Development of a co-dominant SCAR marker linked to the Ph-3 gene for Phytophthora infestans resistance in tomato (Solanum lycopersicum)

Random amplified polymorphism DNA (RAPD) and bulk segregant analysis (BSA) approaches were used to characterize the molecular marker linked to the Phytophthora infestans resistance gene Ph-3 in tomato. A total of 800 RAPD primers were screened. One RAPD marker UBC#602 was identified to be tightly linked to the Ph-3 gene. The marker was successfully converted into a co-dominant sequence characterized amplified region (SCAR) marker. The SCAR marker SCU602 was used to analyze 96 F₂ progenies and fitted the expected 1:2:1 Mendelian segregation ratio. Forty one tomato inbred lines were screened using the SCAR marker in comparison with a reference marker linked to the Ph-3 gene and both markers gave the same results. SCU602 was further validated for association to resistance and its potential in MAS in 72 tomato lines and cultivars. The marker identified three genotypes harbouring the resistance allele. This SCAR marker can be used in breeding programs for the selection of the Ph-3 gene for Phytophthora infestans resistance.     

分子标记辅助选择小麦抗白粉病兼抗赤霉病聚合体

 Sumai 3, a wheat variety resistant to Fusarium head blight(FHB), was crossed with Neimai 9, a commercial wheat cultivar with the resistance to powdery mildew.The SCAR(sequence characterized amplified region) markers of powdery mildew resistance gene Pm21 and four SSR(simple sequence repeats)markers flanking the major FHB resistance QTL(Qfhs.ndsu-3BS) in Sumai 3 were used to detect the resistance loci by marker assisted selection(MAS) in the plants of the F₂ population.Identification of resistance to both powdery mildew and FHB in field showed that 12 plants resistant to both diseases were obtained.In addition, the agronomic traits of these plants were better than those of Sumai 3, and are perhaps the excellent parental materials for wheat breeding.     

Generation and Molecular Mapping of a Sequence Characterized Amplified Region Marker Linked with the Bct Gene for Resistance to Beet curly top virus in Common Bean

ABSTRACT A random amplified polymorphic DNA (RAPD) marker directly linked (0.0 cM) with a resistance gene was identified in a snap bean recombinant inbred population (Moncayo x Primo) consisting of 94 F(5:7) recombinant inbred lines that had uniform segregation for disease reaction to Beet curly top virus (BCTV) across three field locations. Resistance was conditioned by a single dominant allele tentatively designated Bct. Seven hundred and fifty decamer primers were screened to obtain the linked RAPD marker that was then converted to a sequence characterized amplified region (SCAR) marker SAS8.1550. The SCAR mapped within a cluster of resistance genes on linkage group B7 of the core map. A survey of 103 BCTV-resistant and -susceptible snap and dry bean genotypes was conducted using SAS8.1550. Results showed that the SCAR would be highly useful for marker-assisted selection of Bct in snap and dry bean originating from the Andean gene pool. Marker-assisted selection for Bct will expedite the development of BCTV-resistant cultivars and minimize the need for cumbersome pathogen tests.     

Identification and development of molecular markers linked to Phytophthora root rot resistance in pepper (Capsicum annuum L.)

Phytopthora root rot in pepper (C. annuum) is caused by Phytophthora capsici L., which exhibits a high level of pathogenic diversity. Resistance to this disease is conditioned by a number of quantitative trait loci. Pyramiding resistance alleles is desirable and could be simplified by the use of molecular markers tightly linked to the resistance genes. The purpose of this study was development of molecular markers linked to Phytophthora root rot resistance. An F₈ recombinant inbred line (RIL) population derived from a cross between YCM334 and a susceptible cultivar ‘Tean’ was used in combination with bulk segregant analysis utilizing RAPD and conversion of AFLP markers linked to Phytophtora root rot resistance into sequence-characterized amplified region (SCAR) markers. In conversion: one marker was successfully converted into a co-dominant SCAR marker SA133_4 linked to the trait. In bulked segregant analysis (BSA): three RAPD primers (UBC484, 504, and 553) produced polymorphisms between DNA pools among 400 primers screened. Genetic linkage analysis showed that the SCAR and RAPD markers were located on chromosome 5 of pepper. Quantitative trait locus (QTL) analysis showed that the SA133_4 and UBC553 were linked to Phytophtora root rot resistance. These markers were correctly identified as resistant or susceptible in nine promising commercial pepper varieties. These markers will be beneficial for marker-assisted selection in pepper breeding.     

Characterization of a resistance locus (Pfs-1) to the spinach downy mildew pathogen (Peronospora farinosa f. sp. spinaciae) and development of a molecular marker linked to Pfs-1

Downy mildew is a destructive disease of spinach worldwide. There have been 10 races described since 1824, six of which have been identified in the past 10 years. Race identification is based on qualitative disease reactions on a set of diverse host differentials which include open-pollinated cultivars, contemporary hybrid cultivars, and older hybrid cultivars that are no longer produced. The development of a set of near-isogenic open-pollinated spinach lines (NILs), having different resistance loci in a susceptible and otherwise common genetic background, would facilitate identification of races of the downy mildew pathogen, provide a tool to better understand the genetics of resistance, and expedite the development of molecular markers linked to these disease resistance loci. To achieve this objective, the spinach cv. Viroflay, susceptible to race 6 of Peronospora farinosa f. sp. spinaciae, was used as the recurrent susceptible parent in crosses with the hybrid spinach cv. Lion, resistant to race 6. Resistant F(1) progeny were subsequently backcrossed to Viroflay four times with selection for race 6 resistance each time. Analysis of the segregation data showed that resistance was controlled by a single dominant gene, and the resistance locus was designated Pfs-1. By bulk segregant analysis, an amplified fragment length polymorphism (AFLP) marker (E-ACT/M-CTG) linked to Pfs-1 was identified and used to develop a co-dominant Sequence characterized amplified region (SCAR) marker. This SCAR marker, designated Dm-1, was closely linked ( approximately 1.7 cM) to the Pfs-1 locus and could discriminate among spinach genotypes that were homozygous resistant (Pfs-1Pfs-1), heterozygous resistant (Pfs-1pfs-1), or homozygous susceptible (pfs-1pfs-1) to race 6 within the original mapping population. Evaluation of a wide range of commercial spinach lines outside of the mapping population indicated that Dm-1 could effectively identify Pfs-1 resistant genotypes; the Dm-1 marker correctly predicted the disease resistance phenotype in 120 out of 123 lines tested. In addition, the NIL containing the Pfs-1 locus (Pfs-1Pfs-1) was resistant to multiple races of the downy mildew pathogen indicating Pfs-1 locus may contain a cluster of resistance genes.     

A Molecular Marker Correlated with Selected Virulence Against the Tomato Resistance Gene Mi in Meloidogyne incognita, M. javanica, and M. arenaria

ABSTRACT Root-knot nematodes of the genus Meloidogyne are economically important pathogens of a wide range of crops. The tomato resistance gene Mi typically confers resistance to the three major species, M. incognita, M. javanica, and M. arenaria. However, virulent populations completely overcoming the Mi resistance still occur. In an attempt to develop molecular markers for virulence against Mi and gain insights into the genetic relationships among virulent populations of different species and origins, random amplified polymorphic DNA (RAPD) analyses of laboratory-selected virulent, field virulent, and avirulent populations of M. incognita, M. javanica, and M. arenaria were carried out. A RAPD marker, specific for selected virulent populations, was identified, and subsequently, converted to a sequence characterized amplified region (SCAR). Sequence characterization of the SCAR locus showed that alleles from laboratory- and field-selected virulent populations were highly similar to each other and clearly different from alleles from natural virulent and avirulent populations. This result suggests that the genetic mechanism for virulence against Mi may be similar among selected virulent populations of the three Meloidogyne spp., but different between selected and natural virulent populations. Based on the nucleotide polymorphisms at the SCAR locus, codominant and dominant polymerase chain reaction-based markers were developed enabling rapid diagnosis of selected virulent genotypes in M. incognita, M. javanica, and M. arenaria.     

Identification of a major gene (Mex-1) from Coffea canephora conferring resistance to Meloidogyne exigua in Coffea arabica

Among the most damaging root-knot nematode species, Meloidogyne exigua is especially common in Latin America and constitutes a major agronomic constraint in all major coffee-growing ( Coffea arabica ) areas. Growing nematode-resistant coffee represents the most promising option for control of the pest. The present study aimed to determine the mode of inheritance of the M. exigua resistance transferred into C. arabica from a related species, Coffea canephora , and to identify associated molecular markers. Segregation data analysis of F ₂ progeny derived from a cross between the resistant introgression line T5296 and the susceptible accession Et6 showed that the resistance to M. exigua is controlled by a simply inherited major gene (designated the Mex -1 locus). The gall index distribution exhibited by the F ₂ individuals suggested incomplete dominant expression. Fourteen AFLP markers were found associated with the resistance to M. exigua and a localized genetic map of the chromosome segment carrying Mex -1 was constructed. Furthermore, the association of the identified AFLP markers with Mex -1 was confirmed by analysis of a set of genotypes involving 28 introgression Arabica lines either resistant or susceptible to M. exigua in field conditions. These results represent an important starting point to enhance backcross breeding programmes and to perform an early selection of resistant seedlings.     

小麦条锈菌新菌系V26的SCAR检测标记

 建立小麦条锈菌生理小种的快速分子检测技术体系对小麦条锈菌的监测和防治策略的制定具有重要价值。条锈菌V26是近年来出现的,对我国目前小麦抗病育种中普遍应用的抗条锈病基因Yr26具有毒性的新菌系。该菌系的出现,对我国当前小麦生产、抗病育种都造成了严重威胁。本研究选用189条随机引物对CYR29、CYR31、CYR32、CYR33、T4、Su11-4和V26等7个条锈菌生理小种(菌系)进行了扩增,筛选V26的特异性RAPD片段,并对其进行克隆和测序。根据测序结果,设计并合成SCAR特异性引物, 将V26的RAPD标记转化为稳定的SCAR标记。使得对该菌系的快速检测成为可能,同时也将会为条锈菌新小种的监测提供更为准确的科学依据。     

Evaluation of carrot resistance to alternaria leaf blight in controlled environments

The objective of this study was to find a technique for plant resistance screening to alternaria leaf blight (ALB), caused by the fungus Alternaria dauci , in controlled environments. Glasshouse and laboratory screening methods were compared using three cultivars and F₂ genotypes segregating for ALB resistance evaluated against self-pollinated F₃ field-grown plants. Plant disease was assessed through a disease index obtained from the size and number of symptoms on carrot leaves. The results indicated the value of glasshouse evaluation and the inadequacy of detached leaf and hypocotyl assays for carrot screening for ALB resistance. Spearman's rank correlation, applied to results obtained with both F₂ plants and their progeny, indicated that the optimal evaluation stage for ALB resistance in carrot is 20 days after inoculation. This test was powerful enough to be used as a prescreening test in breeding programmes.     

Identification and location of Stb9, a gene for resistance to septoria tritici blotch in wheat cultivars Courtot and Tonic

This study reports the discovery of a gene for resistance to septoria tritici blotch (STB) in two spring wheat cultivars, Courtot and Tonic. The gene, named Stb9 , confers resistance to Mycosphaerella graminicola isolate IPO89011. It was mapped by quantitative trait loci (QTL) analysis using an existing map of Courtot × Chinese Spring and was located between markers Xfbb226 (3·6 cM) and XksuF1b (9 cM) on the long arm of chromosome 2B. Markers linked to Stb9 in Courtot were then shown to be linked to resistance to IPO89011 in F₃ families of Tonic × Longbow. Allelism tests in which Tonic was crossed with Courtot confirmed that Tonic has a gene for resistance to IPO89011 at or very close to the Stb9 locus. SSR markers flanking Stb9 may be used in marker-assisted selection to introgress this gene into winter cultivars or in spring wheat breeding programmes outside Europe.     

Quantitative trait loci associated with isolate specific and isolate nonspecific partial resistance to Phoma macdonaldii in sunflower

Black stem, caused by Phoma macdonaldii , is one of the most important diseases of sunflower in the world. Quantitative trait loci (QTLs) implicated in partial resistance to two single pycnidiospore isolates of P. macdonaldii (MP8 and MP10) were investigated using 99 recombinant inbred lines (RILs) from the cross between sunflower parental lines PAC2 and RHA266. The experimental design was a randomized complete block with three replications. High genetic variability and transgressive segregation were observed among RILs for partial resistance to P. macdonaldii isolates. QTL-mapping was performed using a recently developed high-density SSR/AFLP sunflower linkage map. A total of 10 QTLs were detected for black stem resistance. The phenotypic variance explained by each QTL (R²) was moderate, ranging from 6 to 20%. Four QTLs were common between two isolates on linkage group 5 and 15 whereas the others were specific for each isolate. Regarding isolate-specific and isolate-nonspecific QTLs detected for partial resistance, it is evident that both genetic effects control partial resistance to the disease isolates. This confirms the need to consider different isolates in the black stem resistance breeding programmes. The four SSR markers HA3700, SSU25, ORS1097 and ORS523_1 encompassing the QTLs for partial resistance to black stem isolates could be good candidates for marker assisted selection.     

Response of interspecific Brassica juncea/Brassica rapa hybrids and their advanced progenies to Albugo candida (white blister)

Transfer of factors for resistance to white blister disease caused by Albugo candida between Brassica species involving two genotypes each of B. juncea and B. rapa was studied in hybrids. More hybrids were obtained by in vivo than in vitro techniques, although an in vitro phase was a prerequisite for the establishment of in vivo hybrids. Hybrids were identified by PCR-based inter-simple sequence repeat (ISSR) markers with both male and female species-specific bands being identified. There was a positive correlation between disease severity and number of days after sowing ( r  > 0·93), the highest being towards pod formation and plant maturity at 110 days after sowing. The plants from F₂ and BC₁ progeny showed higher resistance to A. candida than either of the parents. Plants of B. juncea and B. rapa with high field resistance (disease index < 1·0) were selected from BC₂ and F₂BC₁ generations. The frequency of plants classified as resistant in BC₂ progeny ranged from 4·5 to 39·0% in cross-combinations involving B. juncea genotypes as female parent, compared with 100% in the reciprocal cross involving B. rapa as female parent.     

感染“中四”小麦条锈菌T4菌系SCAR检测标记的开发

正条锈病是由小麦条锈菌(Puccinia striiformis f.sp.tritici)引起的世界范围内小麦上最重要的病害之一。利用抗病品种是防治该病最经济、有效的措施。但是由于小麦条锈菌的高度变异性,品种抗病性很容易被条锈菌新小种所克服。因此,持续监测条锈菌生理小种的动态变化,及时发现新小种,对     

Genetic analysis of partial resistance to bacterial leaf streak (Xanthomonas campestris pv. cerealis) in wheat

Genetic variability of partial resistance to bacterial leaf streak was investigated in hexaploid winter wheat ( Triticum aestivum. ), using 16 parental genotypes and 48 pure lines (F10) derived from a composite cross programme. Two experiments were undertaken in a controlled growth chamber. Seeds of all genotypes were grown under controlled conditions using a randomized block design with three replications. Each replication consisted of a row of 20 seedlings of each parent and pure line. An Iranian strain of bacterial leaf streak was used for the inoculation of 12-day-old seedlings. In a third experiment, eight genotypes from parents and F10 pure lines representing a large variability for partial resistance were inoculated with four other Iranian strains of bacterial leaf streak. A large genetic variability was observed amongst the 64 genotypes for partial resistance to the disease. Partial resistance heritability estimates were rather high (70%), indicating that the resistance factors may be transmitted by crossing. Amongst all genotypes investigated, 'DC²-30-N2' and 'IBPT-66' displayed the highest partial resistance to the disease. Significant correlations between strains in the third experiment show that a genotype resistant or susceptible to one strain will have similar reactions with other strains. No significant genetic gain was observed for partial resistance in the best pure line of the 48 lines studied, when compared with the best parental line. Increasing the number of pure lines is likely to result in the identification of genotypes that might prove to be more resistant.     

抗黄矮病小麦新品系的分子标记检测研究

本研究对以小麦-中间偃麦草异附加系L1和小麦-中间偃麦草部分双二倍体‘无芒中4’为抗源选育出的抗黄矮病小麦新品系进行分子检测和抗病性鉴定.通过应用RAPD、SSR、SCAR 3种分子标记OPF15、Xgwm37、SC-W37进行分子检测,并采用人工接种和大田自然感病的方式进行抗黄矮病鉴定,筛选到了‘93646’、‘2003-2’等高抗黄矮病的小麦新品系.分子检测抗黄矮病基因与田间抗病鉴定结果基本一致,应用的3种PCR标记都可以检测出抗病材料,但SCAR标记SC-W37特异性强、稳定性好,可在小麦抗黄矮病育种早代选择过程中发挥重要作用.     

Development of a Specific Polymerase Chain Reaction-Based Assay for the Identification of Fusarium oxysporum f. sp. ciceris and Its Pathogenic Races 0, 1A, 5, and 6

ABSTRACT Specific primers and polymerase chain reaction (PCR) assays that identify Fusarium oxysporum f. sp. ciceris and each of the F. oxysporum f. sp. ciceris pathogenic races 0, 1A, 5, and 6 were developed. F. oxysporum f. sp. ciceris- and race-specific random amplified polymorphic DNA (RAPD) markers identified in a previous study were cloned and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. Each cloned RAPD marker was characterized by Southern hybridization analysis of Eco RI-digested genomic DNA of a subset of F. oxysporum f. sp. ciceris and nonpathogenic F. oxysporum isolates. All except two cloned RAPD markers consisted of DNA sequences that were found highly repetitive in the genome of all F. oxysporum f. sp. ciceris races. F. oxysporum f. sp. ciceris isolates representing eight reported races from a wide geographic range, nonpathogenic F. oxysporum isolates, isolates of F. oxysporum f. spp. lycopersici, melonis, niveum, phaseoli, and pisi, and isolates of 47 different Fusarium spp. were tested using the SCAR markers developed. The specific primer pairs amplified a single 1,503-bp product from all F. oxysporum f. sp. ciceris isolates; and single 900- and 1,000-bp products were selectively amplified from race 0 and race 6 isolates, respectively. The specificity of these amplifications was confirmed by hybridization analysis of the PCR products. A race 5-specific identification assay was developed using a touchdown-PCR procedure. A joint use of race 0- and race 6-specific SCAR primers in a single-PCR reaction together with a PCR assay using the race 6-specific primer pair correctly identified race 1A isolates for which no RAPD marker had been found previously. All the PCR assays described herein detected up to 0.1 ng of fungal genomic DNA. The specific SCAR primers and PCR assays developed in this study clearly identify and differentiate isolates of F. oxysporum f. sp. ciceris and of each of its pathogenic races 0, 1A, 5, and 6.     

Transgressive segregation for resistance to Pyrenophora teres in barley

Populations of F₁, F₂, F₁× parent 1 (BC1), F₁× parent 2 (BC2), and F₃ resulting from two crosses between four susceptible varieties of barley used in national and international breeding programmes were tested at the seedling stage for their resistance to an isolate of Pyrenophora teres which is virulent to the parental varieties. Infection type, average lesion size, and number of lesions per unit leaf area were used to assess disease reaction. In the two crosses, F₂ and especially F₃ generations (produced by self pollination of selected resistant F₂ plants) showed more resistance expressed by infection type and average lesion size than either parent. The frequency distributions of F₂ and F₃ generations derived from these crosses were continuous and showed transgressive segregation for resistance. The results indicated that the four cultivars used in this study possessed additive genes for resistance to P. teres. Thus useful resistance could be obtained following crossing of susceptible cultivars and selection in succeeding generations.     

大白菜抗芜菁花叶病毒基因EST-PCR-RFLP分子标记的研究

 本试验以高抗芜菁花叶病毒C₃株系(TuMV-C₃)的高代自交系A₁56-2和感病自交系P9805杂交后代的F₂代为群体,根据大白菜的抗性相关的表达序列标签(EST)设计引物,利用分离群体分群分析法(BSA),筛选出2个与TuMV-C₃株系抗病基因紧密连锁的EST-PCR-RFLP分子标记BS300及BS160,遗传距离均为6.5 cM,为大白菜分子辅助育种、抗病基因克隆以及研究抗病基因编码特性等奠定基础。     

Resistance to Sphaerotheca pannosa in roses induced by 2,6-dichloroisonicotinic acid

The susceptible rose cv. Madelon and the partially resistant cv. Sonia both responded with reduced development of rose powdery mildew when they were treated with the synthetic inducer 2,6-dichloroisonicotinic acid (INA). The EC₅₀ for number of colonies cm⁻² was approximately 0.4 mg L⁻¹ in both cultivars when treated 4 days prior to inoculation. However, conspicuous differences were observed with respect to number of spores per cm². For sporulation, the EC₅₀ was 0.37 mg L⁻¹ in cv. Madelon and only 0.08 mg L⁻¹ in cv. Sonia. A comparison with the pathosystems cucumber/ Sphaerotheca fuliginea and red cabbage/ Peronospora parasitica is made and the importance of the observed phenomenon for the selection of parents in a breeding programme for (partial) resistance is discussed.     

Heterodera filipjevi许昌群体SCAR标记的建立

菲利普孢囊线虫Heterodera filipjevi是禾谷类作物上重要的病原线虫之一,严重影响禾谷类作物的产量和品质。本课题组前期研究发现菲利普孢囊线虫许昌群体是一个新的致病型,其在小麦上的致病力强于其他多个群体,危害更为严重。本研究旨在建立菲利普孢囊线虫许昌群体的快速、准确的分子检测体系,为Heterodera filipjevi许昌群体的监测和防控及抗病品种的选育利用奠定基础。本研究采用随机扩增多态性DNA(RAPD)和序列特征扩增区域(SCAR)的方法,对黄淮麦区5个重要小麦孢囊线虫致病型共9个线虫群体进行RAPD分析和SCAR标记转化,并通过增加除黄淮麦区外的线虫群体验证所获得的致病型相关分子标记的特异性和有效性。本研究共筛选了331条RAPD引物,筛选出2个菲利普孢囊线虫许昌群体相关的RAPD标记,引物S86可以扩增出1条约550 bp的多态性片段,引物S178可以扩增出1条约1 200 bp的多态性片段,并将这2个RAPD标记成功转化为SCAR标记。SCAR标记的特异性检测结果表明这两个SCAR标记只在许昌群体上有特异性扩增,可以用于许昌群体的分子检测。