Epistatic determinism of durum wheat resistance to the wheat spindle streak mosaic virus

Key message

The resistance of durum wheat to the Wheat spindle streak mosaic virus (WSSMV) is controlled by two main QTLs on chromosomes 7A and 7B, with a huge epistatic effect.

Abstract

Wheat spindle streak mosaic virus (WSSMV) is a major disease of durum wheat in Europe and North America. Breeding WSSMV-resistant cultivars is currently the only way to control the virus since no treatment is available. This paper reports studies of the inheritance of WSSMV resistance using two related durum wheat populations obtained by crossing two elite cultivars with a WSSMV-resistant emmer cultivar. In 2012 and 2015, 354 recombinant inbred lines (RIL) were phenotyped using visual notations, ELISA and qPCR and genotyped using locus targeted capture and sequencing. This allowed us to build a consensus genetic map of 8568 markers and identify three chromosomal regions involved in WSSMV resistance. Two major regions (located on chromosomes 7A and 7B) jointly explain, on the basis of epistatic interactions, up to 43% of the phenotypic variation. Flanking sequences of our genetic markers are provided to facilitate future marker-assisted selection of WSSMV-resistant cultivars.

     

Development of PCR markers linked to resistance to wheat streak mosaic virus in wheat

Wheat streak mosaic virus (WSMV), vectored by the wheat curl mite (Acer tulipae), is an important disease of wheat (Triticum aestivum L.) in the North American Great Plains. Resistant varieties have not been developed for two primary reasons. First, useful sources of resistance have not been available, and second, field screening for virus resistance is laborious and beyond the scope of most breeding programs. The first problem may have been overcome by the development of resistance to both the mite and the virus by the introgression of resistance genes from wild relatives of wheat. To help address the second problem, we have developed polymerase chain reaction (PCR) markers linked to the WSMV resistance gene Wsm1. Wsm1 is contained on a translocated segment from Agropyron intermedium. One sequence-tagged-site (STS) primer set (WG232) and one RAPD marker were found to be linked to the translocation containing Wsm1. The diagnostic RAPD band was cloned and sequenced to allow the design of specific PCR primers. The PCR primers should be useful for transferring Wsm1 into locally adapted cultivars.This is Journal Series No. J-4041 of the Montana Agricultural Experiment Station     

Molecular cytogenetic analysis of Agropyron elongatum chromatin in wheat germplasm specifying resistance to wheat streak mosaic virus

Summary Three lines derived from wheat (6x) x Agropyron elongatum (10x) that are resistant to wheat streak mosaic virus (WSMV) were analyzed by chromosome pairing, banding, and in situ hybridization. Line CI15321 was identified as a disomic substitution line where wheat chromosome 1D is replaced by Ag. elongatum chromosome 1Ae-1. Line 87-94-1 is a wheat-Ag. elongatum ditelosomic addition 1Ae-1L. Line CI15322 contains an Ag. elongatum chromosome, 1Ae-2, that substitutes for chromosome 1D. The short arm of 1Ae-2 paired with the short arm of 1Ae-1 at metaphase I (MI) in 82% of the pollen mother cells (PMCs). However, the long arms of these two chromosomes did not pair with each other. In CI15322, the long arm of chromosome 4D has an Agropyron chromosome segment which was derived from the distal part of 1Ae-1L. This translocation chromosome is designated as T4DS·4DL-1L. T4DS·4DL-1Ae-1L has a 0.73 m distal part of the long arm of 4D replaced by a 1.31 m distal segment from 1Ae-1L. The major WSMV resistance gene(s) in these lines is located on the distal part of 1Ae-1L.Contribution No. 92-599-J from the Kansas Agricutural Experiment Station, Kansas State University, Manhattan, Kansas, USA     

Wheat curl mite resistance: interactions of mite feeding with wheat streak mosaic virus infection

The majority of plant viruses are dependent on arthropod vectors for spread between plants. Wheat streak mosaic virus (family Potyviridae, genus Tritimovirus, WSMV) is transmitted by the wheat curl mite, Aceria tosichella Keifer, and this virus and vector cause extensive yield losses in most major wheat (Triticum aestivum L.)-growing regions of the world. Many cultivars in use are susceptible to this vector-virus complex, and yield losses of 10-99% have been documented. wheat curl mite resistance genes have been identified in goat grass, Aegilops tauschii (Coss) Schmal., and transferred to hexaploid wheat, but very few varieties contain effectively wheat curl mite resistance, due to virulent wheat curl mite populations. However, wheat curl mite resistance remains an effective strategy to reduce losses due to WSMV. The goal of our project was to identify the most effective, reproducible, and rapid method for assessing wheat curl mite resistance. We also wanted to determine whether mite resistance is affected by WSMV infection, because the pathogen and pest commonly occur together. Single and group wheat curl mite infestations produced similar amounts of leaf rolling and folding on wheat curl mite-susceptible wheat varieties that were independent of initial wheat curl mite infestation. This finding will allow accurate, efficient, large-scale screening of wheat germplasm for wheat curl mite resistance by infesting plants with sections of wheat leaf tissue containing mixed stages of wheat curl mite. The wheat curl mite-resistant breeding line 'OK05312' displayed antibiosis (reduced wheat curl mite population development). The effect of WSMV infection on wheat curl mite reproduction was genotype-dependent. Mite populations increased on infected wheat curl mite- and WSMV-susceptible plants compared with uninfected plants, but WSMV infection had no significant effect on wheat curl mite populations on resistant plants. OK05312 is a strong source of wheat curl mite resistance for wheat breeding programs.     

Resistance to wheat streak mosaic virus in transgenic wheat expressing the viral replicase (NIb) gene

Wheat (Triticum aestivum L. cv. Hi-Line) immature embryos were transformed with the replicase gene (NIb) of wheat streak mosaic virus (WSMV) by the biolistic method. Six independent transgenic plant lines were analyzed for transgene expression and for resistance to mechanical inoculation of WSMV at R3 or R4 generation. Four out of the six lines showed various degree of resistance to WSMV. These lines had initially milder symptoms than controls, and the new growth ranged from milder symptoms, a substantial delay in symptom development, or asymptomatic. Two lines displayed higher resistance with very mild virus symptoms after inoculation and the new growth of 72% and 32% plants from these lines were asymptomatic and had no detectable virus through the plant life cycle. Interestingly, five out of the six transgenic lines had no detectable transgene mRNA expression by RNA gel blot hybridization. The only line that had detectable transgene mRNA did not show delay in the symptom development but had overall milder symptom to the virus.     

Structure and temporal dynamics of populations within wheat streak mosaic virus isolates

Variation within the Type and Sidney 81 strains of wheat streak mosaic virus was assessed by single-strand conformation polymorphism (SSCP) analysis and confirmed by nucleotide sequencing. Limiting-dilution subisolates (LDSIs) of each strain were evaluated for polymorphism in the P1, P3, NIa, and CP cistrons. Different SSCP patterns among LDSIs of a strain were associated with single-nucleotide substitutions. Sidney 81 LDSI-S10 was used as founding inoculum to establish three lineages each in wheat, corn, and barley. The P1, HC-Pro, P3, CI, NIa, NIb, and CP cistrons of LDSI-S10 and each lineage at passages 1, 3, 6, and 9 were evaluated for polymorphism. By passage 9, each lineage differed in consensus sequence from LDSI-S10. The majority of substitutions occurred within NIa and CP, although at least one change occurred in each cistron except HC-Pro and P3. Most consensus sequence changes among lineages were independent, with substitutions accumulating over time. However, LDSI-S10 bore a variant nucleotide (G(6016)) in NIa that was restored to A(6016) in eight of nine lineages by passage 6. This near-global reversion is most easily explained by selection. Examination of nonconsensus variation revealed a pool of unique substitutions (singletons) that remained constant in frequency during passage, regardless of the host species examined. These results suggest that mutations arising by viral polymerase error are generated at a constant rate but that most newly generated mutants are sequestered in virions and do not serve as replication templates. Thus, a substantial fraction of variation generated is static and has yet to be tested for relative fitness. In contrast, nonsingleton variation increased upon passage, suggesting that some mutants do serve as replication templates and may become established in a population. Replicated mutants may or may not rise to prominence to become the consensus sequence in a lineage, with the fate of any particular mutant subject to selection and stochastic processes such as genetic drift and population growth factors.     

Identification of RFLP markers for resistance to wheat spindle streak mosaic bymovirus (WSSMV) disease.

Wheat spindle streak mosaic bymovirus (WSSMV) causes an economically important disease of winter wheat in Europe and North America. Artificial inoculation with this virus to identify resistant wheat genotypes is difficult. This study was conducted to identify restriction fragment length polymorphism (RFLP) markers associated with resistance to this disease. A population, consisting of 104 F5 recombinant inbred lines from a cross between hexaploid Triticum aestivum cultivars 'Geneva' (resistant) and 'Augusta' (susceptible), was evaluated for WSSMV symptoms under field conditions for four years. Two linked markers on the long arm of chromosome 2D, Xbcd1095 and Xcdo373, were determined to be associated with WSSMV resistance by bulked segregant analysis of the 10 most resistant and 10 most susceptible lines. Marker Xcdo373 accounted for 79% and Xbcd1095 for 73% of the phenotypic variation. Our results suggest that resistance to WSSMV in this population is qualitative in nature and is controlled by few genes. These markers should be useful in the development of wheat cultivars resistant to WSSMV and perhaps also to wheat yellow mosaic bymovirus (WYMV).     

Soilborne wheat mosaic virus movement protein and RNA and wheat spindle streak mosaic virus coat protein accumulate inside resting spores of their vector, Polymyxa graminis

To study virus-vector interactions between Soilborne wheat mosaic virus (SBWMV) or Wheat spindle streak mosaic virus (WSSMV) and Polymyxa graminis Ledingham, P. graminis was propagated in plants grown hydroponically. P. graminis accumulated to high levels in several barley cultivars tested. Multiple developmental stages of P. graminis could be identified in infected barley roots. Accumulation of SBWMV and WSSMV inside P. graminis sporosori in the roots of soil-grown winter wheat and hydroponically grown barley was compared to determine if data obtained from plants naturally infected plants and plants infected by manual inoculation were similar. WSSMV coat protein (CP), SBWMV RNAs, SBWMV movement protein but not SBWMV CP were detected in both soil-grown winter wheat and hydroponically grown barley roots. These data are the first direct evidence that SBWMV and WSSMV are internalized by P. graminis.     

Identification of alien chromatin specifying resistance to wheat streak mosaic and greenbug in wheat germ plasm by C-banding and in situ hybridization

Summary The chromosome constitutions of eight wheat streak mosaic virus (WSMV)-resistant lines, three of which are also greenbug resistant, derived from wheat/ Agropyron intermedium/Aegilops speltoides crosses were analyzed by C-banding and in situ hybridization. All lines could be traced back to CI15092 in which chromosome 4A is substituted for by an Ag. intermedium chromosome designated 4Ai-2, and the derived lines carry either 4Ai-2 or a part of it. Two (CI17881, CI17886) were 4Ai-2 addition lines. CI17882 and CI17885 were 4Ai-2-(4D) substitution lines. CI17883 was a translocation substitution line with a pair of 6AL.4Ai-2S and a pair of 6AS.4Ai-2L chromosomes substituting for chromosome pairs 4D and 6A of wheat. CI17884 carried a 4DL.4Ai-2S translocation which substituted for chromosome 4D. CI17766 carried a 4AL.4Ai-2S translocation substituting for chromosome 4A. The results show that the 4Ai-2 chromosome is related to homoeologous group 4 and that the resistance gene(s) against WSMV is located on the short arm of 4Ai-2. In addition, CI17882, CI17884, and CI17885 contained Ae. speltoides chromosome 7S substituting for chromosome 7A of wheat. The greenbug resistance gene Gb5 was located on chromosome 7S.Contribution No. 90-515-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kan., USA     

Molecular cytogenetic discrimination and reaction to wheat streak mosaic virus and the wheat curl mite in Zhong series of wheat--Thinopyrum intermedium partial amphiploids.

Thinopyrum intermedium (2n = 6x = 42, JJJsJsSS) is potentially a useful source of resistance to wheat streak mosaic virus (WSMV) and its vector, the wheat curl mite (WCM). Five partial amphiploids, namely Zhong 1, Zhong 2, Zhong 3, Zhong 4, and Zhong 5, derived from Triticum aestivum x Thinopyrum intermedium crosses produced in China, were screened for WSMV and WCM resistance. Zhong 1 and Zhong 2 had high levels of resistance to WSMV and WCM. The other three partial amphiploids, Zhong 3, 4, and 5, were resistant to WSMV, but were susceptible to WCM. Genomic in situ hybridization (GISH) using a genomic DNA probe from Pseudoroegneria strigosa (SS, 2n = 14) demonstrated that two partial amphiploids, Zhong 1 and Zhong 2, have almost the identical 10 Th. intermedium chromosomes, including four Js, four J, and two S genome chromosomes. Both of them carry two pairs of J and a pair of Js genome chromosomes and two different translocations that were not observed in the other three Zhong lines. The partial amphiploids Zhong 3, 4, and 5 have another type of basic genomic composition, which is similar to a reconstituted alien genome consisting of four S and four Js genome chromosomes of Th. intermedium (Zhong 5 has two Js chromosomes plus two Js-W translocations) with six translocated chromosomes between S and Js or J genomes. All three lines carry a specific S-S-Js translocated chromosome, which might confer resistance to barley yellow dwarf virus (BYDV-PAV). The present study identified a specific Js2 chromosome present in all five of the Zhong lines, confirming that a Js chromosome carries WSMV resistance. Resistance to WCM may be linked with J or Js chromosomes. The discovery of high levels of resistance to both WSMV and WCM in Zhong 1 and Zhong 2 offers a useful source of resistance to both the virus and its vector for wheat breeding programs.     

The N-terminal region of wheat streak mosaic virus coat protein is a host- and strain-specific long-distance transport factor

Understanding the genetics underlying host range differences among plant virus strains can provide valuable insights into viral gene functions and virus-host interactions. In this study, we examined viral determinants and mechanisms of differential infection of Zea mays inbred line SDp2 by Wheat streak mosaic virus (WSMV) isolates. WSMV isolates Sidney 81 (WSMV-S81) and Type (WSMV-T) share 98.7% polyprotein sequence identity but differentially infect SDp2: WSMV-S81 induces a systemic infection, but WSMV-T does not. Coinoculation and sequential inoculation of SDp2 with WSMV-T and/or WSMV-S81 did not affect systemic infection by WSMV-S81, suggesting that WSMV-T does not induce a restrictive defense response but that virus-encoded proteins may be involved in differential infection of SDp2. The viral determinant responsible for strain-specific host range was mapped to the N terminus of coat protein (CP) by systematic exchanges of WSMV-S81 sequences with those of WSMV-T and by reciprocal exchanges of CP or CP codons 1 to 74. Green fluorescent protein (GFP)-tagged WSMV-S81 with CP or CP residues 1 to 74 from WSMV-T produced similar numbers of infection foci and genomic RNAs and formed virions in inoculated leaves as those produced with WSMV-S81, indicating that failure to infect SDp2 systemically is not due to defects in replication, cell-to-cell movement, or virion assembly. However, these GFP-tagged hybrids showed profound defects in long-distance transport of virus through the phloem. Furthermore, we found that four of the five differing amino acids in the N terminus of CP between the WSMV-S81 and WSMV-T isolates were collectively involved in systemic infection of SDp2. Taken together, these results demonstrate that the N-terminal region of tritimoviral CP functions in host- and strain-specific long-distance movement.     

Inheritance of resistance to zucchini yellow mosaic virus and watermelon mosaic virus in watermelon

High resistance to zucchini yellow mosaic virus-China strain (ZYMV-CH) and moderate resistance to watermelon mosaic virus (WMV) were found in a selection of PI 595203 (Citrullus lanatus var. lanatus), an Egusi type originally collected in Nigeria. Mixed inoculations showed primarily that these two viruses have no cross-protection. This fact may explain the high frequency of mixed infection often observed in commercial fields. When plants were inoculated with a mixture of the two viruses, the frequency of plants resistant to ZYMV was lower than expected, indicating that WMV infection may reduce the ability of a plant to resist ZYMV. We studied inheritance of resistance to ZYMV-CH and WMV, using crosses between a single-plant selection of PI 595203 and the ZYMV-susceptible watermelon inbreds 9811 and 98R. According to virus ratings of the susceptible parents, the resistant parent, and the F1, F2, and BC1 generations, resistance to ZYMV-CH was conferred by a single recessive gene, for which the symbol zym-CH is suggested. The high tolerance to WMV was controlled by at least two recessive genes.     

RNA interference-mediated resistance to maize dwarf mosaic virus

Maize dwarf mosaic virus (MDMV) is a widespread pathogen that causes serious yield loss to maize crops. A hairpin RNA expression vector was constructed herein to overcome the low efficiency of cultural protection against MDMV and to improve the MDMV resistance mediated by a shorter transgenic inverted-repeat sequence. This expression vector contained a 451 bp inverted-repeat sequence, homologous to the protease gene (P1) of MDMV. It was used for the Agrobacterium tumefaciens-mediated transformation of maize calli induced from a susceptible inbred line. A total of 17 T₂ transgenic lines were identified by both specific PCR amplification and Southern blot hybridization. Of these lines, 15 were evaluated for MDMV resistance in inoculation field trials under two environments. The relative replication levels of the P1 gene were analyzed by quantitative real-time (qRT)-PCR. Results demonstrated that all of the 15 T₂ lines showed an enhanced resistance to MDMV in comparison with that of the non-transformed parent line. Six lines were deemed to be ‘resistant’ with an average disease index below 25 %, which was not significantly different from that of the resistant control. The relative replication levels of the virus gene were significantly reduced in these resistant T₂ transgenic lines. The efficiency of virus gene silencing was directly related to the transgene copy numbers presented in these transgenic lines.     

Properties of lucerne transient streak virus, and evidence of its affinity to southern bean mosaic virus

A virus obtained from naturally infected lucerne ( Medicago sativa ) in New Zealand reacted with antiserum to an Australian isolate of lucerne transient streak virus (LTSV). Some plants infected with New Zealand isolates showed yellow flecks along lateral veins of leaves; symptoms were transient in some lucerne plants but persistent in others. A New Zealand isolate (LTSV-NZ) infected 14 of 39 plant species tested by mechanical inoculation, but was not transmitted by five aphid species. In sap of Nicotiana clevelandii , LTSV-NZ was infective after storage for 4 wk at 20 ^oC, diluting to 10^-5, or heating for 10 min at 70 ^oC. Purified virus preparations contained a single electrophoretic component and a single sedimenting component (s_20w= 112 S ) which formed a single buoyant density component in CsCl (1.37 g cm^-3) but two density components in Cs₂SO₄ (1.26 and 1.32 g cm^-3). LTSV-NZ particles were stable in 10 ITIM EDTA at pH 5, but not at pH 8, being degraded into two sedimenting components of 105 S and 92 S. Particles contained c. 18% RNA in the form of one single-stranded RNA molecule of mol. wt 1–4 times 10⁶, and a polypeptide of mol. wt c. 32 400. LTSV-NZ was serologically unrelated to 24 other isometric plant viruses. However, its properties are similar to those of southern bean mosaic virus and allied viruses. The present cryptogram of LTSV is R/l: 1–4/(18):S/S:S/*.