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.     

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.     

Inheritance of mefenoxam resistance in Phytophthora nicotianae populations from a plant nursery

Three sexual crosses involving isolates of P. nicotianae with differing sensitivity to mefenoxam were established to study the inheritance of mefenoxam resistance. Mefenoxam sensitivity was determined by measuring the mycelial growth on both mefenoxam-amended clarified V8 agar and non-amended agar and then calculating the relative growth. When both parents had the same phenotype (both were resistant or both were sensitive), all F ₁ progeny had the parental phenotype and no segregation for a major effect gene was observed in sensitivity to mefenoxam. However, variations in the mycelial growth of progeny indicated the segregation of minor-effect genes. When the cross involving the mefenoxam-resistant isolate 3A4 and a sensitive parent, the F ₁ progeny segregated for mefenoxam resistance in a ratio of 1:1 (resistant: sensitive), indicating that the mefenoxam resistance is controled by a single dominant gene. Mating type was not linked to the mefenoxam-resistance gene locus. One RAPD marker linked in trans to the MEX locus was obtained by bulked segregant analysis using RAPD markers and was converted to a sequence characterized amplified region marker (SCAR). The SCAR maker identified in this study is a limited but useful tool for differentiating homozygous resistant isolates from sensitive isolates of P. nicotianae.     

西瓜抗小西葫芦黄花叶病毒基因的连锁分子标记研究

 小西葫芦黄花叶病毒中国株系(Zucchini yellow mosaic virus Chinese strain,ZYMV-CH)是危害我国西瓜的主要病毒。本实验以抗病毒病西瓜野生种质P.I.595203与感病的普通西瓜自交系98R为亲本,采用单粒传方式得到109个E代株系,分别对亲本、F₁及109个F₃代株系群体进行了苗期抗ZYMV-CH接种鉴定,通过F₃代群体的抗感分离情况,推测得到F₂代各单株的基因型,采用集团分离分析法(bulked segregant analysis,BSA)在F₂代建立抗感基因池,以亲本、F₁和抗感基因池为模板,对640条RAPD引物进行PCR扩增筛选,其中引物AK13在亲本、F₁和抗感基因池之间扩增出一条多态性片段(644bp),在F₂代群体上验证该多态性条带与ZYMV-CH的抗性基因呈现连锁关系,遗传连锁距离为8cM,定名为AK13_-644,该连锁标记在ZYMV-CH抗性转育后代自交系上得到了验证。最终将此RAPD标记成功转化成SCAR标记SCAK13_-644,该标记可以作为西瓜抗病毒病辅助选择的分子标记。     

Potato <Emphasis Type="Italic">R1</Emphasis> resistance gene confers resistance against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in transgenic tomato plants

Tomato is challenged by several pathogens which cause loss of production. One such pathogen is the oomycete Phytophthora infestans which is able to attack all the aerial parts of the plant. Although a wide range of resistance sources are available, genetic control of this disease is not yet successful. Pyramiding R-genes through genetic transformation could be a straightforward way to produce tomato and potato lines carrying durable resistance to P. infestans. In this work the R1 potato gene was transferred into tomato lines. The tomato transgenic lines were analyzed by using q-RT-PCR and progeny segregation to determine the gene copy number. To test the hypothesis that R1 represents a specifically regulated R-gene, transgenic tomato plants were inoculated with P. infestans isolate 88133 and IPO. All the plants containing the R1 gene were resistant to the late blight isolate IPO-0 and susceptible to isolate 88133. These results provide evidence for specific activation of the R1 gene during pathogen challenge. Furthermore, evidence for enhancement of PR-1 gene expression during P. infestans resistance response was obtained.     

Occurrence of Tomato spotted wilt virus (TSWV) in Jordan

Tomato spotted wilt virus was recorded for the first time in Jordan on tomato plants. Severe disease symptoms were observed in different tomato farms in the Jordan Valley. Using a specific primer pair a fragment of the capsid protein gene of the virus has been amplified by RT-PCR and IC-RT-PCR. The amplified PCR product was cloned and sequenced. Sequence analysis revealed that the Jordanian isolate of TSWV shared high nucleotide similarities with other isolates from different countries. The sequence of the capsid protein gene was deposited in GenBank under the accession number AY646682 . The response of different tomato breeding lines and hybrids, previously developed for resistance against Tomato yellow leaf curl virus (TYLCV) were tested for their reaction to TSWV infection. All tested lines and hybrids were susceptible to TSWV infection. This has been confirmed at the molecular level by using the SCAR 421 marker linked to the TSWV resistance gene Sw-5 .     

番茄抗晚疫病Ph-3基因RAPD标记 的克隆与CAPS标记的建立

目前,番茄晚疫病(Phytophthora infestans)在世界各番茄主产区普遍发生,并造成严重的经济损失,已经成为番茄生产的主要障碍之一.防御番茄晚疫病最有效的方法是培育抗病品种.依靠常规方法耗时、耗力,同时由于人为鉴定标准的差异,会直接影响鉴定结果,延长育种周期,分子标记技术的发展为解决此问题提供了可能.分子标记可被用于在苗期进行大量的抗病鉴定,不受时间、地域的限制,从而加速育种工作进程[1].Qiu等[2]已找到1条与抗晚疫病Ph-3基因连锁的RAPD标记,本研究旨在将此RAPD标记转化为稳定的CAPS标记,为抗晚疫病分子辅助选择育种(MAS)奠定良好的基础.     

N605ab,a specific molecular marker for <Emphasis Type="Italic">Phytophthora infestans</Emphasis>, distinguishes three genotypes in Japan

Random amplified polymorphic DNA (RAPD) analysis using the OPG-06 primer generated specific patterns for Japanese genotypes US-1, JP-1, and a new A1 (JP-2, JP-3, and JP-4) of Phytophthora infestans. N605, a specific RAPD fragment, was cloned and sequenced. PCR primers BD1/BD2 were constructed based on the N605 sequence and were used to clarify the genotypes. PCR products using the BD1/BD2 primers (N605ab marker) easily distinguished the new A1 from US-1 and JP-1. This technique provides a simple and effective method for rapid genotype discrimination that can be used in ecological experiments and forecasts for the occurrence of late blight.     

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.     

结球甘蓝抗TuMV基因的RAPD和SCAR标记研究

 One hundred and forty-four F₂ individuals from a cross combination between 1047 (susceptible) and A21 (resistant) were analyzed for the presence of random amplified polymorphic DNA (RAPD) markers linked to Turnip mosaic virus (TuMV) resistant gene in cabbage by using bulked segregant analysis (BSA).Two polymorphic markers were screened out of 200 random primers.These two RAPD fragments were linked to the resistant gene at 7.7 cM and 8.38 cM respectively and converted to SCAR markers successfully.     

Characterization of Egyptian <Emphasis Type="Italic">Phytophthora infestans</Emphasis> population using simple sequence repeat markers

The plant pathogenic oomycete, Phytophthora infestans, is the causal agent of late blight disease in tomato and potato. For characterizing Egyptian P. infestans isolates by DNA marker analysis, 40 isolates of P. infestans were collected from different locations in Egypt during two growing seasons (2012/2013 and 2013/2014). The 40 isolates were grouped into seven genotypes, in which 24 alleles were detected. The identified genotypes were not completely associated with geographic location and sample collection years. These results provide genetic and geographical information for developing a program to manage late blight disease.     

What is the evidence for sexual reproduction of Phytophthora infestans in Europe?

The biology of late blight of potato and tomato, caused by Phytophthora infestans, changed when sexual reproduction by the pathogen became possible in many parts of the world, including Europe. In northern Europe, especially Scandinavia, there is increasing evidence that the pathogen is reproducing sexually on a regular basis, although in other regions further south or to the west it appears to reproduce primarily in a clonal manner. The presence of both mating types, the production of viable oospores, and observations of fields with soilborne sources of inoculum are consistent with sexual reproduction. Studies with different marker systems have revealed a population structure without any dominating clonal lineages in Scandinavia, and that is most easily explained by sexual reproduction. Phytophthora infestans recovered from the soil can also be linked to parental genotypes using likelihood‐based methods when codominant markers are used. A synthesis of all the available data points to a second centre of sexual reproduction in northern Europe.     

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.     

Pyramiding Ty‐2 and Ty‐3 genes for resistance to monopartite and bipartite tomato leaf curl viruses of India

Breeding resistance to whitefly‐transmitted begomoviruses is an important goal of tomato breeding programmes worldwide. So far, resistance to begomoviruses in tomato has been achieved using wild species, and at least five resistance genes (Ty genes) have been studied. The present study was undertaken to combine Ty‐2 and Ty‐3 and to determine the effect of pyramiding on infection of tomato by three diverse begomovirus species. The diagnostic ability of the markers linked to Ty genes was assessed and marker‐assisted selection was used to develop pyramided tomato lines from the crosses between Ty stocks. Five stable pyramided tomato lines that differ in fruit morphology and yield potential were developed. The horticultural performance of pyramided lines in field trials showed that the yield and horticultural traits are well maintained in the plants. The response of these lines was assessed using agroinoculation and field tests in a disease hotspot. The pyramided lines and Ty‐3‐carrying lines exhibited a high level of resistance to the monopartite and two bipartite begomoviruses tested. The pyramided tomato lines developed in this study could be important genetic resources for sustainable tomato production in areas affected by tomato leaf curl virus disease. The combined results of disease resistance tests also showed that Ty‐3 is critical for achieving broad‐spectrum resistance. The limitations of relying on a single gene and the importance of pyramiding are discussed in the light of available evidence on frequent recombination in begomoviruses.     

Use of microsatellite marker Pi26 for rapid discrimination of five Japanese genotypes of Phytophthora infestans

Microsatellite markers were tested to rapidly discriminate five Japanese genotypes (US-1, JP-1, JP-2, JP-3, and JP-4) of Phytophthora infestans. Collected from 1958 to 2007, 111 isolates of Japanese P. infestans were examined using a fluorescent-labeled primer and capillary electrophoresis. Microsatellite marker Pi26 generated specific products for each genotype without any differences in terms of isolation area or year for a particular genotype. The Pi26 marker is a powerful tool for obtaining information on the structure of Japanese populations of P. infestans.     

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.     

Phytophthora species causing crown and root rot of tomato in southern Italy*

Phytophthora capsici, Phytophthora cryptogea and Phytophthora nicotianae were isolated from tomato plants with symptoms of crown and root rot in plastic‐house crops in Sicilia and Calabria (southern Italy). The species were identified primarily on the basis of morphological and cultural characteristics. The identification was confirmed using molecular methods, polyacrylamide gel electrophoresis (PAGE) of mycelial proteins and polymorphism of DNA sequences amplified by polymerase chain reaction using random primers (RAPD‐PCR). P. capsici caused significant losses in tomato crops that had succeeded capsicum crops. P. cryptogea was found to be the most frequent species causing basal stem rot of tomato, a disease of increasing importance in commercial tomato crops in plastic houses in Sicilia. P. nicotianae was common in plastic houses where poor drainage resulted in standing water.     

Effects of host and pathogen genotypes on inducibility of resistance in tomato (Solanum lycopersicum) to Phytophthora infestans

Thirteen tomato (Solanum lycopersicum) accessions were tested for inducibility of resistance against two isolates of Phytophthora infestans using BABA (dl ‐3‐amino butyric acid) as the inducing agent. In a more detailed trial, six of the accessions were assessed for inducibility of resistance to six P. infestans isolates on three leaves of different age per plant. Plants were inoculated 1 week after treatment with BABA. Area under the disease progress curve (AUDPC), sporulation capacity (SC) and infection efficiency (IE) were all affected by treatment with BABA. On leaves of all ages AUDPC was most affected by induction (43–100% reduction on the youngest leaves) followed by SC (14–100%) and IE (0–100% reduction). Tomato genotypes varied significantly in inducibility of resistance against P. infestans and the degree of induction generally decreased with increasing leaf age, whilst the absolute susceptibility with respect to AUDPC and SC rarely changed. The level of induction was not always related to the resistance level of the tomato accession and it was significantly influenced by the pathogen isolate used for challenge inoculation. The results show that inducibility of resistance is a selectable trait that is, however, isolate‐specific.     

Molecular mapping of the crown rust resistance gene rpc1 in barley

ABSTRACT Crown rust of barley, caused by Puccinia coronata var. hordei, occurs sporadically and sometimes may cause yield and quality reductions in the Great Plains region of the United States and Canada. The incompletely dominant resistance allele Rpc1 confers resistance to P. coronata in barley. Two generations, F(2) and F(2:3), developed from a cross between the resistant line Hor2596 (CIho 1243) and the susceptible line Bowman (PI 483237), were used in this study. Bulked segregant analysis combined with random amplified polymorphic DNA (RAPD) primers were used to identify molecular markers linked to Rpc1. DNA genotypes produced by 500 RAPD primers, 200 microsatellites (SSRs), and 71 restriction fragment length polymorphism (RFLP) probes were applied to map Rpc1. Of these, 15 RAPD primers identified polymorphisms between resistant and susceptible bulks, and 62 SSR markers and 32 RFLP markers identified polymorphisms between the resistant and susceptible parents. The polymorphic markers were applied to 97 F(2) individuals and F(2:3) families. These markers identified 112 polymorphisms and were used for primary linkage mapping to Rpc1 using Map Manager QT. Two RFLP and five SSR markers spanning the centromere on chromosome 3H and one RAPD marker (OPO08-700) were linked with Rpc1 and, thus, used to construct a 30-centimorgan (cM) linkage map containing the Rpc1 locus. The genetic distance between Rpc1 and the closest marker, RAPD OPO08-700, was 2.5 cM. The linked markers will be useful for incorporating this crown rust resistance gene into barley breeding lines.     

偃麦草属E染色体组特异SCAR标记对Lr19的特异性和稳定性研究

 小麦抗叶锈基因Lr19来源于长穗偃麦草,表现优良的抗叶锈性,国内外发现对Lr19有毒性的小麦叶锈菌菌株的报道较少。本研究以TcLr19和Thatcher亲本以及TcLr19×Thatcher F₂代单株构建的分离群体为材料,建立了与Lr19共分离的稳定的SCAR分子标记,命名为Y₁₉SCAR₉₈₂。对49个小麦抗叶锈近等基因系材料的稳定性检测结果表明,其重复性好且为Lr19特异的分子标记。对120个小麦品种检测的结果表明,该标记可有效应用于小麦抗叶锈分子辅助选择育种。