Societal Costs of Late Blight in Potato and Prospects of Durable Resistance Through Cisgenic Modification

In the European Union almost 6 Mha of potatoes are grown representing a value of close to €6,000,000,000. Late blight caused by Phytophthora infestans causes annual losses (costs of control and damage) estimated at more than €1,000,000,000. Chemical control is under pressure as late blight becomes increasingly aggressive and there is societal resistance against the use of environmentally unfriendly chemicals. Breeding programmes have not been able to markedly increase the level of resistance of current potato varieties. New scientific approaches may yield genetically modified marker-free potato varieties (either trans- and/or cisgenic, the latter signifying the use of indigenous resistance genes) as improved variants of currently used varieties showing far greater levels of resistance. There are strong scientific investments needed to develop such improved varieties but these varieties will have great economic and environmental impact. Here we present an approach, based on (cisgenic) resistance genes that will enhance the impact. It consists of five themes: the detection of R-genes in the wild potato gene pool and their function related to the various aspects in the infection route and reproduction of the late blight causing pathogen; cloning of natural R-genes and transforming cassettes of single or multiple (cisgenic) R-genes into existing varieties with proven adaptation to improve their value for consumers; selection of true to the wild type and resistant genotypes with similar qualities as the original variety; spatial and temporal resistance management research of late blight of the cisgenic genetically modified (GM) varieties that contain different cassettes of R-genes to avoid breaking of resistance and reduce build-up of epidemics; communication and interaction with all relevant stakeholders in society and transparency in what research is doing. One of the main challenges is to explain the different nature and possible biological improvement and legislative repercussions of cisgenic GM-crops in comparison with transgenic GM-crops. It is important to realize that the present EU Directive 2001/18/EC on GM crops does not make a difference between trans- and cisgenes. These rules were developed when only transgenic GM plants were around. We present a case arguing for an updating and refinement of these rules in order to place cisgenic GM-crops in another class of GM-plants as has been done in the past with (induced) mutation breeding and the use of protoplast fusion between crossable species.     

Cisgenesis Does Not Solve the Late Blight Problem of Organic Potato Production: Alternative Breeding Strategies

Average potato yields in Dutch organic farming systems vary from 15 to 29 Mg/ha and are limited by low input of nitrogen and severe late blight attacks caused by the oomycete Phytophthora infestans. Under Dutch late blight regulations it is mandatory to kill the haulm at 7% infestation. The late blight attacks have been so early in the organic potato production of the past few years that its acreage is now gradually decreasing whereas consumer demand is increasing. Agronomic control strategies have limited success. First priority lies in breeding for highly resistant varieties to safeguard organic potato production in the Netherlands. Cisgenesis, however, is not an option for the organic sector. Although the product of cisgenesis does not contain genes from non-crossable species it is a result of a genetic engineering process which is excluded from use in organic agriculture. As the principles and standards of organic agriculture are process-based, cisgenesis does not comply with the norms and standards of organic agriculture. The arguments of the organic sector go well beyond the alleged risks of the gene technology and relate to the technology itself. Breeding at DNA-level, instead of at whole-plant level, violates the integrity of life as described in the concept of naturalness. The Dutch organic sector is now aiming at increasing the traditional breeding activities including the participation of farmer-breeders in close cooperation with the formal breeding companies. Additional selection methods need to be developed to include required traits other than late blight resistance, such as nutrient efficiency. Recently two varieties have been released with high resistance against late blight based on introgressing genes from Solanum bulbocastanum. Organic agriculture can benefit from marker assisted breeding to achieve adequate pyramiding of different, new sources of resistance.     

Applied Biotechnology to Combat Late Blight in Potato Caused by <Emphasis Type="Italic">Phytophthora Infestans</Emphasis>

Potato is an important crop, grown worldwide. It suffers from many pests and diseases among which late blight, caused by the oomycete Phytophthora infestans, is the worst. The disease is still causing major damage in many potato production areas and control is only possible by applying fungicides frequently. The knowledge on the molecular biology and genetics of the interaction between the plant and the oomycete is developing rapidly. These are relevant fields of study, currently dominated by the discovery of many resistance genes and numerous effector proteins and the analysis of their specific mode of action. These studies may yield essential information needed for the development of durable resistance. The long-term and worldwide effort to breed for resistance so far has had little effect. A novel breeding approach may change this. It is based on cisgenic modification (CM) consisting of marker-free pyramiding of several resistance genes and their spatial and temporal deployment yielding dynamic varieties that contain potato genes only. It is envisioned that this CM approach with potato’s own genes will not only prove societally acceptable but may also result in simplifications in the legislation on use of the CM approach. Various parties in the potato research arena intend to cooperate in this novel approach in a number of developing countries where potato substantially contributes to food security. The use of resources such as land, water and energy improves when the effect of late blight is markedly reduced.     

Progress in Mapping and Cloning Qualitative and Quantitative Resistance Against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Potato and Its Wild Relatives

Cultivated potato is susceptible to many pests and pathogens, none of which is more of a threat to potato agriculture than the late blight disease, caused by the oomycete Phytophthora infestans (Mont.) de Bary. To date all efforts to thwart this most adaptive of pathogens have failed, and early attempts to deploy ‘R genes’ introgressed from the wild Mexican hexaploid Solanum demissum ended in abject failure. With the advent of facile gene mapping and cloning, allied to knowledge of plant resistance gene structure, renewed efforts are leading to mapping and isolation of new sources of late blight resistance in potato wild species, many of which are being performed under the auspices of the BIOEXPLOIT project (Sub-project 2). We document recent advances in late blight resistance gene mapping and isolation, and postulate how these genes, allied to knowledge of pathogen effectors and their recognition specificity, may greatly enhance our chances of halting the progress of late blight disease in potato crops worldwide.     

br-2玉米矮生系导入热带种质的应用研究

根据基因重组原理,利用隐性单基因br-2矮生基因导入热带种质,使抗病、窄叶片、优品质等性状与br-2矮生基因有机结合,有效克服矮生系节间短、叶片密集、授粉不良等缺点,成功选育配合力高的单基因br-2矮生系南381,由此组配的玉米单交种隆单9号在西南及南方大面积推广应用。     

植物基因组CNV研究进展及其对麦类作物相关研究的启示

拷贝数变异(Copy number variation,CNV)是作物基因组中广泛存在的一种遗传变异,与作物的适应性、抗逆性等许多重要农艺性状相关。本文主要就CNV的发生机制、研究方法,尤其是植物中CNV与表型、CNV与植物适应性进化、CNV与重要农艺性状的关联分析等方面的研究进展进行了概述,并就植物CNV的研究对麦类作物相关研究的启示进行了深入剖析,以期为我国麦类作物开展CNV相关研究提供参考信息。     

小麦-华山新麦草7Ns异附加系的分子细胞遗传学研究

为了给有效利用华山新麦草基因提供新的种质材料,利用细胞遗传学、分子标记等技术,结合田间农艺性状考察,对从小麦-华山新麦草七倍体材料H8911-99与硬粒小麦D4286杂交F4代分离群体中选育的杂交后代12DH25进行了鉴定。华山新麦草基因组特异SCAR标记鉴定表明,12DH25含有华山新麦草遗传物质;有丝分裂和花粉母细胞减数分裂中期I染色体数为2n=44=22Ⅱ,基因组原位杂交(GISH)出现两条杂交信号,且能配对,表明两条外源染色体是华山新麦草的一对同源染色体。选取定位于小麦7个部分同源群上的28对STS标记对12DH25及其亲本基因组DNA进行扩增,定位于小麦第7同源群上的STS标记BE591127和BG274576能在12DH25中扩增出华山新麦草特征条带,将12DH25附加的华山新麦草染色体归属于小麦第7部分同源群。由此确定矮秆材料12DH25是一个稳定的小麦-华山新麦草7Ns二体异附加系。     

Yield benefit and underlying cost of insect-resistance transgenic rice: Implication in breeding and deploying transgenic crops

The rapid development of transgenic biotechnology has greatly promoted the commercialization of genetically modified (GM) crops including the insect-resistant crops worldwide. Apart from the enormous yield benefits brought by the GM crops, the cryptic fitness cost associated with transgenes has also been detected under experimental conditions although it is considered to be rare. To estimate the yield benefit and cost of insect-resistant GM rice, we studied field performances of three insect-resistant GM rice lines, involving their non-GM parental variety as comparison. Great benefits as estimated by the yield-related traits were observed in the GM rice lines when high insect pressure was recorded, but a cryptic yield loss was detected when the level of insect pressure was extremely low. Given the fact that cryptic yield loss presented in the GM rice lines under the low insect pressure, a strategic field deployment should be required when insect-resistant GM rice are commercialized to circumvent the unnecessary yield losses. This is probably true for other insect-resistant GM crops. Effective biotechnology and breeding measures are also needed to particularly minimize the potential underlying cost of an insect-resistance transgene before commercial production of the GM crops.     

Resistance to <Emphasis Type="Italic">Potato virus Y</Emphasis> in a Multitrait Potato Breeding Scheme without Direct Selection in Each Generation

In an experimental breeding scheme to improve late blight (Phytophthora infestans) and white potato cyst nematode (Globodera pallida) resistance of tetraploid potato over three generations of crossing and selection, 15 clones survived the final selection, and these were derived from 15 great-grandparents. There was no direct selection for resistance to Potato virus Y (PVY), but 14 out of the 15 great-grandparents were resistant to PVY and three had extreme resistance. Thirteen of the 15 descendants had PVY resistance and one extreme resistance. This was within the range expected for a random (unselected) sample from the genotypes of the great-grandparents. Hence, we found no evidence for any positive or negative association between PVY resistance and the attributes selected. The conclusion is that laborious selection is not required in every generation when many parents have PVY resistance, including some with more than one copy of a PVY resistance gene or resistance at more than one locus. However, in the future, determining the major virus resistance genes present in potential parents in each generation using diagnostic molecular markers would prevent susceptible × susceptible crosses being made and maximise the number of resistant × resistant ones.     

宁夏小麦品种慢锈基因Lr34/Yr18的分子检测

Lr34/Yr18是重要的小麦慢叶锈/慢条锈基因,可用于小麦锈病抗性改良。为了明确Lr34/Yr18基因在宁夏小麦中的分布特点,利用STS标记csLV34对111份宁夏小麦品种中慢锈基因Lr34/Yr18的等位变异进行了分子检测,并且进行了成株期条锈病抗性鉴定。结果表明,20份材料携带Lr34/Yr18基因,占18.0%。不同来源的小麦品种中Lr34/Yr18基因分布频率不同,农家品种中分布频率最高,占90.9%;引进品种中所占比例为14.3%;育成品种中所占比例最低,仅占7.7%。含有Lr34/Yr18基因的品种对条锈病具有较好的抗性,可作为今后宁夏小麦抗锈病育种的重要抗源。     

野生稻有利基因转移研究进展

稻属有22个种,包括20个野生稻种和2个栽培稻种,现已命名AA、 BB、 CC、 BBCC、 CCDD、 EE、 FF、 GG和HHJJ 9个基因组。野生稻是一个重要的有利基因库,包含对各种生物胁迫的抗性和非生物胁迫的耐性基因。就野生稻的有利性状、野生稻有利基因转移研究进展作了概述,并对野生稻有利基因分子标记定位研究,野生稻有利基因转移的遗传机制以及野生稻有利基因利用前景进行了讨论。     

Advances in Functional Genomics and Genetic Modification of Potato

The challenges facing potato breeding have actually changed very little over the years with resistance to pests and pathogens remaining high on the agenda together with improvements in storability, reduction in blemishes, and novelty and consistency in cooking/processing qualities. The need to expand the range of targets for potato improvement is being driven by requirements for reduced agrochemical usage and by predictions of the effects of changing climates. Thus fertiliser and water use efficiency are moving up the political agenda. Genetic variation present in germplasm collections needs to be harnessed to provide the genes and alleles required. This paper provides examples of the functional genomics tools and approaches being developed and deployed to provide new options for advancing the breeding of next generation crops. Whilst genetic modification (GM) approaches remain contentious in Europe, this paper will also provide some recent examples of the range of potential impacts that GM approaches could make. It will also consider the value of so-called intragenic or cisgenic approaches to potato genetic engineering.     

The BIOEXPLOIT Project

The EU Framework 6 Integrated Project BIOEXPLOIT concerns the exploitation of natural plant biodiversity for the pesticide-free production of food. It focuses on the pathogens Phytophthora infestans, Septoria tritici, Blumeria graminis, Puccinia spp. and Fusarium spp. and on the crops wheat, barley, tomato and potato. The project commenced in October 2005, comprises 45 laboratories in 12 countries, and is carried out by partners from research institutes, universities, private companies and small-medium enterprises. The project has four strategic objectives covered in eight sub-projects. These objectives relate to (i) understanding the molecular components involved in durable disease resistance, (ii) exploring and exploiting the natural biodiversity in disease resistance, (iii) accelerating the introduction of marker-assisted breeding and genetic engineering in the EU plant breeding industry, and (iv) coordinating and integrating resistance breeding research, providing training in new technologies, disseminating the results, and transferring knowledge and technologies to the industry.     

小麦抗条锈基因Yr1和Yr2分子检测体系研究

为建立小麦抗条锈病基因分子检测体系,分别以Yr1和Yr2紧密连锁的标记为检测标记,以Sull-1、CYR29、CYR32、CYR33和V26/CM42为小麦条锈菌鉴别菌株,构建Yr1和Yr2分子检测体系,并对181份小麦高代系材料进行了抗病鉴定和Yr1和Yr2基因分析。结果表明,gwm372和gwm382可作为Yr1的检测标记;wmc364可作为Yr2的检测标记;5个鉴别菌株对Yr1和Yr2具有较好的区分能力。Yr1和Yr2基因在181份小麦高代系材料中比例较低,表明这两个基因在我国小麦抗病育种过程中丢失严重。     

Development of rice introgression lines with brown planthopper resistance and KDML105 grain quality characteristics through marker-assisted selection

‘Khao Dawk Mali 105’ (KDML105), a Thai aromatic rice cultivar, has been accepted in markets as a prime jasmine rice with premium prices. It has been extensively used as a parental line to develop new cultivars for rainfed lowland areas in Thailand because of its favorable quality and fragrance. However, this cultivar is highly susceptible to brown planthopper (BPH), a phloem sap-feeding insect pest of rice. The main objective of this study was to combine KDML105 essential grain quality traits with BPH resistance from the donor cultivar, ‘Rathu Heenati’. The linkage drag between Bph3 and Wx^a alleles was successfully broken by phenotypic and marker-assisted selections. All introgression lines (ILs) developed in this study showed a broad spectrum resistance against BPH populations in Thailand and had KDML105 grain quality standards. Finally this study was revealed that the ILs can be directly developed into BPH resistant varieties or can be used as genetic resources of BPH resistance to improve rice varieties with the Wx^b allele in rice breeding programs.     

Development and Molecular Characterization of Wheat-Aegilops peregrina Introgression Lines with Resistance to Leaf Rust and Stripe Rust

A wild non-progenitor species from wheat (Triticum aestivum L.) tertiary gene pool, Aegilops peregrina (Hack.) Maire & Weiller accession pau3519 (UUSS), was used for introgression of leaf rust (caused by Puccinia triticina) and stripe rust (caused by Puccinia striiformis f. sp. tritici) resistance in bread wheat. The accession was crossed and backcrossed with hexaploid wheat line Chinese Spring Ph^I to develop two homozygous BC₂F₆ wheat-Ae. peregrina introgression lines (ILs), viz., IL pau16058 and IL pau16061, through induced homoeologous recombination. Homozygous lines were screened against six Puccinia triticina and two Puccinia striiformis f. sp. tritici pathotypes at the seedling stage and a mixture of prevalent pathotypes of both species at the adult plant stage. IL pau16061 showed resistance to leaf rust only, whereas IL pau16058 was resistant to both leaf and stripe rust pathotypes throughout plant life. Molecular profiling of these ILs with simple sequence repeat (SSR) markers indicated that alien introgressions were mainly terminal and very few were interstitial. Identification of linked markers with advanced genomic technologies will aid in marker-assisted pyramiding of alien genes in cultivated wheat background.     

转小麦 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因拟南芥植株的主要抗旱性研究

核氧还蛋白（nucleoredoxin,NRX）可通过还原目标蛋白的二硫键来调控其生物活性,在植物的生长发育和抗逆境胁迫中发挥着重要作用。蛋白质二硫键异构酶（protein disulfide isomerase,PDI）、h型硫氧还蛋白（h-type thioredoxin,TRXh）和蛋白磷酸酶2A催化亚基（protein phosphatase 2A catalytic subunit,PP2Ac）是小麦核氧还蛋白TaNRX1的互作蛋白。为了明确TaNRX1互作蛋白的抗旱性功能,本研究在拟南芥中过表达了小麦 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因,对野生型和转基因拟南芥的表型和抗旱相关生理指标进行了鉴定。结果表明,干旱胁迫处理后,转 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因拟南芥的根长、存活率、脯氨酸含量均大于野生型,离体叶片失水率、丙二醛（MAD）含量均小于野生型。二氨基联苯胺（diaminobenzidine,DAB）对H₂O₂组织定位染色结果表明,干旱胁迫处理后,转 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因拟南芥的H₂O₂含量均低于野生型。上述结果说明,TaNRX1的互作蛋白基因 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D增强了拟南芥对于干旱胁迫的抵抗能力。本研究可为小麦抗旱育种提供候选基因和理论基础。     

先玉335、郑单958和京科968抗虫能力的比较研究

以先玉335、郑单958、京科968为材料,以玉米螟(Ostrinia furnacalis)、黏虫(Mythimna separata)和禾谷缢管蚜(Rhopalosiphum padi)为供试昆虫,探究玉米品系大喇叭口期对玉米螟和黏虫及抽雄期对禾谷缢管蚜抗性差异,测定其主要抗虫化合物丁布类物质的含量。结果发现,京科968对玉米螟及禾谷缢管蚜抗性均高于另外两个品系;对黏虫的抗性,郑单958和京科968高于先玉335。大喇叭口期京科968中丁布类物质含量最高,抽雄期郑单958和京科968中主要丁布成分含量均高于先玉335,上述组织中丁布合成相关基因在郑单958及京科968中的表达量也均高于先玉335。本研究揭示杂交玉米抗虫性与抗性相关化合物含量及其相关基因表达间存在正向相关关系,为抗虫玉米分子育种提供理论参考。     

高抗白粉病和条锈病小麦-卵穗山羊草衍生后代1003的分子细胞遗传学鉴定

卵穗山羊草中蕴含着许多小麦改良所需的优良基因,是小麦重要的三级基因库。为了解其更多遗传特性,本研究利用细胞学、原位杂交、分子标记、形态学和抗病性鉴定等技术对小麦-卵穗山羊草SY159的衍生后代1003进行鉴定。细胞学鉴定结果表明,1003含有44条染色体,减数第一次分裂中期含有22个二价体且配对良好,减数第一次分裂后期含有44条染色体且均等分离;基因组原位杂交(Genomic in situ hybridization,GISH)分析显示,1003含有42条小麦染色体和2条卵穗山羊草染色体;EST和PLUG分子标记分析表明,导入的染色体属于7M染色体;荧光原位杂交(Fluorescence in situ hybridization,FISH)分析表明,1003中含有38条与中国春标准核型相一致的染色体,4A、5A和7M的FISH信号有变异;苗期抗病性鉴定结果表明,1003对白粉病生理小种E09免疫,对条锈病生理小种条中23(CYR23)高抗;形态学调查表明,1003的农艺性状介于双亲之间,千粒重高于双亲。因此,1003是一个具有白粉病和条锈病抗性的小麦-卵穗山羊草二体异附加系,可为小麦品种改良和抗病育种提供新的种质资源。     

一个新疆杂草黑麦居群的细胞学分析

新疆杂草黑麦(Secale cereale subsp.segetale Zhuk.)是我国发现的一种稀有野生黑麦资源,具有极强的抗病、抗逆性和优异的丰产性状。为系统了解该特殊种质资源的生物学特征,并为这一珍贵种质资源应用于远缘杂交育种、牧草种质开发及其生物多样性保护提供基础的细胞学依据,选取新疆杂草黑麦的一个居群89R38,观察分析其花粉母细胞的减数分裂行为规律及其根尖细胞的核型特点。结果表明,89R38的绝大多数花粉母细胞减数分裂中染色体的行为正常,在终变期同源染色体配对可形成7个二价体,而在少数花粉母细胞减数分裂中(2.06%)观察到落后染色体、染色体桥等异常行为;成熟花粉粒的醋酸洋红染色反应检测其花粉粒的育性为99.08%;核型公式为2n=2x=14=12m+2sm,核型为1A型。