Genetic and physical fine mapping of Scmv2, a potyvirus resistance gene in maize

Sugarcane mosaic virus (SCMV) is an important virus pathogen both in European and Chinese maize production, causing serious losses in grain and forage yield in susceptible cultivars. Two major resistance loci confer resistance to SCMV, one located on chromosome 3 (Scmv2) and one on chromosome 6 (Scmv1). We developed a large isogenic mapping population segregating in the Scmv2, but not the Scmv1 region, to minimize genetic variation potentially affecting expression of SCMV resistance. We fine mapped Scmv2 to a region of 0.28 cM, covering a physical distance of 1.3426 Mb, and developed six new polymorphic SSR markers based on publicly available BAC sequences within this region. At present, we still have three recombinants left between Scmv2 and the nearest polymorphic marker on either side of the Scmv2 locus. The region showed synteny to a 1.6 Mb long sequence on chromosome 12 in rice. Analysis of the public B73 BAC library as well as the syntenic rice region did not reveal any similarity to known resistance genes. However, four new candidate genes with a possible involvement in movement of virus were detected.     

Analysis of sugarcane mosaic virus resistance in maize in an isogenic dihybrid crossing scheme and implications for breeding potyvirus-resistant maize hybrids.

The gene action of 2 sugarcane mosaic virus (SCMV) resistance loci in maize, Scmv1 and Scmv2, was evaluated for potyvirus resistance in an isogenic background. All 4 homozygous and 5 heterozygous isogenic genotypes were produced for introgressions of the resistant donor (FAP1360A) alleles at both loci into the susceptible parent (F7) genetic background using simple sequence repeat markers. For SCMV and maize dwarf mosaic virus (MDMV), virus symptoms appeared rapidly in the 3 homozygous genotypes, with susceptibility alleles fixed at 1 or both loci. Although the 9 isogenic genotypes revealed a high level of resistance to Zea mosaic virus (ZeMV), the same 3 homozygous genotypes were only partially resistant. This indicates that 1 resistance gene alone is not sufficient for complete resistance against SCMV, MDMV, and ZeMV. Scmv1 showed strong early and complete dominant gene action to SCMV, but it gradually became partially dominant. Scmv2 was not detected at the beginning, showing dominant gene action initially and additive gene action at later stages. Both genes interacted epistatically (for a high level of resistance, at least 1 resistance allele at each of both loci is required). This implies that double heterozygotes at the 2 loci are promising for producing SCMVresistant hybrids. Results are discussed with respect to prospects for isolation of SCMV and MDMV resistance genes.     

The Pic19 NBS-LRR gene family members are closely linked to Scmv1, but not involved in maize resistance to sugarcane mosaic virus

Sugarcane mosaic virus (SCMV) is the causal pathogen for a severe mosaic virus disease of maize worldwide. In our previous research, the maize resistance gene analog (RGA) Pic19 and its three cognate BAC contigs were mapped to the same region as the SCMV resistance gene Scmv1. Here we report the isolation and characterization of the Pic19R gene family members from the inbred line FAP1360A, which shows complete resistance to SCMV. Two primer pairs were designed based on the conserved regions among the known Pic19 paralogs and used for rapid amplification of cDNA ends of FAP1360A. Six full-length cDNAs, corresponding to the Pic19R-1 to -6 paralogs, were obtained. Three of them (Pic19R-1 to -3) had uninterrupted coding sequences and were, therefore, regarded as candidates for the Scmv1 gene. A total of 18 positive BAC clones harboring the Pic19R-2 to -5 paralogs were obtained from the FAP1360A BAC library and assembled into two BAC contigs. Two markers, tagging Pic19R-2 and -3 and Pic19R-4, were developed and used to genotype a high-resolution mapping population segregating solely for the Scmv1 locus. Although closely linked, none of these three Pic19R paralogs co-segregated with the Scmv1 locus. Analysis of the Pic19R family indicated that the Pic19R-1 paralog is identical to the known Rxo1 gene conferring resistance to rice bacterial streak disease and none of the other Pic19R paralogs seems to be involved in resistance to SCMV.     

Identification of genetically linked RGAs by BAC screening in maize and implications for gene cloning,mapping and MAS

The resistance gene analogue (RGA) pic19 in maize, a candidate for sugarcane mosaic virus (SCMV) resistance gene (R gene) Scmv1, was used to screen a maize BAC library to identify homologous sequences in the maize genome and to investigate their genomic organisation. Fifteen positive BAC clones were identified and could be classified into five physically independent contigs consisting of overlapping clones. Genetic mapping clustered three contigs into the same genomic region as Scmv1 on chromosome 6S. The two remaining contigs mapped to the same region as a QTL for SCMV resistance on chromosome 1. Thus, RGAs mapping to a target region can be successfully used to identify further-linked candidate sequences. The pic19 homologous sequences of these clones revealed a sequence similarity of 94-98% on the nucleotide level. The high sequence similarity reveals potential problems for the use of RGAs as molecular markers. Their application in marker-assisted selection (MAS) and the construction of high-density genetic maps is complicated by the existence of closely linked homologues resulting in 'ghost' marker loci analogous to 'ghost' QTLs. Therefore, implementation of genomic library screening, including genetic mapping of potential homologues, seems necessary for the safe application of RGA markers in MAS and gene isolation.     

Development of RGA-CAPS markers and genetic mapping of candidate genes for sugarcane mosaic virus resistance in maize

Three previously published resistance gene analogues (RGAs), pic13, pic21 and pic19, were mapped in relation to sugarcane mosaic virus (SCMV) resistance genes ( Scmv1, Scmv2) in maize. We cloned these RGAs from six inbreds including three SCMV-resistant lines (D21, D32, FAP1360A) and three SCMV-susceptible lines (D145, D408, F7). Pairwise sequence alignments among the six inbreds revealed a frequency of one single nucleotide polymorphism (SNP) per 33 bp for the three RGAs, indicating a high degree of polymorphism and a high probability of success in converting RGAs into codominant cleaved amplified polymorphic sequence (CAPS) markers compared to other sequences. SNPs were used to develop CAPS markers for mapping of the three RGAs in relation to Scmv1 (chromosome 6) and Scmv2 (chromosome 3), and for pedigree analyses of resistant inbred lines. By genetic mapping pic21 was shown to be different from Scmv2, whereas pic19 and pic13 are still candidates for Scmv1 and Scmv2, respectively, due to genetic mapping and consistent restriction patterns of ancestral lines.     

Saturation of two chromosome regions conferring resistance to SCMV with SSR and AFLP markers by targeted BSA

Quantitative trait loci (QTLs) and bulked segregant analyses (BSA) identified the major genes Scmv1 on chromosome 6 and Scmv2 on chromosome 3, conferring resistance against sugarcane mosaic virus (SCMV) in maize. Both chromosome regions were further enriched for SSR and AFLP markers by targeted bulked segregant analysis (tBSA) in order to identify and map only markers closely linked to either Scmv1 or Scmv2. For identification of markers closely linked to the target genes, symptomless individuals of advanced backcross generations BC5 to BC9 were employed. All AFLP markers, identified by tBSA using 400 EcoRI/ MseI primer combinations, mapped within both targeted marker intervals. Fourteen SSR and six AFLP markers mapped to the Scmv1 region. Eleven SSR and 18 AFLP markers were located in the Scmv2 region. Whereas the linear order of SSR markers and the window size for the Scmv2 region fitted well with publicly available genetic maps, map distances and window size differed substantially for the Scmv1 region on chromosome 6. A possible explanation for the observed discrepancies is the presence of two closely linked resistance genes in the Scmv1 region.     

Responses of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) near isogenic lines carrying <Emphasis Type="Italic">Wsm1</Emphasis>, <Emphasis Type="Italic">Wsm2,</Emphasis> and <Emphasis Type="Italic">Wsm3</Emphasis> to three viruses in the Potyviridae

Genes on chromosomes six (Wsm1), three (Wsm2) and ten (Wsm3) in the maize (Zea mays L.) inbred line Pa405 control resistance to Wheat streak mosaic virus (WSMV), and the same or closely linked genes control resistance to Maize dwarf mosaic virus (MDMV) and Sugarcane mosaic virus (SCMV). Near isogenic lines (NIL) carrying one or two of the genes were developed by introgressing regions of the respective chromosomes into the susceptible line Oh28 and tested for their responses to WSMV, MDMV, and SCMV in the field and greenhouse. F₁ progeny from NIL × Oh28 were also tested. Wsm1, or closely linked genes, provided resistance to all three viruses, as determined by symptom incidence and severity. Wsm2 and Wsm3 provided resistance to WSMV. Wsm2 and/or Wsm3 provided no resistance to MDMV, but significantly increased resistance in plants with one Wsm1 allele. NIL carrying Wsm1, Wsm2, or Wsm3 had similar SCMV resistance in the field, but NIL with Wsm2 and Wsm3 were not resistant in the greenhouse. Addition of Wsm2 to Wsm1 increased SCMV resistance in the field. For all viruses, symptom incidence was higher in the greenhouse than in the field, and relative disease severity was higher in the greenhouse for WSMV and MDMV. An Italian MDMV isolate and the Ohio SCMV infected the Wsm1 NIL, while the Ohio MDMV and Seehausen SCMV isolates did not. Our results indicate that the three genes, or closely linked loci, provide virus resistance. Resistance conferred by the three genes is influenced by interactions among the genes, the virus species, the virus isolate, and the environment.     

Proteomic and Phytohormone Analysis of the Response of Maize (Zea mays L.) Seedlings to Sugarcane Mosaic Virus

Background

Sugarcane mosaic virus (SCMV) is an important virus pathogen in crop production, causing serious losses in grain and forage yields in susceptible cultivars. Control strategies have been developed, but only marginal successes have been achieved. For the efficient control of this virus, a better understanding of its interactions and associated resistance mechanisms at the molecular level is required.

Methodology/Principal Findings

The responses of resistant and susceptible genotypes of maize to SCMV and the molecular basis of the resistance were studied using a proteomic approach based on two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS/MS) analysis. Ninety-six protein spots showed statistically significant differences in intensity after SCMV inoculation. The classification of differentially expressed proteins showed that SCMV-responsive proteins were mainly involved in energy and metabolism, stress and defense responses, and photosynthesis. Most of the proteins identified were located in chloroplasts, chloroplast membranes, and the cytoplasm. Analysis of changes in phytohormone levels after virus inoculation suggested that salicylic acid, abscisic acid, jasmonic acid, and azelaic acid may played important roles in the maize response to SCMV infection.

Conclusions/Significance

Among these identified proteins, 19 have not been identified previously as virus-responsive proteins, and seven were new and did not have assigned functions. These proteins may be candidate proteins for future investigation, and they may present new biological functions and play important roles in plant-virus interactions. The behavioural patterns of the identified proteins suggest the existence of defense mechanisms operating during the early stages of infection that differed in two genotypes. In addition, there are overlapping and specific phytohormone responses to SCMV infection between resistant and susceptible maize genotypes. This study may provide important insights into the molecular events during plant responses to virus infection.     

Genetic basis of resistance to sugarcane mosaic virus in European maize germplasm

 Sugarcane mosaic virus (SCMV) causes considerable damage to maize (Zea mays L.) in Europe. The objective of the present study was to determine the genetic basis of resistance to SCMV in European maize germplasm and to compare it with that of U.S. inbred Pa405. Three resistant European inbreds D21, D32, and FAP1360A were crossed with four susceptible inbreds F7, KW1292, D408, and D145 to produce four F₂ populations and three backcrosses to the susceptible parent. Screening for SCMV resistance in parental inbreds and segregating generations was done in two field trials as well as under greenhouse conditions. RFLP markers umc85, bnl6.29, umc10, umc44, and SSR marker phi075 were used in F₂ populations or F₃ lines to locate the resistance gene(s) in the maize genome. Segregation in the F₂ and backcross generations fitted to different gene models depending on the environmental conditions and the genotype of the susceptible parent. In the field tests, resistance in the three resistant European inbreds seems to be controlled by two to three genes. Under greenhouse conditions, susceptibility to SCMV in D32 appears to be governed by one dominant and one recessive gene. Allelism tests indicated the presence of a common dominant gene (denoted as Scm1) in all three resistant European inbreds and Pa405. Marker analyses mapped two dominant genes: Scm1 on chromosome 6S and Scm2 on chromosome 3. Received: 17 November 1997 / Accepted: 25 November 1997     

Possible involvement of maize Rop1 in the defence responses of plants to viral infection

The expression of host genes can be altered during the process of viral infection. To investigate the viral infection-induced up-regulated gene expression changes of maize at different time intervals post-inoculation with Sugarcane mosaic virus (SCMV), a suppression subtractive hybridization cDNA library was constructed. A total of 454 cDNA clones were identified to be viral infection-induced up-regulated genes. The influence of Rop1 on the infection of maize by SCMV was investigated. The results showed that transient silencing of the ZmRop1 gene through virus-induced gene silencing enhanced the accumulation and systemic infection of SCMV and another potyvirus (Pennisetum mosaic virus) in maize plants, whereas transient over-expression of ZmRop1 in maize protoplasts reduced SCMV accumulation. Furthermore, it was demonstrated that the heterologous expression of ZmRop1 impaired Potato virus X infection in Nicotiana benthamiana plants. These data suggest that ZmRop1 may play a role in plant defence responses to viral infection.     

Molecular Variability and Distribution of Sugarcane Mosaic Virus in Shanxi,China

Background

Sugarcane mosaic virus (SCMV) is responsible for large-scale economic losses in the global production of sugarcane, maize, sorghum, and some other graminaceous species. To understand the evolutionary mechanism of SCMV populations, this virus was studied in Shanxi, China. A total of 86 maize leaf samples (41 samples in 2012 and 45 samples in 2013) were collected from 4 regions of Shanxi.

Results

Double-antibody sandwich (DAS)-ELISA and RT-PCR showed 59 samples (30 samples in 2012 and 29 samples in 2013) to be positive for SCMV, from which 10 new isolates of SCMV were isolated and sequenced. The complete genomes of these isolates are 9610 nt long, including the 5′ and 3′ non-coding regions, and encode a 3063-amino acid polyprotein. Phylogenetic analyses revealed that 24 SCMV isolates could be divided on the basis of the whole genome into 2 divergent evolutionary groups, which were associated with the host species. Among the populations, 15 potential recombination events were identified. The selection pressure on the genes of these SCMV isolates was also calculated. The results confirmed that all the genes were under negative selection.

Conclusions

Negative selection and recombination appear to be important evolutionary factors shaping the genetic structure of these SCMV isolates. SCMV is distributed widely in China and exists as numerous strains with distinct genetic diversity. Our findings will provide a foundation for evaluating the epidemiological characteristics of SCMV in China and will be useful in designing long-term, sustainable management strategies for SCMV.     

Characterization of Sugarcane Mosaic Virus Scmv1 and Scmv2 Resistance Regions by Regional Association Analysis in Maize

Sugarcane Mosaic Virus (SCMV) causes one of the most severe virus diseases in maize worldwide, resulting in reduced grain and forage yield in susceptible cultivars. In this study, two association panels consisting of 94 inbred lines each, from China and the U.S., were characterized for resistance to two isolates: SCMV-Seehausen and SCMV-BJ. The population structure of both association panels was analyzed using 3072 single nucleotide polymorphism (SNP) markers. The Chinese and the U.S. panel were both subdivided into two sub-populations, the latter comprised of Stiff Stalk Synthetic (SS) lines and Non Stiff Stalk Synthetic (NSS). The relative kinships were calculated using informative 2947 SNPs with minor allele frequency ≥ 5% and missing data ≤ 20% for the Chinese panel and 2841 SNPs with the same characteristics were used for the U.S. panel. The Scmv1 region was genotyped using 7 single sequence repeat (SSR) and sequence-tagged site (STS) markers, and 12 SSR markers were used for the Scmv2 region in the U.S. panel, while 5 of them were used for the Chinese panel. For all traits, a MLM (Mix Linear Model) controlling both population structure and relative kinship (Q + K) was used for association analysis. Three markers Trx-1, STS-11, and STS-12 located in the Scmv1 region were strongly associated (P = 0.001) with SCMV resistance, and explained more than 16.0%, 10.6%, and 19.7% of phenotypic variation, respectively. 207FG003 located in the Scmv2 region was significantly associated (P = 0.001) with SCMV resistance, and explained around 18.5% of phenotypic variation.     

Identification of maize lethal necrosis disease causal viruses in maize and suspected alternative hosts through small RNA profiling

Maize lethal necrosis disease (MLND) is a devastating viral disease of maize caused by double infection with Maize chlorotic mottle virus (MCMV) and any one of the Potyviridae family members. Management of MLND requires effective resistance screening and surveillance tools. In this study, we report the use of small RNA (sRNA) profiling to detect MLND causal viruses and further the development of alternative detection markers for use in routine surveillance of the disease-causing viruses. Small RNAs (sRNAs) originating from five viruses namely MCMV, Sugarcane mosaic virus (SCMV), Maize streak virus (MSV), Maize-associated totivirus (MATV) and Maize yellow mosaic virus (MYMV) were assembled from infected maize samples collected from MLND hot spots in Kenya. The expression of the identified viral domains was further validated using quantitative real-time PCR. New markers for the detection of some of the MLND causal viruses were also developed from the highly expressed domains and used to detect the MLND-causative viruses in maize and alternative hosts. These findings further demonstrate the potential of using sRNAs especially from highly expressed viral motifs in the detection of MLND causal viruses. We report the validation of new sets of primers for use in detection of the most common MLND causal viruses MCMV and SCMV in East Africa.     

玉米抗甘蔗花叶病毒分子标记开发

由甘蔗花叶病毒引起的玉米矮花叶病是我国黄淮海地区玉米生产的重要病害,开发抗矮花叶病基因分子标记是开展抗病分子标记辅助育种的基础。本文基于玉米6．00-6．01区域的“一致性抗甘蔗花叶病毒QTL区间”寻找抗病基因的功能保守域,依据序列多态性开发出抗病分子标记InDel-130和InDel-110,在已知抗性的102份玉米自交系中进行验证。通过分析标记抗病带型和感病带型中的抗病和感病自交系数目,卡平方测验表明标记InDel-130在供试自交系中与抗病性的表现独立无关．而标记InDel-110与甘蔗花叶病毒抗性高度相关,为共显性标记,可用于玉米抗甘蔗花叶病毒种质筛选和分子标记辅助育种。     

Genetic analysis of resistance to six virus diseases in a multiple virus-resistant maize inbred line

Key message

Novel and previously known resistance loci for six phylogenetically diverse viruses were tightly clustered on chromosomes 2, 3, 6 and 10 in the multiply virus-resistant maize inbred line, Oh1VI.

Abstract

Virus diseases in maize can cause severe yield reductions that threaten crop production and food supplies in some regions of the world. Genetic resistance to different viruses has been characterized in maize populations in diverse environments using different screening techniques, and resistance loci have been mapped to all maize chromosomes. The maize inbred line, Oh1VI, is resistant to at least ten viruses, including viruses in five different families. To determine the genes and inheritance mechanisms responsible for the multiple virus resistance in this line, F₁ hybrids, F₂ progeny and a recombinant inbred line (RIL) population derived from a cross of Oh1VI and the virus-susceptible inbred line Oh28 were evaluated. Progeny were screened for their responses to Maize dwarf mosaic virus, Sugarcane mosaic virus, Wheat streak mosaic virus, Maize chlorotic dwarf virus, Maize fine streak virus, and Maize mosaic virus. Depending on the virus, dominant, recessive, or additive gene effects were responsible for the resistance observed in F₁ plants. One to three gene models explained the observed segregation of resistance in the F₂ generation for all six viruses. Composite interval mapping in the RIL population identified 17 resistance QTLs associated with the six viruses. Of these, 15 were clustered in specific regions of chr. 2, 3, 6, and 10. It is unknown whether these QTL clusters contain single or multiple virus resistance genes, but the coupling phase linkage of genes conferring resistance to multiple virus diseases in this population could facilitate breeding efforts to develop multi-virus resistant crops.     

Response of maize (Zea mays L.) lines carrying Wsm1, Wsm2, and Wsm3 to the potyviruses Johnsongrass mosaic virus and Sorghum mosaic virus

Maize dwarf mosaic disease is one of the most important viral diseases of maize (Zea mays L.) throughout the world. It is caused by several virus species in the family Potyviridae, genus Potyvirus, including Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), Johnsongrass mosaic virus (JGMV) and Sorghum mosaic virus (SrMV). Resistance to another member of the family Potyviridae, Wheat streak mosaic virus (WSMV), is conferred by three alleles (Wsm1, Wsm2, Wsm3) in the maize inbred line Pa405, and these or closely linked genes were previously shown to confer resistance to the potyviruses MDMV and SCMV. In this study, we assessed whether Wsm alleles are linked to resistance to JGMV and SrMV. Near isogenic lines (NILs) carrying one or two of the Wsm alleles introgressed into the susceptible line Oh28 and F1 progeny from NIL × Oh28 were tested for their response to JGMV and SrMV. Our results indicate that Wsm1 provides resistance to both JGMV and SrMV in a dose-dependent manner. Wsm2 and Wsm3 each provide limited resistance, and combining Wsm alleles enhances that resistance.