Hammond-Kosack K

Kanyuka K

Shytikova Yu V

Zlatska AV

English

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 1Sources for resistance to Soil-Borne Cereal Mosaic Virus (SBCMV) among cultivated accessions of common wheat and its wild relatives  Zlatska AV1, Shytikova Yu.V1, Kanyuka K2, Hammond-Kosack K2 1 Ukrainian Institute for Plant Varieties Examination, 15 Henerala Rodimtseva Str., Kyiv 03041, Ukraine.  e-mail: zlatska@hotmail.com, azlatska@sops.gov.ua; 2 2Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK  INTRODUCTION  Soil-borne cereal mosaic virus (SBCMV) and related viruses are considered to be one of the most important pathogens of winter wheat in Europe, America and Asia [1], which can reduce grain yield of susceptible wheat variety on infected fields by up to 50-80% [2; 3]. SBCMV is exclusively vectored by the Polymyxa graminis [4] - eukaryotic zoosporic microorganism parasitising the plant roots. Viruses survive in soil in P.graminis resting spores that may remain viable for decades until suitable conditions for their germination will appear [5]. Using of resistant crop varieties currently the only practical, environmentally friendly and sustainable means of control [6]. Therefore, identification a new genetic source of resistance to SBCMV and its vector among world collection of common wheat varieties as well as wheat wild and cultivated relatives is a very important task. Preliminary investigations suggest that the soil-borne viruses of cereals are quite rare in Eastern European countries and particular in Ukraine. However, this could be partially explained by the fact that the disease is difficult to detect under the field condition otherwise an alternative explanation may be that the most commonly used Ukrainian wheat varieties are resistant to either viruses, or their vector. Last hypothesis is supported by the data from USA published on the Internet (see http://www.ars-grin.gov/npgs/) that show several older wheat varieties of the Ukrainian origin to be field resistant to Soil-borne wheat mosaic virus (SBWMV) (a virus related to the European SBCMV). Several years ago in Rothamsted Research  a gene, Sbm1, for resistance to SBCMV was identified using the population of doubled haploids developed by crossing of resistant common wheat variety Cadenza and susceptible Avalon. This gene was mapped to the gene-rich region on 5DL chromosome by application of microsatellite markers (barc110, barc144 and wmc765) and allele specific primers have been developed [7, 8]. In a separate study performed in Kansas State University on KS96WGRC40 a resistance to Soil-borne wheat mosaic virus was also detected on the long arm of chromosome 5D. This gene was transferred into common wheat gene pool from Aegilops taushii (line TA2397) [9]. Therefore we infer that this particular region of chromosome 5D is of great importance for investigation of resistance of wheat and wheat wild relatives to the group of Soil-borne mosaic viruses transferred by P.graminis. The aims of present study were: (i) to screen collection of common wheat varieties (old and current), which  were cultivated in Ukraine, in respect to resistance to SBCMV; (ii) to study polymorphism between UK and Ukrainian varieties and analysis of diversity among accessions by application of target microsatellites (barc110, barc144 and wmc765) and allele specific primers (S12M61F2 and S12M61R2) linked to Sbm1 (resistance to SBCMV) as well as cfd 10 which is linked to SBWMV resistance;(iii) to test collection of  wild and cultivated wheat relatives kindly provided by National Centre of Plant Genetic Resources of Ukraine as potential new source of resistance to SBCMV and its vector and (iv) to study ability of specific P. graminis ribotypes to invalid roots of different monocotyledonous genotypes.  MATERIAL AND METHODS Plant material. 53 accessions of common wheat varieties. 80 accessions of wheat cultivated and wild relatives, 39 accessions of genus Triticum and 22 of genus Aegilops, 1 accession of Dasypyrum villosum and 18 amphidiploids. These were kindly provided from collections of National Centre of Plant Genetic Resource of Ukraine (Kharkiv), Institute of Plant Physiology and Genetics NASU (Kyiv) and Institute of Genetics and Breeding UAAS (Odesa) Detection of SBCMV. Detection of SBCMV have been done by ELISA method according to the protocol published previously [7] (Fig. 1).   Fig.1 Left picture – plants tested under controlling conditions; Right picture - results of ELISA testing PCR. Plant DNA extraction was done according the standard method. Allele specific primers S12M61F2 and S12M61R2 of gene SbmCz1 of resistance to SBCMV recently were developed by Kanyuka (unpublished) by CAPS method. Microsatellite analysis and CAPS have been done as it was described in Bass et all., [8] and Hall et al [9]. Following microsatellite markers have been used barc110, barc144, wmc765 and cfd 10. Detection of P. graminis. To identify P. graminis resting spores roots were stained with 0.1 % acid fuchsin [10] and then were examined  2 under the light microscope. Detection of specific P. graminis ribotypes in plant roots by conventional PCR have been carried out according to protocol of Ward et al [11].  RESULTS AND DISCUSSIONS  Among accessions which have been tested three main groups according to their reaction of resistance to SBCMV were identified: 1. Highly susceptible accessions (group 1, table 1); 2. Resistant with high ELISA A405nm values in root tissue (high concentration of virus in the roots) (group 4, table 1); 3. Resistant with low ELISA A405nm values in root tissue (group 5 table 1). A number of accessions were heterogeneous by those characteristics (groups 2, 3 and 6 table 1). The resistant variety Cadenza and susceptible Avalon were used as controls. Table 1. Resulta of ELISA testing of plant accessions Group Absorbance values in ELISA, A405nm after 7 weeks after 9 weeks roots 1 2 1 2 1 2 1 1,400-1,682 1,200-1,400 1,100-1,800 0,900-1,500 - - 2 1,200-1,500 0,008-0,037 1,070-1,400 0,004-0,016 - 0,450-0,700 3 0,005-0,019 1,200-1,550 0,004-0,015 0,956-1,400 0,184-0,204 - 4 0,009-0,013 0,005-0,014 0,005-0,036 0,012-0,041 0,548-1,792 0,448-1,200 5 0,009-0,012 0,009-0,016 0,005-0,023 0,009-0,025 0,002-0,087 0,035-0,167 6 0,006-0,024 0,008-0,028 0,002-0,015 0,010-0,032 0,011-0,045 1,225-1,900 Group 1 was formed by Beltskaya 32, Priboi, Odeska 267, Tira, Strumok, Odeska chervonokolosa, Ukrainka 0246 (two accessions), Mironovskaya 808, Myronivska 61, Myronivska ostysta, Myronivska 33, Myronivska ranniostygla, Kyivska ostysta, Smuglyanka, Lutestsns 7, Poliska 90, Kopylivchanka, Khersonska bezosta, Ivanivska ostysta, Bezostaya 1, Kavkaz, Skyfianka, Lutescens 329, Kishinevskaya, Avalon Group 2:  Krymka (accession1), Odesskaya 51, Albatros odesky, Odeska 162, Selyanka, Povaga, Myronivska 27, Myronivska 65, Podolyanka, Tsyganka, Yatran 60, Kharus; Species of genus Triticum (AABB): T.dicoccoides IU016033, T.dicoccum UA 0300005, T.dicoccum v. serbicum UA0300009, T.dicoccum v. volgense UA0300014, T.persicum v. osseticum IR 00204, T. persicum v.rubiginosum UA 0300065; (AA): T.monococcum UA 0300104; Species of genus Aegilops: Ae. umbellulata IU 015892 (UU), Ae.biuncialis (Crimea 2) (UUMM),  Ae.neglecta IU 015928 (UUMM) and amphidiploids: АD Ae.ventricosa-T.dicoccum UA 0500021. Group 3: Krymka (accession 1), Obrii, Panna; Hexaploid Species of genus Triticum AABBDD: T.spelta v .caeruleum , UA 0300074;  Tetraploid species of genus Triticum AABB: T.dicoccum v. aeruginosum haussknechtianum UA 03000008, T. persicum v.rubiginosum UA 0300065; species of genus Aegilops: Ae. umbellulata IU 015905 UU, Ae. recta NCPGR UUMMDD and amphidiploids: MIT 346 (T.durum v.muticoitalicum-Ae.taushii k-346). Group 4: Ukrainka odes`ka, Kuyalnik, Cadenza; Species of genus Triticum AABBDD: T.spelta v.arduini IR00157, T.spelta v.baulander v.duhamelianum UA 0300101, AABB: T.dicoccum v. volgense UA0300031, T.turanicum IR 000162, T. persicum v. persicum UA 0300062; species of genus Aegilops: Ae.triuncialis (NCPGR) UUCC, Ae.columnaris IU 015916 UUMM, and amphidiploids: AD T.durum-Hordeum chilense IU 024318, Haynatricum (Dasypyrum villosum-T.dicoccum) UA 0500018. Group 5::Odesskaya 4; Diploid an d Tetraploid species of genus Triticum: T.boeoticum d1814/96 (AA), T.petropavlovlovskii UA 0300106 (AABB), Other wheat related species: Ae. tauschii 23/2001 (DD ), Dasypyrum villosum NCPGR (VV). Group 6:: Odesskaya 16, Nikoniya, Remeslivna; species of genus Aegilops: Ae. vavilovii IU 015922 DDMMSS, Ae. geniculata 26/93 UUMM. Those accessions of groups 3, 5 and 6 which had low ELISA A405nm values in root tissue have been tested in respect to presence in their roots plasmodium or resting spores of P. graminis using cytological methods and were confirmed to be present in all of those accessions. It is known that ribotypes I of P. graminis isolates were obtained from barley and  most ribotypes II isolates reported have been from wheat and other cereals [11], therefore we tested roots of some wheat wild relatives accessions and detected that P. graminis of ribotype I can affect not only barley roots but roots of Dasypyrum villosum  (Fig 2).  Fig 2. Detection  P.graminis ribotype 1 in roots of some accessions: 1 – 100 bp marker, 2 – cv. Nikoniya, 3- cv Cadenza , 4 – T.spelta v caeruleum, 5 – Ae. geniculata, 6 – Ae. umbellulata, 7 – Ae. taushii, 8 - Dasypyrum villosum, 9 – T. boeoticum, 10 – Ae. columnaris, 11 – Positive control on ribotype 1, 12 – Positive control on ribotype II The recently developed allele specific molecular marker S12M61F2 and S12M61R2 of gene Sbm1  (resistance source to SBCMV in UK variety Cadenza Kanyuka unpublished) was applied for investigation  of resistant varieties (table 1) in order to reveal presence of this gene (allele) in genome of those cultivars (Fig 3). It was observed that resistance of tested varieties was controlled by the same gene; however a few new alleles of this gene were identified which are different from that detected in Cadenza and their sequences should be studied in future.  The Sbm1 locus was mapped to the gene-rich region on 5DL (Fig. 4, A). There are three SSR markers (barc110, barc144 and wmc765) which are closely linked to this locus and exhibit polymorphism between resistant UK variety Cadenza (alleles 184bp, 239bp, 213bp respectively) and susceptible Avalon (alleles 175bp, 223bp, 199bp respectively). Sbwmv, the other gene inferred to be  related to Sbm1 (Fig. 4), which  3controlled resistance to SBWMV in US line KS96WGRC40, is closely linked to the cfd 10 locus.  1     2    3     4    5    6     7     8    9   10   11   12  13 14  15  16  17  18  19   20  21  22 Fig.3 1, 11 – marker 200,300,400bp. (from the down to the top) 2-3 Remeslivna, 4-5 Ukrainka odesska, 6-7 Odesskaya 4,8-9 odesskaya 16, 10 Avalon; 12-14 Donskaya polukarlikovaya, 15-16 Kolumbiya, 17 – Povaga, 18-19 - Nikoniya, 20-22 Kuyalnyk A B Fig. 4. Chromosome location of Sbm1 (A), and Sbwmv (B) We performed the screening of 30 resistant and susceptible varieties representing different regions by utilization of four SSR markers linked to Sbm1 and Sbwmv genes in order (i) to study polymorphism between UK, and US and to compare with varieties tested in our work and (ii) the analysis of diversity among accessions. It was revealed that both UK and Ukrainian resistant and susceptible varieties have the same alleles of SSR markers for barc144 and wmc765, therefore polymorphism is low. Application of cfd 10 SSR marker did not show any differences between susceptible and resistant to SBCMV accessions, therefore not useful for MAS. For barc110 polymorphism was higher, at least 6 alleles were identified, however they did not separate precisely resistant and susceptible varieties. The highest frequency of varieties with resistant alleles is among cultivars of Southern region and the lowest appeared to be in the Northern. Mainly cultivars inherited their resistance from old landrace Krymka planted at the beginning of XX(20th Century) and  can be found in pedigree of a lot of cultivars all over the world [12].  Our results show that resistance to SBCMV accessions can be found among cultivated and wild wheat relatives with genomes (subgenomes) AA, DD, UU, VV and probably MM. Some tested Ukrainian varieties appeared to be resistant to SBCMV, especially the old cultivar Odesskaya 4 which has genetic system which prevents multiplication of P. graminis and virus in root tissues of wheat. Resistance to SBCMV in tested varieties is controlled by the same gene detected for UK variety Cadenza; however different alleles. P. graminis with ribotype I can affect not only barley roots, but as well the roots of Dasypyrum villosum. We have initiated work on association mapping of the loci carried by the genes Sbm1 and Sbwmv in order to better understanding the genetic control of resistance to soil-borne viruses. REFERENCES 1. Myers L.D. et al (1993) Temperature-influenced virus movement in expression of resistance to soilborne wheat mosaic virus in hard red winter wheat (Triticum aestivum). Phytopathology, 83: 548-551. 2. Bayles R.A. et al (2002) Tolerance of Wheat Varieties to Soil-Borne Wheat Mosaic Virus (SBWMV). London, UK: Home-Grown Cereals Authority. HGCA Project Report. No. 278.  3. Budge G. et al., (2002) Assessing Resistance of UK Winter Wheat Varieties to Soil-Borne Wheat Mosaic Virus and Wheat Spindle Streak Mosaic Virus. London, UK: Home-Grown. Cereals Authority. HGCA Project Report. No. 293.  4. Rao A.S., et al.,  (1969) Relation of soil-borne wheat mosaic virus and its fungal vector, Polymyxa graminis. Phytopathology 59, 581-7.  5. Brakke M.K. et al. (1988) Experiences with soil-borne wheat mosaic virus in North America and elsewhere. In: Cooper J.I. Asher M.J.C., eds. Developments in Applied Biology II. Viruses with Fungal Vectors. Wellesbourne, UK: Association of Applied Biologists, 183-202.  6. Kanyuka K. et al (2003) Polymyxa graminis and the cereal viruses it transmits: a research challenge. Molecular Plant Pathology 4: 393-406.  7. Kanyuka K. et al. (2004) A controlled environment test for resistance to Soil-borne cereal mosaic virus (SBCMV) and its use to determine the mode of inheritance of resistance in wheat cv. Cadenza and for screening Triticum monococcum genotypes for sources of SBCMV resistance. Plant Pathology 53: 154-160.  8. Bass C. et al. (2006) The Sbm 1 locus conferring resistance to Soil-borne cereal mosaic virus maps to gene-rich region on 5DL in wheat. Genome 49. P.1140-1148. 9. Hall M.D. et al. (2007) Mapping Septoria Leaf Blotch and Soil-Borne Mosaic Virus Resistance Genes Derived from Aegilops tauschii. Abstracts of XV International Plant and Animal Genome conference. P312 P. 179. 10. Hooper D.J. (1986) Preserving and staining nematodes in plant tissues. In: Southey J.F. (ed.) Laboratory Methods for Work with Plant and soil Nematodes. London, U.K.: MAFF, pp. 81-85. 11. Ward E. et al. (2005) The use of conventional and quantitative real-time PCR assays for Polymyxa graminis to examine host plant resistance, inoculum levels and intraspecific variation. New Phytologist : 3. - P. 875-885. 12. Rabinovich S., Fedak G.,Lukow O. (2000) High-molecular weight glutenins of common winter wheat of European countries and their links with glutenins of old and current varieties of Ukraine. Cytology and genetics. 34. P.104-120 

Sources for resistance to Soil-Borne Cereal Mosaic Virus (SBCMV) among cultivated accessions of common wheat and its wild relatives

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Sources for resistance to Soil-Borne Cereal Mosaic Virus (SBCMV) among cultivated accessions of common wheat and its wild relatives

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