Mapping and Comparative Analysis of QTL for Crown Rust Resistance in an Italian x Perennial Ryegrass Population

ABSTRACT Crown rust (Puccinia coronata f. sp. lolli) is a serious fungal foliar disease of perennial ryegrass (Lolium perenne L.) and Italian ryegrass (L. multiflorum Lam.), which are important forage and turf species. A number of quantitative trait loci (QTL) for crown rust resistance previously were identified in perennial ryegrass under growth chamber or greenhouse conditions. In this study, we conducted a QTL mapping for crown rust resistance in a three-generation Italian x perennial ryegrass interspecific population under natural field conditions at two locations over 2 years. Through a comparative mapping analysis, we also investigated the syntenic relationships of previously known crown rust resistance genes in other ryegrass germplasms and oat, and genetic linkage between crown rust resistance QTL and three lignin genes: LpOMT1, LpCAD2, and LpCCR1. The interspecific mapping population of 156 progeny was developed from a cross between two Italian x perennial ryegrass hybrids, MFA and MFB. Because highly susceptible reactions to crown rust were observed from all perennial ryegrass clones, including two grandparental clones and eight clones from different pedigrees tested in this study, two grandparent clones from Italian ryegrass cv. Floregon appeared to be a source of the resistance. Two QTL on linkage groups (LGs) 2 and 7 in the resistant parent MFA map were detected consistently regardless of year and location. The others, specific to year and location, were located on LGs 3 and 6 in the susceptible parent MFB map. The QTL on LG2 was likely to correspond to those previously reported in three unrelated perennial ryegrass mapping populations; however, the other QTL on LGs 3, 6, and 7 were not. The QTL on LG7 was closely located in the syntenic genomic region where genes Pca cluster, Pcq2, Pc38, and Prq1b resistant to crown rust (P. coronata f. sp. avenae) in oat (Avena sativa L.) were previously identified. Similarly, the QTL on LG3 was found in a syntenic region with oat genes resistant to crown rust isolates PC54 and PC59. This indicates that the ortholoci for resistance genes to different formae speciales of crown rust might be present between two distantly related grass species, ryegrass and oat. In addition, we mapped four restriction fragment length polymorphism loci for three key ryegrass lignin genes encoding caffeic acid-O-methyltransferase, cinnamyl alcohol dehydrogenase, and cinnamoyl CoA-reductase on LG7. These loci were within a range of 8 to 17 centimorgans from the QTL on LG7, suggesting no tight linkage between them. The putative ortholoci for those lignin biosynthesis genes were identified on segments of rice (Oryza sativa L.) chromosomes 6 and 8, which are the counterparts of ryegrass LG7. Results from the current study facilitate understanding of crown rust resistance and its relationship with lignin biosynthesis, and also will benefit ryegrass breeders for improving crown rust resistance through marker-assisted selection.     

小麦品种天选43抗条锈性遗传分析及抗病基因的分子标记

 天选43是由8845-01-01-1-1和抗源材料贵农22杂交选育而成的普通小麦品种,对我国目前所有条锈菌生理小种均表现良好抗性。为明确其抗条锈性遗传基础,本研究选用当前条锈菌流行小种CYR32和CYR33,对天选43与感病品种铭贤169杂交F₁、F₂和F₃代群体进行遗传分析,同时应用460对SSR引物对接种CYR32的天选43/铭贤169 F₂代150个单株群体进行抗病基因定位。结果表明,天选43对CYR32抗性由1对显性基因控制,而对CYR33抗性由1对隐性基因控制。筛选到10个与抗CYR32基因连锁的SSR标记Xwmc134、Xgwm413、Xbarc187、Xwmc406、Xcfd65、Xgwm18、Xbarc181、Xbarc137、Xwmc419和Xgwm230,两侧距离目的基因最近的标记为Xgwm18和Xgwm413,遗传距离分别为0.8 cM和3.4 cM,并初步将其抗病基因定位于小麦染色体1BS上,暂命名为YrTx43。基因来源、抗病遗传分析、分子标记检测及染色体位点分析表明,YrTx43很可能是与Yr24、Yr26具有等位性的抗条锈基因。     

普通小麦-柔软滨麦草易位系M852-1抗条锈基因的遗传分析和分子作图

 M852-1是经杂交和回交培育的普通小麦-柔软滨麦草易位系,苗期对我国小麦条锈菌流行小种均表现良好抗性。为明确其抗条锈性遗传规律,本研究选用条锈菌流行小种（类型）CYR29、CYR32、CYR33和Su11-7的单孢菌系对其与铭贤169杂交F₁、F₂、F₃及BC₁代群体进行遗传分析, 同时应用420对SSR引物对接种CYR32的M852-1/铭贤169 F₂代144个单株作图群体进行抗病基因定位。结果表明,M852-1对供试小种均表现免疫或近免疫,对CYR29的抗锈性由1对显性基因控制,对CYR32、CYR33和Su11-7的抗锈性均由1对隐性基因控制。筛选到3个与抗CYR32基因连锁的SSR标记Xbarc124、Xbarc200和Xgwm429,遗传距离分别为6.3、5.6 和 9.7 cM。根据SSR标记锚定性将该基因定位于小麦2BS染色体,暂命名为YrM852。基因来源、分子标记检测及染色体位点分析表明,YrM852很可能是1个不同于目前已知抗条锈病基因的新基因。     

普通小麦-华山新麦草易位系9020-17-25-6抗条锈性遗传分析及分子标记

为明确普通小麦-华山新麦草易位系9020-17-25-6的抗条锈病基因及其遗传特点,利用中国条锈菌小种CYR29对9020-17-25-6、铭贤169及其杂交后代F1、F2、F3代进行苗期抗条锈性鉴定及遗传分析,选取48条RGAP引物和491对SSR引物对接种CYR29的F2代群体进行筛选,寻找与抗病基因连锁的分子标记。结果表明:9020-17-25-6对CYR29具有良好的抗条锈性,由1对显性基因独立控制,暂定名为Yr Hua9020。筛选到2个RGAP标记(M1和M2)和位于染色体3AS上的4个SSR标记(Xwmc11、Xwmc532、Xcfd79、Xgwm2)与Yr Hua9020连锁,与目的基因的遗传距离分别为6.9、9.5、17.8、12.2、7.2和17.8 c M。与已定位于3A染色体上的抗条锈病基因的比较研究表明,Yr Hua9020是一个与已知基因不同的新的抗条锈病基因。     

大麦抗条纹病基因的定位分析

为发掘大麦中抗条纹病的新基因,采用三明治法通过人工接种大麦条纹病菌Pyrenophora graminea强致病力菌株QWC对甘啤2号(免疫)与Alexis(高感)杂交F_1代及F_2代分离群体进行抗性遗传分析,利用群体分离分析法鉴定与抗病基因连锁的SSR标记,并通过QTL IciMapping软件构建遗传连锁图谱完成对抗病基因的定位。结果显示,甘啤2号与Alexis杂交F_1代对大麦条纹病菌强致病力菌株QWC表现为免疫,F_2代表现3∶1抗感分离,表明甘啤2号对菌株QWC的抗性由1个显性抗性基因控制,将该抗病基因暂命名为Rdg3;该基因位于大麦7H染色体上的SSR标记Bmag206和Bmag7之间,与二者的遗传距离分别为1.78 cM和2.86 cM。经与已定位于7H染色体上的抗病基因比较,发现Rdg3是一个新的抗条纹病基因,可作为大麦抗病育种的新种质资源。     

EPSPS gene amplification in glyphosate-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) from Arkansas

BACKGROUND: Resistance to glyphosate in weed species is a major challenge for the sustainability of glyphosate use in crop and non‐crop systems. A glyphosate‐resistant Italian ryegrass population has been identified in Arkansas. This research was conducted to elucidate its resistance mechanism. RESULTS: The investigation was conducted on resistant and susceptible plants from a population in Desha County, Arkansas (Des03). The amounts of glyphosate that caused 50% overall visual injury were 7 to 13 times greater than those for susceptible plants from the same population. The EPSPS gene did not contain any point mutation that has previously been associated with resistance to glyphosate, nor were there any other mutations on the EPSPS gene unique to the Des03 resistant plants. The resistant plants had 6‐fold higher basal EPSPS enzyme activities than the susceptible plants, but their I₅₀ values in response to glyphosate were similar. The resistant plants contained up to 25 more copies of EPSPS gene than the susceptible plants. The level of resistance to glyphosate correlated with increases in EPSPS enzyme activity and EPSPS copy number. CONCLUSION: Increased EPSPS gene amplification and EPSPS enzyme activity confer resistance to glyphosate in the Des03 population. This is the first report of EPSPS gene amplification in glyphosate‐resistant Italian ryegrass. Other resistance mechanism(s) may also be involved. Copyright © 2012 Society of Chemical Industry     

小偃6号成株期高温抗条锈性遗传分析

为揭示小偃6号抗病机制和培育持久抗病品种,采用常规杂交分析方法,在小麦抽穗期利用小麦条锈菌小种CYR30、CYR32和Su11-4对小偃6号、铭贤169及其杂交F1、F2、F2∶3接种,平均气温达到21℃时对小偃6号进行了抗条锈性调查和遗传分析。结果显示,接种CYR30、CYR32时,F1代表现高感,F2代群体中抗感分离比例符合1 R∶15 S的理论比例。接种Su11-4时,F1代表现高抗,F2代群体中抗感分离比例符合3R∶1S的理论比例。研究表明小偃6号对CYR30、CYR32的抗病性均由2对隐性基因累加作用控制,对Su11-4的抗病性由1对显性基因控制。     

Assessing new SSR markers for utility and informativeness in genetic studies of brown rust fungi on wheat,triticale, and rye

The aim of the present study was to validate new simple-sequence repeat (SSR) markers and use them to assess genetic variability among 24 isolates of Puccinia triticina collected from wheat (Pt-wheat) and triticale (Pt-triticale), and 15 isolates of P. recondita f. sp. secalis (Prs) collected from rye. The Pt and Prs isolates were tested for virulence on a set of 35 Thatcher wheat near-isogenic lines, eight rye lines with known resistance genes, and 53 triticale cultivars with uncharacterized leaf rust resistance. Molecular genotypes were determined using a newly developed set of 34 SSR microsatellite primer pairs. All SSR markers tested on Pt isolates successfully amplified fragments of appropriate size. When tested on the Prs isolates, 21 out of the 34 Pt SSRs amplified expected fragments. Sixteen of these 21 SSRs were polymorphic, providing for the first time microsatellite markers to study genetic variation in Prs. Based on virulence data, variation among Prs isolates was low, probably due to the small number of rye differential lines available. Much higher variation for virulence was observed within the collection of Pt isolates from wheat and triticale, and two separate groups were established with mixed host origin. Substantial genetic variation was detected among the isolates studied with the SSR markers, assuming two different models of SSR evolution (infinite alleles model and stepwise mutation model). The newly developed set of SSR markers proved their effectiveness in detecting genetic variation and should be useful in further population genetics investigations of the two pathogens.     

Genetic differentiation of the wheat leaf rust fungus Puccinia triticina in Europe

The objective of this study was to determine whether genetically differentiated groups of Puccinia triticina are present in Europe. In total, 133 isolates of P. triticina collected from western Europe, central Europe and Turkey were tested for virulence on 20 lines of wheat with single leaf rust resistance genes, and for molecular genotypes with 23 simple sequence repeat (SSR) markers. After removal of isolates with identical virulence and SSR genotype within countries, 121 isolates were retained for further analysis. Isolates were grouped based on SSR genotypes using a Bayesian approach and a genetic distance method. Both methods optimally placed the isolates into eight European (EU) groups of P. triticina SSR genotypes. Seven of the groups had virulence characteristics of isolates collected from common hexaploid wheat, and one of the groups had virulence characteristics of isolates from tetraploid durum wheat. There was a significant correlation between the SSR genotypes and virulence phenotypes of the isolates. All EU groups had observed values of heterozygosity greater than expected and significant fixation values, which indicated the clonal reproduction of urediniospores in the overall population. Linkage disequilibria for SSR genotypes were high across the entire population and within countries. The overall values of R_ST and F_ST were lower when isolates were grouped by country, which indicated the migration of isolates within Europe. The European population of P. triticina had higher levels of genetic differentiation compared to other continental populations.     

春小麦品种墨波成株期抗条锈基因遗传解析

为明确春小麦品种墨波成株期抗条锈性遗传基础,以墨波与感病品种Taichung29(T29)杂交创建F_(2∶3)分离群体,通过青海西宁市和海东市2个试验点2年田间病圃鉴定,应用植物数量性状主基因+多基因混合遗传模型单个分离世代分析方法对墨波/T29 F2群体的抗性遗传效应进行了分析。结果发现群体单株/家系的病害严重度和反应型在2个试验点均未呈现连续性分布,但是在不同区段内,群体株系间又表现出较明显的连续性变异,初步推测,墨波成株期对小麦条锈病抗性具有由主效基因和微效基因共同控制的特征;遗传分析结果表明,墨波的成株期抗条锈性最优遗传模型均为2对主基因遗传,并受微效基因影响,在海东市试验点用反应型数据分析得到的最优遗传模型为C-6模型2MG-EEAD,即2对等显性主基因遗传,在海东市及西宁市试验点用严重度数据分析得到的最优遗传模型均为C-1模型2MG-ADI,即2对主基因加性-显性-上位性遗传。     

Mendelian inheritance of rust resistance to Melampsora larici‐epitea in crosses between Salix sachalinensis and S. viminalis

Two F₁, two F₂ and two backcross (BC) full‐sib families of Salix sachalinensis × S. viminalis were tested for resistance to two pathotypes of Melampsora larici‐epitea in leaf‐disc inoculation experiments. Two single‐pustule isolates, VM and ST, belonging to pathotypes LET1 and LET5, respectively, were used in the tests. Disease was scored based on the number of uredinia, uredinial diameter and inoculum densities. Both F₁ families were completely resistant to both VM and ST. Resistance to VM segregated at a 9:6:1 ratio in the F₂ families and at a 1:2:1 ratio in the BC families, suggesting that two independently segregating genes controlled rust resistance, with resistance dominant over susceptibility. This also indicates incomplete dominance of the resistance alleles over the susceptibility to VM. For ST, the equivalent ratios were 3:1 and 1:1, showing that a single dominant gene was responsible for rust resistance. The broad sense heritabilities were >0·91 for uredinial diameter and 0·1–0·33 for the number of uredinia. There were significant overall correlations between data from inoculations with VM and those from inoculations with ST in the number of uredinia, uredinial diameter and disease scores (Spearman’s rank correlation coefficients = 0·31–0·75).     

Identification of allele specific AFLP markers linked with bacterial wilt [Ralstonia solanacearum (Smith) Yabuuchi et al.] resistance in hot peppers (Capsicum annuum L.)

Even though the bacterial wilt is identified as the most destructive disease in hot peppers world-wide, robust molecular markers that facilitate marker assisted selection are absent till date. Kerala Agricultural University (India) has released two hot peppers named Ujwala and Anugraha which show high level field resistance to this pathogen. The variety Anugraha was developed through backcross breeding between a high yielding but highly susceptible variety Pusa Jwala with the highly resistant Ujwala, using Pusa Jwala as a recurrent parent. Thus, Pusa Jwala and Anugraha are near isogenic lines (NILs) differing for the resistance to bacterial wilt only and the resistance is governed by a homozygous recessive (rr) gene action. The F₁s of Anugraha × Pusa Jwala were selfed to generate the segregating F₂ population. The F₂ population has been field screened, 10 highly susceptible and 10 most resistant plants were identified and DNA from these plants were bulked separately. Bulked segregant analysis with AFLP primer combination EcoACT + MseCAC was done using the DNA from donor parent Ujwala, susceptible parent Pusa Jwala, resistant parent Anugraha, bulked susceptible F₂ and bulked resistant F₂ plants. On resolution using capillary electrophoresis system in genetic analyzer, the AFLP products have yielded three polymorphic bands (103, 117, and 161 bp) which were linked with the resistant recessive allele and three polymorphic bands (183, 296, 319 bp) linked with the dominant susceptible allele of the bacterial wilt resistance gene. The results were confirmed through co-segregation analysis in most resistant and susceptible plants of F₂ segregating population.     

Genetic structure and diversity of Phakopsora pachyrhizi isolates from soyabean

Simple sequence repeat (SSR) markers were used to classify 116 isolates of Phakopsora pachyrhizi, the cause of soyabean rust, collected from infected soyabean leaves in four agroecological zones in Nigeria. A high degree of genetic variation was observed within the sampled populations of P. pachyrhizi. Eighty‐four distinct genotypes were identified among three of the four agroecological zones. Nei’s average genetic diversity across geographical regions was 0·22. Hierarchical analysis of molecular variance showed low genetic differentiation among all populations of P. pachyrhizi. The majority (> 90%) of the genetic diversity was distributed within each soyabean field, while approximately 6% of the genetic diversity was distributed among fields within geographic regions. Low population differentiation was indicated by the low F_ST values among populations, suggesting a wide dispersal of identical genotypes on a regional scale. Phylogenetic analysis indicated a strictly clonal structure of the populations and five main groups were observed, with group II accounting for 30% of the entire population. Because of the asexual reproduction of P. pachyrhizi, single‐step mutations in SSR genotypes are likely to account for the genetic differences within each group.     

抗条锈病小麦种质WS4-8的抗性鉴定及遗传

为明确小麦体细胞无性系4-8(WS4-8)抗条锈病的遗传稳定性及抗性遗传特点,采用基因推导、抗性鉴定、遗传分析等方法对其进行了抗条锈性的鉴定和等位性分析。结果表明,WS4-8所携带的抗性基因与已知抗性基因不同;WS4-8的条锈病抗性表现优异,遗传稳定;用CY33小种对WS4-8和铭贤169的正交、反交组合F1和F2代植株人工接种鉴定表明,F1全部抗病,F2群体符合3R∶1S单基因控制的抗性遗传规律,WS4-8对CY33的抗性由1对显性核基因控制;用CY33对WS4-8分别与Yr5/6×Avocet S、Yr10/6×Avocet S、Yr15/6×Avocet S及92R137(Yr26)组配的杂交组合F1及F2代植株人工接种鉴定表明,F1全部抗病,而F2中有感病植株,说明WS4-8所携带的抗条锈病基因与Yr5、Yr10、Yr15、Yr26不等位。研究表明,WS4-8的抗条锈性是由1对显性核基因控制,与已知抗性基因不同,可能是一个新的抗条锈病基因。     

Validation of a QTL for resistance to ascochyta blight linked to resistance to fusarium wilt race 5 in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Ascochyta blight caused by Ascochyta rabiei and fusarium wilt caused by Fusarium oxysporum. f. sp. ciceris are the two most serious diseases of chickpea (Cicer arietinum). Quantitative trait loci (QTL) or genes for ascochyta blight resistance and a cluster of resistance genes for several fusarium wilt races (foc1, foc3, foc4 and foc5) located on LG2 of the chickpea map have been reported independently. In order to validate these results and study the linkage relationship between the loci that confer resistance to blight and wilt, an intraspecific chickpea recombinant inbred lines (RIL) population that segregates for resistance to both diseases was studied. A new LG2 was established using sequence tagged microsatellite sites (STMS) markers selected from other chickpea maps. Resistance to race 5 of F. oxysporum (foc5) was inherited as a single gene and mapped to LG2, flanked by the STMS markers TA110 (6.5 cM apart) and TA59 (8.9 cM apart). A QTL for resistance to ascochyta blight (QTL_AR3) was also detected on LG2 using evaluation data obtained separately in two cropping seasons. This genomic region, where QTL_AR3 is located, was highly saturated with STMS markers. STMS TA194 appeared tightly linked to QTL_AR3 and was flanked by the STMS markers TR58 and TS82 (6.5 cM apart). The genetic distance between foc5 and QTL_AR3 peak was around 24 cM including six markers within this interval. The markers linked to both loci could facilitate the pyramiding of resistance genes for both diseases through MAS.     

Genotypic Diversity of the Wheat Leaf Blotch Pathogen Mycosphaerella Graminicola (anamorph) Septoria Tritici in Germany

The population structure and genotypic diversity of Mycosphaerella graminicola from six natural field populations in Germany were studied with molecular markers. To reveal the potential effects of plant host resistance on the pathogen population, hierarchical samples were taken from susceptible and resistant cultivars. A total of 203 single spore isolates was subjected to molecular marker analysis using the amplified fragment length polymorphism technique (AFLP). Among the 203 isolates analyzed, 142 different multilocus haplotypes (MLH) were identified revealing a high degree of genotypic diversity of the M. graminicola population. On average, a F _ST value of 0.04 was found, indicating a low genetic differentiation with only 4% of the genetic variation between the local populations but leaving 96% of the genetic variation within the populations. According to the low F _ST value, a high migration rate of Nm 12 was found. The observed high within-population diversity, and the significant migration between populations, prevented genetic isolation and differentiation of putative geographically separated populations. Furthermore, plant host resistance had no obvious effect on the population structure and diversity of M. graminicola. Genotypic variability can be attributed to sexual recombination which appears to have a considerably larger influence on the population structure. Gene flow on this scale could have significant implications for plant breeding and fungicide spraying programmes.     

Genetic analysis of host–pathogen incompatibility between Lolium isolates of Pyricularia oryzae and wheat

Lolium isolate TP2 of Pyricularia oryzae, causal agent of gray leaf spot of perennial ryegrass (Lolium perenne), is virulent on perennial ryegrass, but avirulent on wheat cultivars. Genetic analysis of wheat F₂ populations revealed that the resistance of wheat cultivars Chinese Spring, Shin-chunaga, and Norin 4 to TP2 was conditioned by two genes, R1 and R2. R1 was highly effective, while R2 was less effective. The strong resistance gene R1, designated Rmg6, was mapped on chromosome 1D using microsatellite markers. For revealing genetic mechanisms of avirulence, TP2 was crossed with Triticum isolate Br48. Segregation analysis of their F₁ progenies revealed that the avirulence of TP2 on the three wheat cultivars was conditioned by two unlinked genes, one (A1) highly effective and the other (A2) less effective. These results suggest that the incompatibility between TP2 and the common wheat cultivars is conditioned by two gene pairs; the Rmg6–A1 interaction results in strong resistance, and the R2–A2 interaction results in moderate resistance.     

小麦-滨麦易位系M8657-1抗条锈病基因遗传分析和分子标记

 M8657-1, one of the wheat translocation lines derived from Leymus mollis Trin. Hara, is possessed of effective resistance at all stages to Su-ll and other dominant races of Puccinia striiformis f. sp. tritici in China. Seedlings of the parents, F₁, and F₂ progeny derived from the cross of M8657-1 (resistant) Mingxian169 (susceptible) were inoculated with Su-ll in greenhouse to identify and map the probable new stripe rust resistance gene. The results suggested that the stripe rust resistance in M8657-1 was conferred by a pair of recessive genes. Simple sequence repeat (SSR) technique was used to detect molecular marker associated with the resistance gene:208 pairs of wheat SSR primers were used to screen the two parents, as well as resistant and susceptible bulks and then three SSR markers were selected for genotyping the F₂ population. The geue, temporarily designated as YrLml, was found to be located on the chromosome 7DL and flanked by three SSR markers GDM67, WMC150 and WMC671, with the genetic distance of 5.0, 9.7 and 11.8cM, respectively.     

普通小麦-柔软滨麦草易位系M97抗条锈基因的遗传分析和分子作图

为了明确M97抗条锈性遗传规律,在苗期用7个小麦条锈菌系对M97与感病品种铭贤169的杂交后代F1、F2、F3和BC1代进行抗条锈性遗传分析,并对M97抗Sun11-4的抗条锈基因进行SSR分子标记。M97对Sun11-4和Sun11-11的抗病性均由1对显性基因控制,对CY29、CY30、CY33的抗病性由1显1隐2对基因共同控制,对CY31的抗病性由2对显性基因独立或重叠作用控制。以接种Sun11-4的F2代分离群体构建作图群体,筛选到Xwmc222、Xwmc147、Xbarc229和Xwmc339等4个与抗病基因连锁的SSR标记,其遗传距离分别为3.4、4.8、7.6和12.1 cM。将该抗病基因定位于小麦1DS染色体,且该基因不同于已知的抗条锈基因,暂命名为YrM97。用YrM97两侧遗传距离最近的2个标记Xwmc222和Xwmc147对42个黄淮麦区主栽小麦品种进行分子检测,仅有9.5%的品种具有与YrM97相同的标记位点。