety breeding. The integration of genome-wide association study and genomic selection tools with speed breeding shortened the breeding cycle and aided in rapid improvement of genetic gain. In this review, we emphasized the progress made through classical breeding as well as the limitations and usefulness of current genomic methods in improving drought tolerance. We briefly addressed methods for identifying genetic determinants for drought tolerance and deploying them through genomics-assisted breeding programmes to develop high-yielding drought-tolerant rice cultivars.     

Construction of a high-density reference linkage map of tea (Camellia sinensis)

A few linkage maps of tea have been constructed using pseudo-testcross theory based on dominant marker systems. However, dominant markers are not suitable as landmark markers across a wide range of materials. Therefore, we developed co-dominant SSR markers from genomic DNA and ESTs and constructed a reference map using these co-dominant markers as landmarks. A population of 54 F₁ clones derived from reciprocal crosses between ‘Sayamakaori’ and ‘Kana-Ck17’ was used for the linkage analysis. Maps of both parents were constructed from the F₁ population that was taken for BC₁ population. The order of most of the dominant markers in the parental maps was consistent. We constructed a core map by merging the linkage data for markers that detected polymorphisms in both parents. The core map contains 15 linkage groups, which corresponds to the basic chromosome number of tea. The total length of the core map is 1218 cM. Here, we present the reference map as a central core map sandwiched between the parental maps for each linkage group; the combined maps contain 441 SSRs, 7 CAPS, 2 STS and 674 RAPDs. This newly constructed linkage map can be used as a basic reference linkage map of tea.     

Genomic prediction for yields,processing and nutritional quality traits in cultivated potato (Solanum tuberosum L.)

Current potato breeding approaches are hampered by several factors including costly seed tubers, tetrasomic inheritance and inbreeding depression. Genomic selection (GS) demonstrated interesting results regardless of the ploidy level, and can be harnessed to circumvent these problems. In this work, three GS models were evaluated using 50,107 informative SilicoDArT markers and 11 traits in two values for cultivation and use (VCU) potato trials. Two key breeding problems modelled included predicting the performance of (i) new and unphenotyped clones (cross‐validation) and (ii) a VCU using another as training set (TS). GS models performed comparably. Cross‐validation accuracy was high for D35, D45, DMW and BVAL, in ascending order. Prediction accuracies of the VCUs were highly correlated, but the best prediction was obtained for the smaller VCU using the bigger as TS. Cross‐validation and VCU prediction accuracies were higher when bigger TSs were used. The findings herein indicate that GS can be attractively integrated in potato breeding, particularly in early clonal generations to predict and select for traits with low heritability which would otherwise require more testing years, environments and resources.     

Expressed sequence tags from organ-specific cDNA libraries of tea (Camellia sinensis) and polymorphisms and transferability of EST-SSRs across Camellia species

Tea is one of the most popular beverages in the world and the tea plant, Camellia sinensis (L.) O. Kuntze, is an important crop in many countries. To increase the amount of genomic information available for C. sinensis, we constructed seven cDNA libraries from various organs and used these to generate expressed sequence tags (ESTs). A total of 17,458 ESTs were generated and assembled into 5,262 unigenes. About 50% of the unigenes were assigned annotations by Gene Ontology. Some were homologous to genes involved in important biological processes, such as nitrogen assimilation, aluminum response, and biosynthesis of caffeine and catechins. Digital northern analysis showed that 67 unigenes were expressed differentially among the seven organs. Simple sequence repeat (SSR) motif searches among the unigenes identified 1,835 unigenes (34.9%) harboring SSR motifs of more than six repeat units. A subset of 100 EST-SSR primer sets was tested for amplification and polymorphism in 16 tea accessions. Seventy-one primer sets successfully amplified EST-SSRs and 70 EST-SSR loci were polymorphic. Furthermore, these 70 EST-SSR markers were transferable to 14 other Camellia species. The ESTs and EST-SSR markers will enhance the study of important traits and the molecular genetics of tea plants and other Camellia species.     

茶树莽草酸代谢途径相关基因研究进展

莽草酸生物合成是连接植物初生代谢和次生代谢的关键节点。目前,莽草酸合成代谢途径关键基因的克隆、功能验证及表达调控的研究取得了一定成果,对调控茶树生长发育、提高茶叶产量和品质等研究均有重要意义。为此,本综述就莽草酸代谢途径与茶叶产量品质形成的相关性、相关调控基因研究进展,以及其他植物中该途径相关基因研究进展等进行了总结,以期为茶树遗传育种基础理论及高效育种技术的研发等提供参考。     

植物关联分析的研究进展及其在茶树分子标记辅助育种上的应用前景

茶树是我国重要的经济和饮料作物,由于自交不亲和和长期的异花授粉使其高度异质杂合,选育一个良种耗时长,提高早期选育效率和准确鉴定目标性状成为茶树育种家关心的重大问题之一。关联分析是研究分子标记或候选基因与目标性状关系的一种新方法,现已广泛应用于植物等位基因发掘、功能基因验证以及功能性标记的开发和应用等各方面的研究中,为茶树遗传育种研究提供了新思路。本文主要介绍了关联分析的基本原理、策略和及其在植物中的应用现状,并且探讨了其在茶树分子标记辅助育种研究上的应用前景。     

Assessment of genetic diversity of tea (Camellia sinensis (L.) O. Kuntze) by inter-simple sequence repeat polymerase chain reaction

Twenty-five diverse tea (Camellia sinensis(L.) O. Kuntze) cultivars were analyzed using the simple sequence repeat anchored polymorearse chain reaction (SSR-anchored PCR) or Inter SSR-PCR (ISSR). Out of the 45 primers 12were chosen for final study. These amplified a total of 130 bands out of which108 (84%) were polymorphic. A dendrogram was constructed using UPGMA method revealed three distinct clusters of Cambod, Assamand China type, which concur with the known taxonomical classification of tea. These results suggest that the ISSR-PCR method is potentially useful for genetic fingerprinting and molecular taxonomic classification of tea genotypes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genomic selection and enablers for agronomic traits in maize (Zea mays): A review

Maize is a commodity crop providing millions of people with food, feed, industrial raw material and economic opportunities. However, maize yields in Africa are relatively stagnant and low, at a mean of 1.7 t ha⁻¹ compared with the global average of 6 t ha⁻¹. The yield gap can be narrowed with accelerated and precision breeding strategies that are required to develop and deploy high-yielding and climate-resilient maize varieties. Genomic and phenotypic selections are complementary methods that offer opportunities for the speedy choice of contrasting parents and derived progenies for hybrid breeding and commercialization. Genomic selection (GS) will shorten the crop breeding cycle by identifying and tracking desirable genotypes and aid the timeous commercialization of market-preferred varieties. The technology, however, has not yet been fully embraced by most public and private breeding programmes, notably in Africa. This review aims to present the importance, current status, challenges and opportunities of GS to accelerate genetic gains for economic traits to speed up the breeding of high-yielding maize varieties. The first section summarizes genomic selection and the contemporary phenotypic selection and phenotyping platforms as a foundation for GS and trait integration in maize. This is followed by highlights on the reported genetic gains and progress through phenotypic selection and GS for grain yield and yield components. Training population development, genetic design and statistical models used in GS in maize breeding are discussed. Lastly, the review summarizes the challenges of GS, including prediction accuracy, and integrates GS with speed breeding, doubled haploid breeding and genome editing technologies to increase breeding efficiency and accelerate cultivar release. The paper will guide breeders in selection and trait introgression using GS to develop cultivars preferred by the marketplace.     

Breeding for improved drought tolerance in Chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is an important legume crop as a protein source across the world. It is mostly grown on arid and marginal lands where it faces drought stress at different growth stages. Drought stress exerts drastic effects on nutrient uptake, hinders the nodule formation and adversely affects yield and yield components. Generally drought at any growth stage and organizational level is responsible for reduction in economic yield. Significant variability in chickpea germplasm is present on the basis of responses to drought stress in the form of drought escape, drought avoidance and drought tolerance; these mechanisms prevent chickpea crop from harmful effects of drought. Improvement in chickpea germplasm against drought stress could be made by using several breeding approaches, that is introduction, hybridization, mutation breeding, marker‐assisted breeding and omic techniques. These breeding approaches, especially marker‐assisted breeding and omics, are further strengthened with the availability of the chickpea genome sequence. This review highlighted the significance, status and advances in different breeding strategies for improvement of drought tolerance in chickpea.     

茶树赤霉素受体基因CsGID1a的克隆与表达分析

GID1作为赤霉素信号转导的受体,在赤霉素作用中具有重要作用。采用同源克隆方法,利用RT-PCR和RACE技术在茶树中克隆到赤霉素受体基因GID1的cDNA全长,命名为CsGID1a (GenBank登录号为JX235369)。该基因全长1411 bp,开放阅读框1 023 bp,编码341个氨基酸。生物信息学分析显示,CsGID1a编码的蛋白分子量为38.53 kD,理论等电点为5.62;无信号肽位点,是非分泌性蛋白,具有1个跨膜区,基因被定位于细胞核内;CsGID1a氨基酸序列具有激素敏感性脂肪酶(HSL)家族蛋白的HGG、GXSXG功能域以及羧酸酯酶典型的三级结构;与其他物种的GID1相似性均在60％以上,与葡萄的相似性最大达87％、进化关系最近。荧光定量PCR结果显示,高浓度(1.0×10^–5 mol L^–1)GA₃能够下调CsGID1a的表达,5 h内的表达呈下降趋势;随着越冬茶芽萌动进程,CsGID1a表达量逐渐降低,特别在3月初萌发以后变化较大,推测赤霉素及其受体基因可能与茶树越冬芽解休眠相关。     

茶饼病病叶表面微生物多样性及病害真菌的分离鉴定

为了分析茶饼病病叶表面微生物多样性，分离鉴定病叶上真菌微生物。以茶饼病病叶与健康叶片为试材，采用扫描电镜对叶表面进行观察，利用16S和ITS测序分析不同叶片的细菌与真菌群落组成。在茶饼病病害叶片表面检测到113种细菌，252种真菌；健康叶片上检测到237种细菌，161种真菌。病叶细菌的多样性显著低于健康叶片，病叶真菌的多样性高于健康叶片。与健康叶片相比，枝孢属和拟盘多毛孢属真菌在病叶中丰度较低，被孢霉属丰度较高。采用PDA培养基从感病叶片表面分离鉴定获得了枝状枝孢菌，该菌可能在茶饼病的感染过程中发挥着重要作用。茶饼病感染后茶叶表面微生物群落结构发生显著变化，分离鉴定出的枝状枝孢菌可能与茶饼病的致病过程密切相关。     

茶树中性/碱性转化酶基因CsINV10的克隆与表达分析

基于课题组前期对茶树冷驯化系统的转录组测序分析结果,从中挑选出6条与中性/碱性转化酶基因高度相似的EST序列,电子拼接和RT-PCR验证后获得一条全长为2101 bp的核酸序列。该基因包含1923 bp的ORF,编码640个氨基酸,蛋白分子量为71. 8 kD,理论等电点（pI）为5.69。根据BlastX同源性比对显示该基因与荔枝LcNI相似性最高（80%）,为G100家族成员,属于中性/碱性转化酶基因,将其命名为CsINV10（GenBank登录号为KT359348）。对CsINV10氨基酸序列的系统进化树分析显示,其与木薯MeNINV8亲缘关系最近。进一步分析显示,CsINV10的氨基酸序列无N端信号肽,无跨膜结构域,属于亲水性蛋白,并定位在叶绿体上。荧光定量PCR分析表明,CsINV10具有组织表达特异性,在茶树叶和花中的表达量最高,根系中最低。分析发现,低温（4℃）、干旱和盐胁迫分别处理茶树1 d后,成熟叶片中CsINV10的表达呈逐渐上升趋势;而在ABA条件处理下,该基因呈先升高后降低趋势,在处理5 d后基本不表达,表明该基因可能参与茶树对多种逆境胁迫的响应,这为后续进一步研究转化酶基因在茶树抗寒等逆境胁迫中的作用奠定基础。     

茶树己糖激酶基因CsHXK2的启动子克隆及表达特性分析

植物己糖激酶是双功能蛋白,具有磷酸化己糖和介导糖信号的关键性作用。前期研究中,我们从茶树中克隆获得4个己糖激酶基因,其中CsHXK2基因编码492个氨基酸残基,与拟南芥AtHXK3、番茄LeHXK4归为Type A类HXKs。利用RT-PCR技术,克隆获得长度为2029bp的CsHXK2基因启动子。CsHXK2基因可能受到光照、低温、病原菌、糖和多种激素等信号的调控,且可能特异性表达于叶、花、种子、根系、腋芽等组织。CsHXK2蛋白定位于叶绿体内。酵母突变体功能互补试验表明,去除叶绿体转运信号肽的CsHXK2成熟蛋白具有葡萄糖和果糖磷酸化活性。茶树组织特异性表达分析显示,CsHXK2基因在根和茎中表达量最高,而在老叶中表达量最低。CsHXK2基因的表达受低温胁迫而显著下调,经炭疽菌侵染的茶树叶片内CsHXK2基因的表达也受到显著抑制,而外源赤霉素(GA₃)处理的茶树叶片内CsHXK2基因表达显著上调。本研究结果表明,CsHXK2基因在茶树的生长发育过程和逆境胁迫响应中发挥重要的调控作用。