Advances in Localization and Molecular Markers of Wheat Leaf Rust Resistance Genes

Genetic resistance is the most economical method of reducing yield losses caused by wheat leaf rust. To identify the leaf rust resistance genes in commonly used parental germplasm and released cultivars become very important for utilizing the genetic resistance to wheat leaf rust fully. Up to date, about 90 leaf rust resistance genes have been found, of which 51 genes have been located and mapped to special chromosomes, and 56 genes have been designated officially according to the standards set forth in the Catalogue of Gene Symbols for wheat. Twenty-four wheat leaf rust resistance genes have been developed for their molecular markers. It is very important to isolate, characterize, and map leaf rust resistance genes due to the resistance losses of the genes caused by the pathogen continuously.

Identification and Molecular Tagging of Leaf Rust Resistance Gene (Lr24) in Wheat

This research was aimed to develop AFLP markers co-segregated with gene Lr24 and validate the using for marker assisted selection （MAS）. An F2 population developed from the cross between the resistant line TcLr24 and the susceptible line Thatcher was tested for resistance to the Puccinia triticina races BGQQ and SHRT using for genetic analysis and molecular marker. A total of 224 AFLP primer combinations were used to test the resistant and susceptible parents, as well as the resistant bulk and the susceptible bulk. Four AFLP markers, P-AGA/M-CTT289 bp, P-AGC/M-CAC1ss bp, P-AGC/M- CAC162 bp, and P-ACG/M-CGC239 bp, were co-segregated with Lr24. The AFLP fragment from the primer combination P- ACG/M-CGC was cloned, sequenced and converted into a STS marker named as ASTS212. Thatcher backgrounded NILs and 115 varieties were examined by using this STS marker and the marker SCS13026oz developed by Gupta. 5R615, 5R616, IR13, and 1R17 were identified and validated to contain gene Lr24. The marker is dominant and may be useful in identification the resistance gene Lr24 in wheat and wheat breeding programs.

Postulation of seedling leaf rust resistance genes in 84 Chinese winter wheat cultivars

Wheat leaf rust（caused by Puccinia triticina） is one of the most important fungal diseases in China. There are tens of winter wheat cultivars which are approved to be released by the government at a national level and more than 100 wheat cultivars at the provincial level. But there is no information about leaf rust（Lr） genes in these cultivars, which makes it difficult for farmers and breeders to select which cultivars they should plant in their fields and use in their breeding programs. The objective of this paper was to identify the leaf rust resistant genes at seedling stage present in the 84 commercial wheat cultivars from China that have been released in the past few years. A set of 20 near isogenic lines with Thatcher background and 6 lines with known Lr genes were used to test the virulence of 12 races of P. triticina（Pt）. By comparing the infection types（ITs） produced on the 84 cultivars by the 12 Pt races with the ITs on the differential sets, the Lr genes were postulated. In addition, 8 molecular markers of Lr genes such as Lr9, Lr10, Lr19, Lr20, Lr21, Lr24, Lr26 and Lr29, which are closely linked to or co-segregated with the Lr gene, were used for further validation of the genes in the 84 Chinese winter wheat cultivars. Twelve Lr genes, including Lr1, Lr3,（Lr3bg）,（Lr3ka）, Lr11, Lr13, Lr14 a, Lr16, Lr26, Lr27, Lr30 and Lr31 were postulated to be present either singly or in combinations in these Chinese wheat cultivars. Lr3 and Lr26 were detected most often in the tested cultivars, with frequencies of 51.2 and 38.1%, respectively. No wheat Lr genes were detected in 16 cultivars, and 4 cultivars may carry unknown Lr genes other than those used in this study. Lr9, Lr20, Lr21, Lr24, Lr25 and Lr29 were not present in any of the 84 tested accessions.

A SSR Marker for Leaf Rust Resistance Gene Lr19 in Wheat

Microsatellite was carded out in Thatcher, six near-isogenic lines and F2 progeny of TcLr19xThatcher to develop molecular markers for leaf rust resistance gene Lr19. Thirteen primer pairs were screened, of which one primer pair Xgwm44 displayed polymorphsim in the population of TcLr 19, Thatcher, and their F2 generations. One marker closed linked to Lr19 resistance trait was obtained, and was named Xgwm44139bp with the genetic distance 0.9 cM. The research shows that Lr19 has more potential in marker-assisted breeding programs in wheat and provides a step stone for mapping genetic map, physical map and the eventual cloning.

Molecular Markers for Leaf Rust Resistance Gene Lr45 in Wheat Based on AFLP Analysis

Amplified fragment length polymorphism （AFLP） analysis was carried out in Thatcher, near isogenic lines （NILs） canting different genes conferring resistance against wheat leaf rust, and TcLr45 × Thatcher F2 progenies were used to develop markers for Lr45 gene. Sixty AFLP primer combinations were screened and most of them provided clear amplification products, 31 primer combinations displayed polymorphism of TcLr45 in 23 NILs. Two AFLP markers closely linked to the gene Lr45 were acquired： P-AGG/M-GAG261bp, which was found closely linked to the Lr45 locus at a distance of 0.6 cM on one side, and P-ACA/M-GGT105bp, which was found at a distance of 1.3 cM on the other side. The specific hands were cloned and subsequently sequenced. The 261-bp fragment produced by P-AGG/M-GAG showed 86% similarity with the sequence of Vulgate Hort I gene; the 105-bp fragment produced by P-ACA/M-GGT showed 96% similarity with the phosphatidylserine decarboxylase gene of the Triticum monococcum. Both included an open reading frame （ORF）.

Seedling and adult plant resistance to leaf rust in 46 Chinese bread wheat landraces and 39 wheat lines with known Lr genes

Wheat leaf rust,caused by Puccinia triticina(Pt),is an important foliar disease that has an important influence on wheat yield.The most economic,safe and effective way to control the disease is growing resistant cultivars.In the present study,a total of 46 wheat landraces and 34 wheat lines with known Lr(leaf rust resistance)genes were inoculated with 16Pt pathotypes for postulating seedling resistance gene(s)in the greenhouse.These cultivars and five wheat differential lines with adult plant resistance(APR)genes(Lr12,Lr22b,Lr34,Lr35 and Lr37)were also evaluated for identification of slow rusting resistance in the field trials in Baoding,Hebei Province of China in the 2014–2015 and 2015–2016 cropping seasons.Furthermore,10 functional molecular markers closely linked to 10 known Lr genes were used to detect all the wheat genotypes.Results showed that most of the landraces were susceptible to most of the Pt pathotypes at seedling stage.Nonetheless,Lr1 was detected only in Hongtangliangmai.The field experimental test of the two environments showed that 38 landraces showed slow rusting resistance.Seven cultivars possessed Lr34 but none of the landraces contained Lr37 and Lr46.Lr genes namely,Lr9,Lr19,Lr24,Lr28,Lr29,Lr47,Lr51 and Lr53 were effective at the whole plant stage.Lr18,Lr36 and Lr45 had lost resistance to part of pathotypes at the seedling stage but showed high resistance at the adult plant stage.Lr34 as a slowing rusting gene showed good resistance in the field.Four race-specific APR genes Lr12,Lr13,Lr35 and Lr37 conferred good resistance in the field experiments.Seven race-specific genes,Lr2b,Lr2c,Lr11,Lr16,Lr26,Lr33 and LrB had lost resistance.The 38 landraces showed slow rusting resistance to wheat leaf rust can be used as resistance resources for wheat resistance breeding in China.

PCR-based molecular markers linked to the leaf rust resistance gene Lr19 in different bread wheat cultivars

61 varieties of wheat collected in the gene fund of the Research Institute of Crop Husbandry were screened using SCAR-markers associated with the gene of resistance to brown leaf rust, Lr19. As a result of PCR analysis using SCS123 marker the 737 bp locus was detected in 48 genotypes. The expected fragment of the 688 bp was detected in 53 genotypes using the SCS253 marker. The results obtained using both markers indicate that the Lr19 gene is present on 7D chromosomes of 45 genotypes. The existence of the Lr19 gene has not been proven only for 5 from the 61 analyzed wheat genotypes.

Identification of AFLP Markers Linked to Leaf Rust Resistance Genes Using Near Isogenic Lines of Wheat

The present investigation was undertaken to find molecular markers linked to leaf rust resistance genes, Lr9 and Kharchia local mutant KLM4-3B. Preliminary AFLP analysis was carried out with different stocks, a survey of primer combinations with different selective nucleotide indicated that for each primer combination, the number of scorable loci ranged from 34 to 123. Only a limited primer combination used in the set of parental and near isogenic lines showed a high level of polymorphism for AFLP marker. Putative AFLP marker were found to be linked to Lr9, Lr19 and KLM4-3B. The alien genes were readily identified.

Molecular mapping of leaf rust resistance genes in the wheat line Yu 356-9

The Chinese wheat line Yu 356-9 exhibits a high level of resistance to leaf rust. In order to decipher the genetic base of resistance in Yu 356-9, gene postulation, inheritance analyses, and chromosome linkage mapping were carried out. Gene postulation completed using 15 leaf rust pathotypes and 36 isogenic lines indicated that Yu 356-9 was resistant to all pathotypes tested. F1 and F2 plants from the cross Yu 356-9 （resistant）/Zhengzhou 5389 （susceptible） were tested with leaf rust pathotype ＂FHNQ＂ in the greenhouse. Results indicated a 3：1 segregation ratio, indicative of the presence of a single dominant leaf rust resistance gene in Yu 356-9 which was temporarily designated as LrYu. Bulk segregant analysis and molecular marker assays were used to map LrYu. Five simple sequence repeat （SSR） markers on chromosome 2BS were found closely linked to LrYu. Among these markers, Xwmc770 is the most closely linked, with a genetic distance of 5.7 cM.

Status and strategies in breeding for rust resistance in wheat

Wheat along with rice and maize is fulfilling half of the calories demands of the world. Global Wheat production has increased tremendously since green revolution in 1960’s and helped in minimizing hunger and malnutrition. Developing countries, which consume 60% of the global wheat production, have shown a higher yield increase than the developed countries in the past [1]. It was driven by the hunger prevalence in these countries and was attributable to the introduction of high yielding and rusted resistant semi dwarf varieties developed under the collaborative efforts of International and National research systems during the last 50 years. Whereas, climate change and the emergence of new pests and diseases are threatening the food sustainability. The evolution of new races of disease pathogens like stem rust (Ug 99) is of serious concern. In order to feed the ever increasing population we have to increase wheat production at the rate 1.6% which can be achieved by developing high yielding varieties having a good tolerance level for biotic and abiotic stresses.

50个小麦品种抗叶锈基因鉴定

选育抗性品种是小麦叶锈病防治举措中最经济、可行的方法。为进一步挖掘抗病基因,选取河南、河北、山东等8个省小麦产区50个小麦品种。首先在苗期将16个叶锈菌生理小种(THFS、TGTS、THJS、FHKT、FGJN、KHKS、FCJQ、RFKS、THFM、MHGT、KHGS、KBGT、FHGT、PHHT、FHJT、FCJT)接种在36份小麦抗叶锈病近等基因系材料以及50个供试小麦品种上。因各菌种带有不同毒力,可根据表现型的差异,再将已知抗病基因紧密连锁的特异性分子标记与之结合分析,进而推测50份小麦材料中可能含有的抗叶锈病基因。通过基因推导、分子标记以及系谱分析综合鉴定抗锈性基因,结果表明,在50个品种中共检测出9个(Lr1、Lr2c、Lr10、Lr16、Lr26、Lr34、Lr37、Lr45和Lr46)已知抗叶锈性基因和少量未知基因。含有Lr1基因的有淄麦12等22个品种;含有Lr2c基因的有鲁麦14等10个品种;含有Lr10基因的只有莱州9361一个品种;含有Lr16基因的有科农199等25个品种;含有Lr26基因的有徐州24等15个品种;含有Lr34基因的有宝麦3号和京冬8号;含有Lr37基因的有淄麦12、鹤0927和荷9946;含有Lr45基因的有连麦2号等11个品种;含有Lr46基因的有山农19等38个品种。

Isolation and Characterization of NBS-LRR Class Resistance Homologous Gene from Wheat

One resistance gene analog fragment named RGA-CIN14 was isolated from TcLr19 wheat,which contains kinase-2,kinase-3a,and the GLPL motif of the NBS-spanning region,using degenerated primers according to the nucleotide binding site （NBS） conserved domain.Based on the RGA-CIN14,a full-length cDNA,CIN14,which was 2 987 bp encoding 880 amino acids,was obtained by using the method of the rapid amplification cDNA ends （RACE）.Bioinformatics analysis showed that the deduced amino acids of CIN14 protein consisted of a NB-ARC conserved domain and many leucine-rich repeats （LRR） domains.The phylogenetic tree analysis indicated a considerable identity of the protein encoded by CIN14 with that of wheat leaf rust resistance gene Lr1,but a lower similarity with Lr21.The expression profile of the CIN14 gene detected by semi-quantitative RT-PCR showed that the CIN14 gene was not induced by Puccinia triticina and it was a constitutive gene with low abundance in the wheat leaf tissue.The resistance homology sequence was successfully obtained,which provides the shortcut for cloning of the resistance gene in TcLr19 wheat.

小麦叶锈菌2个候选效应因子的克隆及其qRT-PCR分析

小麦叶锈病是由隐匿柄锈菌(Puccinia triticina,Pt)侵染的一种气传性真菌病害,对小麦造成严重的产量损失。Pt产生的吸器是产生效应因子的主要场所,而效应因子是Pt致病的关键。本研究基于早期构建的小麦叶锈菌单胞菌系13-5-28-1(JHKT)在休眠孢子、萌发夏孢子和侵染6 d的转录组文库,在系统筛选候选效应因子的基础上,选取两个候选效应因子Pt12448和Pt34354进行克隆,利用qRT-PCR分析候选效应因子的表达模式,结果发现其中Pt12448基因表现为后期诱导上调,Pt34354基因则为前期诱导上调。研究结果将推进对小麦叶锈菌致病机制的研究进程。

吡唑醚菌酯与氟环唑复配对小麦叶锈病的防治效果及对小麦的安全性

为筛选适用于小麦叶锈病防治的化学药剂,本研究选取吡唑醚菌酯和氟环唑及二者不同比例复配组合,采用喷雾接种法,测定了吡唑醚菌酯和氟环唑单剂及其不同复配比例在作为保护剂使用时对小麦叶锈病的室内防治效果,并测定了室内筛选确定的最佳比例复配药剂对小麦的安全性及对小麦叶锈病的田间防治效果。室内防效试验显示:吡唑醚菌酯、氟环唑及二者不同比例复配对小麦叶锈病病斑扩展有强烈的抑制作用,其中吡唑醚菌酯的抑制作用更强,其EC_(50)值为0.01μg/mL。联合毒力评价表明:所有复配组合均表现出协同相加作用,吡唑醚菌酯·氟环唑质量比50∶133复配时增效系数(SR)最大,为1.50。室内及田间防治试验表明:在所设浓度梯度范围内,防效与浓度呈正相关;在有效成分120 g/hm^(2)剂量下,26%吡唑醚菌酯·氟环唑悬浮剂(SC)的田间防效为85.12%,优于各自单剂处理。安全性评价结果显示:所有处理均能保证不同品种小麦正常生长,未发生药害现象。研究表明,吡唑醚菌酯·氟环唑复配对小麦叶锈病有很好的防治效果,可为生产上防治小麦叶锈病和科学用药提供理论依据。

39份外引小麦种质的抗叶锈病基因检测及其抗性鉴定

为明确39份外引小麦种质的抗叶锈病基因组成,利用与抗病基因紧密连锁或共分离的分子标记对抗叶锈病基因进行检测,并结合田间接种鉴定对种质材料的叶锈病抗性进行评价。结果表明,抗叶锈病基因Lr1、Lr10、Lr20、Lr24、Lr26、Lr34、Lr37和Lr38在39份种质中均有分布,频率分别为30.77%、17.95%、2.56%、5.13%、12.82%、30.77%、23.08%和5.13%,其中携带Lr24和Lr38的种质抗性良好。Lr34和Lr37单独存在时,种质对叶锈病的抗性分别表现为中感和高感,两者聚合时能够提高载体种质的抗性水平,表现为中抗。此外,4份种质材料(KPL-1、OK.95571、莫斯科39和非黑土地24)对叶锈病的抗性表现突出,均达到0;级,是抗叶锈病育种的重要抗源,其中KPL-1和OK.95571分别携带3个和5个抗叶锈病基因;莫斯科39和非黑土地24仅携带Lr1,推测这两份材料含有其他未检测的抗叶锈病基因。综上,较多的抗叶锈病基因聚合有利于提高种质的叶锈病抗性。

甘肃省小麦品种叶锈病抗性鉴定及评价

小麦叶锈病是由专性寄生菌(Puccinia triticina)引起的主要小麦病害之一。选育并种植抗病品种是防控小麦叶锈病最经济、有效和安全的方法。为了掌握甘肃省小麦品种的抗叶锈特点,为全省小麦抗叶锈病育种、品种合理布局以及小麦锈病的综合治理提供参考依据。2016—2017年度和2017—2018年度对1013份甘肃省小麦生产品种、高代材料、后备品种及抗源等不同小麦材料和33份甘肃省主栽小麦品种采用苗期室内接种和成株期田间接种的方法进行抗叶锈病性鉴定。结果表明,小麦种质资源中,苗期表现抗病的材料有301份,分布频率为31.19%;成株期表现抗病的材料380份,分布频率为44.92%。另外,筛选出10份全生育期抗叶锈病的材料,即2006-1-4-1-4-2-7-1-1-8、小黑麦CM-12、010-61-3-1-1、02-129-2-1-1-3-2-1、0439-6-5-1-1-1-1-2-1、9629-03A-3-2-1-1、09-104-1-3-1-1、01-29-7-1-1-2-1-3、SW-14和陇麦491。甘肃省主栽品种中,苗期表现抗病的有5份,分别为兰天22号、天选43号、天选67号、天选72号和中梁38号,分布频率为15.15%;成株期表现抗病的有12份,分别为陇中5号、天选46号和天选48号等,分布频率为36.36%。其中,中梁38号、天选67号和天选72号在全生育期表现出近免疫-中抗,未检测出对供试混合菌表现免疫的品种;此研究可为抗叶锈病鉴定材料在小麦育种中应用提供依据。

40份国内小麦品种抗叶锈性鉴定

为分析40份国内小麦材料所含有的抗叶锈病基因,本研究采用16个叶锈菌生理小种对其进行基因推导,推测待测材料抗叶锈病基因组成,并利用11个与己知抗病基因紧密连锁的特异性分子标记进行验证。进一步选取5个高毒力小种制成混合菌种,在保定试验田进行成株期接菌,筛选可能含有成株慢锈基因的小麦品种。综合系谱分析、基因推导、分子标记检测结果,在待测小麦品种中检测出6个已知抗叶锈病基因(Lr1、Lr11、Lr20、Lr26、Lr30、Lr37)。其中运黑14207等10个品种含有Lr1,禾美988和百农207含有Lr11,豫麦49和百农207含有Lr20,万丰269等23个品种含有Lr26,运黑14207和郑麦103含有Lr30,漯6073等4个品种含有Lr37,并且有的品种含有未知抗叶锈病基因。田间成株期抗性鉴定筛选出漯6073等7个品种具有慢锈性。本研究明确了部分品种的基因构成,发掘了优良抗源材料,可用于抗病育种及基因聚合。

75份国内外小麦品种抗叶锈性鉴定及基因分析

为了挖掘小麦抗叶锈病基因,为我国小麦抗叶锈病遗传育种基因库提供更多的选择。选取了75份国内外小麦材料以及36份已知抗叶锈基因载体品种,将这些材料在苗期分别接种14个不同毒力的叶锈菌生理小种,分别鉴定这些不同毒力的生理小种在小麦材料上的发病严重程度;同时提取所有材料的新鲜叶片DNA,选取已经确定的抗叶锈病基因相关联的分子标记特异性引物对75份供试材料进行分子标记检测。结合以上2种方法,推测75份小麦材料中含有的抗叶锈病基因。结果显示,75份小麦材料中含有Lr1、Lr2a、Lr2c、Lr10、Lr11、Lr14a、Lr16、Lr18、Lr20、Lr26、Lr34、Lr37、Lr46这13种已知抗叶锈病基因,这些基因以单基因或多基因聚合的方式存在于小麦品种中。其中,Lr1基因和Lr46基因占比较大,分别高达43%,56%。这些基因单独存在于小麦材料中,并不能表现出良好的抗叶锈性;当几个或者多个基因共同存在于小麦材料中,可以表现出远远高于单一基因的抗叶锈性,与前人研究相符。通过分子标记检测到小麦材料大白春小麦S3中含有Lr1、Lr10和Lr46基因,温室苗期鉴定发现该材料对本研究的11种叶锈生理小种表现出抗性,是比较良好的抗叶锈病小麦材料。