Authors:Mitja Križman, Jernej Jakše, Mirko Prošek, Dea Baričevič, and Branka Javornik
Agarose gel electrophoresis is a basic separation tool used in molecular biology, mostly for qualitative DNA analysis. There are constraints limiting its use in quantitative analysis, namely low repeatability and a narrow linear range. However, by using an internal standard or internal normalization, repeatability and linear range could be significantly improved. In the work discussed in this paper it was shown that an approximately fivefold improvement in repeatability and an over threefold wider linear range could be achieved by applying internal normalization. Using the proposed approach, genetic markers, for example RAPD and PCR-RFLP, or even microsatellite markers, could be conveniently quantitatively assessed using agarose gel electrophoresis.
Authors:R. Singh, P. Hucl, M. Båga, and R. Chibbar
Pre-harvest sprouting (PHS) in bread wheat (Triticum aestivum L.) is one of the major abiotic constraints influencing production of high quality grain. Selection for pre-harvest sprouting (PHS) resistance in bread wheat (Triticum aestivum L.) in early generations is difficult because it is expressed as a quantitatively inherited trait and subject to environmental effects. The objectives of this study were to validate a major quantitative trait locus (QTL) for PHS resistance on chromosome 4A in bread wheat and to isolate near-isogenic lines for this QTL using marker-assisted selection. A total of 60 Canadian wheat cultivars and experimental lines were screened with three SSR markers in a QTL region for PHS resistance. The SSR markers DuPw004, barc170 and wmc650 explained 67%, 75% and 60% of total variation in germination (%), respectively, among different wheat genotypes. Marker assisted back crossing with DuPw004 reduced the population size in BC1F1 and BC2F1 generation by 41% and 59%, respectively. A survey of pedigrees of different genotypes revealed that the parental line RL4137 is a major source of increased PHS resistance in a number of western Canadian wheat cultivars. Microsatellite markers (DuPw004, barc170 and wmc650) will be useful for plant breeders to pyramid QTL from different PHS resistance sources.
Authors:F. Rafiei Boroujeni, A. Arzani, F. Afshari, and M. Torabi
The objectives of this research were to examine the inheritance of leaf rust resistance genes in the Iranian wheat cultivar ‘Marvdasht’, which is highly resistant to leaf and stripe rusts, and to identify Lr genes present in this cultivar using molecular markers. The genetic basis of resistance to the leaf rust pathogen (Puccinia triticina) in ‘Marvdasht’ was studied in F2:3 populations derived from crosses of Bolani (susceptible cultivar) × Marvdasht. Isolates 84-1 and 85-28 of P. triticina, which are the predominant isolates in Iran, were used to examine the segregation of resistance originating from ‘Marvdasht’. The results indicated that resistance in ‘Marvdasht’ to Puccinia triticina isolate 84-1 was governed by two dominant seedling resistance genes Lr1 and Lr17a. Allelism studies using an F2 population derived from a cross between ‘Falat’ (Seri 82) and Marvdasht indicated that resistance in Marvdasht was not due to the resistance gene Lr26 present in ‘Falat’. With the application of a previously developed molecular marker for Lr1, the STS marker RGA-567-5, the presence of Lr1 was verified in Marvdasht. Based on bulk segregant analysis, Lr17a was mapped to the distal end of chromosome 2AS and was closely linked to microsatellite marker Xbarc212 at a distance of 3.7 cM. In conclusion, the presence of Lr1 and Lr17a was confirmed in the cultivar Marvdasht.
In order to investigate the origin of two 4B-5R wheat-rye translocations a number of microsatellite markers were considered, (a) which were specific for the rye chromosome arm 5RL and (b) which should enable precise characterisation for the gene pool of a ‘Cornell’ wheat derivative. Seven out of eight markers revealed amplification fragments with the rye control, while the marker WMS0186 amplified only a PCR product with the appropriate chromosome arm of the wheat control. The heterogeneity of the DNA fragment patterns among five wheat-rye translocation lines confirms different wheat backgrounds of the American, Danish/UK, Hungarian, and Swedish sources of the 4B-5R translocations. The homogeneous DNA marker patterns of ‘Viking-hairy neck’ and ‘Cornell Sel. 82a1-2-4-7’ are particularly clear. The corresponding molecular markers together with the cytological data, genomic
hybridisation, the physiological investigations, and the historical review support the hypothesis of a common origin of ‘Viking-hairy neck’ and ‘Cornell Sel. 82a1-2-4-7’. The importance of the results for practical breeding and for introgression experiments is discussed.
Authors:M. Sivasamy, M. Aparna, J. Kumar, P. Jayaprakash, V. Vikas, P. John, R. Nisha, Satyaprakash, K. Sivan, and E. Punniakotti
Nearly twenty thousand wheat lines were phenotyped for the presence of leaf tip necrosis (LTN), a phenotypic trait linked to adult plant leaf rust resistance (APR) genes, viz. Lr34, Lr46 and Lr67 having pleiotropic association with multiple disease resistance genes. Thirty-six lines showed varied expression of LTN and moderate level of leaf rust severity at adult plant stage with slow rusting (disease progress at a retarded rate). Seedling resistance test (SRT) revealed susceptible and mixed infection types, a characteristic of adult plant resistance (APR) genes. Further molecular confirmation for the presence of these genes using available microsatellite markers revealed that of the 36 lines, five lines carried Lr46+ alone and five other lines carried Lr67+ alone. Seven lines carried the combination of Lr34+ and Lr67+ while six lines confirmed to carry the combination of Lr46+ and Lr67+. Remarkably three lines carried all the three APR genes, viz. Lr34+, Lr46+ and Lr67+. All these stocks can be a source of APR multiple disease resistance genes. Ten lines were not confirmed to carry any of the genes but still had LTN and SRT results showing an infection type typical of APR genes and these can be the source of identifying newer APR genes. The resistance based on minor APR genes when combined with a few additional minor genes in the background of high yielding cultivars is expected to have high level of race non-specific resistance and to be durable.
Authors:A. Kaushik, S. Jain, V. Bhankar, and R. Jain
Availability of markers that distinguish commercially important premium Basmati rice varieties from cheaper Basmati or non-Basmati indica rice varieties is of interest to exporters, commercial suppliers and consumers to ensure the varietal purity of Basmati rice supplies. In this study, a fingerprint database of 27 rice varieties including commercially important traditional Basmati (TB), cross-bred (evolved) Basmati and some non-Basmati indica rice varieties has been prepared using 50 microsatellite markers. A total of 271 electromorphs (alleles) were detected at 50 SSR (simple sequence repeat) loci, 92 of which were observed only in 2–10 of the 16 Basmati rice varieties. Notably, 7–10 of the sixteen Basmati rice varieties had the same Basmati specific electromorphs at RM562, RM551 and RM547 loci. Alarge number of unique (22) and null (16) electromorphs were noticed in Basmati rice varieties. A combination of four SSR markers (RM224, RM248, RM547 and RM594) can be used to differentiate all the 27 rice varieties. Specific SSR markers have been identified for the authentication of Premium Basmati rice cultivars such as Taraori Basmati (RM547, RM594 and RM511), Basmati 370 (RM252, RM426 and RM527), Karnal Local (RM248, RM423 and RM488) and Pusa 1121 (RM252, RM400 and RM410), and for the detection of adulterant like Sharbati (RM215, RM423 and RM259). SSR database reported here, shall broaden the list of SSR markers already recommended for varietal identification and detection of adulteration in Basmati rice supplies.
Authors:Péter Gergics, Judit Tőke, Ágnes Szilágyi, Ágnes Szappanos, Zoltán Kender, György Barta, Miklós Tóth, Péter Igaz, Károly Rácz, and Attila Patócs
Számos monogénesen öröklődő kórképben a betegséget okozó gén teljes vagy részleges deletiója, illetve kópiaszámának megváltozása patogenetikai tényezőként jön számításba. A direkt DNS-szekvenálás nem alkalmas a gén nagy deletiójának, illetve kópiaszám-változásának kimutatására. Az összefoglalóban a szerzők áttekintik a nagy géndeletio vizsgálómódszereit, és két, monogénesen öröklődő betegségben végzett saját vizsgálataik példáján keresztül bemutatják a módszerek gyakorlati alkalmazásának lehetőségeit. Vázolják a géndeletio-vizsgálat hagyományos (kromoszóma-sávtechnika, Southern-blot, fluoreszcens in situ hibridizáció) és polimeráz láncreakcióra alapozott módszereit (denaturáló nagy felbontóképességű folyadékkromatográfia, kvantitatív valós idejű polimeráz láncreakció, mikroszatellitamarker-analízis, multiplex amplifikálhatópróba-hibridizáció, multiplex ligatióspróba-analízis), valamint a technikai és informatikai haladás legújabb vívmányait (komparatív genomhibridizálás, „array” analízis). Saját vizsgálataikban von Hippel–Lindau-szindrómában szenvedő betegekben kvantitatív valós idejű polimeráz láncreakció és multiplex ligatióspróba-amplifikálás alkalmazásával bemutatják a
illetve congenitalis adrenalis hyperplasiás betegekben a
géndeletio-vizsgálat eredményeit és ezek klinikai jelentőségét.
Authors:M. Molnár-Láng, G Linc, E. D. Nagy, and et al.
New wheat × barley, wheat × Aegilops biuncialis and wheat × rye hybrids were produced with the aim of alien gene transfer from these species into wheat. Amphiploids were produced with the help of colchicine treatment from the last two combinations. The new wheat × barley hybrids were multiplied in tissue culture because of the high degree of sterility and then pollinated with wheat to obtain backcross progenies. Wheat-barley chromosome pairing was detected using genomic in situ hybridization (GISH) in two combinations (Mv9 kr1 × Igri, Asakazekomugi × Manas). In vitro conditions caused an increase in chromosome arm association frequency in both combinations and in fertility in some regenerants. Five wheat-barley translocations were produced in a wheat background and characterized through the combination of cytogenetic and molecular genetic approaches (GISH, FISH and SSR markers). The following translocations were identified: 2DS.2DL-1HS, 3HS.3BL, 6BS.6BL-4HL, 4D-5HS and 7DL.7DS-5HS. Physical mapping of the SSR markers on chromosomes 1H and 5H was carried out using the intragenomic and interspecific translocation breakpoints and the centromere as physical landmarks. Disomic wheat-Aegilops biuncialis additions were produced after backcrossing the wheat-Ae. biuncialis amphiploids. Fluorescence in situ hybridization (FISH) was carried out using two repetitive DNA clones (pSc119.2 and pAs1) on Ae. biuncialis and its two diploid progenitor species to detect chromosome polymorphism. The 7M and 3M disomic chromosome additions were selected and five more lines still need to be characterized. The octoploid triticale (Mv9 kr1 × Lovászpatonai) produced in Martonvásár was crossed with a 1RS.1BL wheat cultivar Matador. GISH analysis detected pairing between the 1RS arm of the translocation chromosome and that of Lovászpatonai rye in 32 % of the pollen mother cells, making it possible to select recombinants from this combination. The new recombinants between the 1RS of Petkus and the 1RS of Lovászpatonai rye cultivars are being analysed with the help of microsatellite markers.