Mapping of new resistance (<Emphasis Type="Italic">Vr2</Emphasis>, <Emphasis Type="Italic">Rm1</Emphasis>) and ornamental (<Emphasis Type="Italic">Di2</Emphasis>, <Emphasis Type="Italic">pl</Emphasis>) Mendelian trait loci in peach

Peach powdery mildew is one of the major diseases of the peach. Various sources of resistance to PPM have thus been identified, including the single dominant locus Vr2 carried by the peach rootstock ‘Pamirskij 5’. To map Vr2, a linkage map based on microsatellite markers was constructed from the F₂ progeny (WP²) derived from the cross ‘Weeping Flower Peach’ × ‘Pamirskij 5’. Self-pollinations of the parents were also performed. Under greenhouse conditions, all progenies were scored after artificial inoculations in two classes of reactions to PPM (resistant/susceptible). In addition to Vr2, WP² segregated for three other traits from ‘Weeping Flower Peach’: Rm1 for green peach aphid resistance, Di2 for double-flower and pl for weeping-growth habit. With their genomic locations unknown or underdocumented, all were phenotyped as Mendelian characters and mapped: Vr2 mapped at the top of LG8, at 3.3 cM, close to the CPSCT018 marker; Rm1 mapped at the bottom of LG1, at a position of 116.5 cM, cosegregating with the UDAp-467 marker and in the same region as Rm2 from ‘Rubira’^®; Di2 mapped at 28.8 cM on LG6, close to the MA027a marker; and pl mapped at 44.1 cM on LG3 between the MA039a and SSRLG3_16m46 markers. Furthermore, this study revealed, for the first time, a pseudo-linkage between two traits of the peach: Vr2 and the Gr locus, which controls the red/green color of foliage. The present work therefore constitutes a significant preliminary step for implementing marker-assisted selection for the four major traits targeted in this study.     

Allelic variation for the rust resistance gene <Emphasis Type="Italic">Lr34</Emphasis>/<Emphasis Type="Italic">Yr18</Emphasis> in Canadian wheat cultivars

The wheat (Triticum aestivum L.) gene Lr34/Yr18 conditions resistance to leaf rust, stripe rust, and stem rust, along with other diseases such as powdery mildew. This makes it one of the most important genes in wheat. In Canada, Lr34 has provided effective leaf rust resistance since it was first incorporated into the cultivar Glenlea, registered in 1972. Recently, molecular markers were discovered that are either closely linked to this locus, or contained within the gene. Canadian wheat cultivars released from 1900 to 2007, breeding lines and related parental lines, were tested for sequence based markers caSNP12, caIND11, caIND10, caSNP4, microsatellite markers wms1220, cam11, csLVMS1, swm10, csLV34, and insertion site based polymorphism marker caISBP1. Thirty different molecular marker haplotypes were found among the 375 lines tested; 5 haplotypes had the resistance allele for Lr34, and 25 haplotypes had a susceptibility allele at this locus. The numbers of lines in each haplotype group varied from 1 to 140. The largest group was represented by the leaf rust susceptible cultivar “Thatcher” and many lines derived from “Thatcher”. The 5 haplotypes that had the resistance allele for Lr34 were identical for the markers tested within the coding region of the gene but differed in the linked markers wms1220, caISBP1, cam11, and csLV34. The presence of the resistance or susceptibility allele at the Lr34 locus was tracked through the ancestries of the Canadian wheat classes, revealing that the resistance allele was present in many cultivars released since the 1970s, but not generally in the older cultivars.     

Molecular mapping of <Emphasis Type="Italic">Pc68</Emphasis>, a crown rust resistance gene in <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

Crown rust, which is caused by Puccinia coronata f. sp. avenae, P. Syd. & Syd., is the most destructive disease of cultivated oats (Avena sativa L.) throughout the world. Resistance to the disease that is based on a single gene is often short-lived because of the extremely great genetic diversity of P. coronata, which suggests that there is a need to develop oat cultivars with several resistance genes. This study aimed to identify amplified fragment length polymorphism AFLP markers that are linked to the major resistance gene, Pc68, and to amplify the F₆ genetic map from Pc68/5*Starter × UFRGS8. Seventy-eight markers with normal segregation were discovered and distributed in 12 linkage groups. The map covered 409.4 cM of the Avena sativa genome. Two AFLP markers were linked in repulsion to Pc68: U8PM22 and U8PM25, which flank the gene at 18.60 and 18.83 centiMorgans (cM), respectively. The marker U8PM25 is located in the linkage group 4_12 in the Kanota × Ogle reference oat population. These markers should be useful for transferring Pc68 to genotypes with good agronomic characteristics and for pyramiding crown rust resistance genes.     

Expression of <Emphasis Type="Italic">Bruguiera gymnorhiza</Emphasis><Emphasis Type="Italic">BgARP1</Emphasis> enhances salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

We have previously reported that expression of salt-responsive genes, including Bruguiera gymnorhiza ankyrin repeat protein 1 (BgARP1), enhances salt tolerance in both Agrobacterium tumefaciens and Arabidopsis. In this report, we further characterized BgARP1-expressing Arabidopsis to elucidate the role of BgARP1 in salt tolerance. BgARP1-expressing plants exhibited more vigorous growth than wild-type plants on MS plates containing 125–175 mM NaCl. Real-time PCR analysis showed enhanced induction of osmotin34 in the 2-week-old transformants under 125 mM NaCl. It was also showed that induction of typical salt-responsive genes, including RD29A, RD29B, and RD22, was blunted and delayed in the 4-week-old transformants during 24 h after 200 mM NaCl treatment. Ion content analysis showed that transgenic plants contained more K⁺, Ca²⁺, and NO₃ ⁻, and less NH₄ ⁺, than wild-type plants grown in 200 mM NaCl. Our results suggest that BgARP1-expressing plants may reduce salt stress by up-regulating osmotin34 gene expression and maintaining K⁺ homeostasis and regulating Ca²⁺ content. These results indicate that BgARP1 is functional on a heterogeneous background.     

Genetic effects of introgression genomic components from Sea Island cotton (<Emphasis Type="Italic">Gossypium barbadense</Emphasis> L.) on fiber related traits in upland cotton (<Emphasis Type="Italic">G</Emphasis>. <Emphasis Type="Italic">hirsutum</Emphasis> L.)

The germplasm with exotic genomic components especially from Sea Island cotton (Gossypium barbadense L. Gb) is the dominant genetic resources to enhance fiber quality of upland cotton (G. hirsutum L., Gh). Due to low efficiency of phenotypic evaluation and selection on fiber quality, genetic dissection of favorable alleles using molecular markers is essential. Genetic dissection on putative Gb introgressions related to fiber traits were conducted by SSR markers with mapping populations derived from a cross between Luyuan343 (LY343), a superior fiber quality introgression line (IL) with genomic components from Gb, and an elite Upland cotton cv. Lumianyan#22 (LMY22). Among 82 polymorphic loci screened out from 4050 SSRs, 42 were identified as putative introgression alleles. A total of 29 fiber-related QTLs (23 for fiber quality and six for lint percentage) were detected and most of which clustered on the putative Gb introgression chromosomal segments of Chr.2, Chr.16, Chr.23 and Chr.25. As expected, a majority of favorable alleles of fiber quality QTLs (12/17, not considering the QTLs for fiber fineness) came from the IL parent and most of which (11/12) were conferred by the introgression genomic components while three of the six (3/6) favorable alleles for lint percentage came from the Gh parent. Validation of these QTLs using an F₈ breeding population from the same cross made previously indicated that 13 out of 29 QTLs showed considerable stability. The results suggest that fiber quality improvement using the introgression components could be facilitated by marker-assisted selection in cotton breeding program.     

Dominant gene for common bean resistance to common bacterial blight caused by <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">axonopodis</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis>

The common bacterial blight pathogen [Xanthomonas axonopodis pv. phaseoli (Xap)] is a limiting factor for common bean (Phaseolus vulgaris L.) production worldwide and resistance to the pathogen in most commercial cultivars is inadequate. Variability in virulence of the bacterial pathogen has been observed in strains isolated from Puerto Rico and Central America. A few common bean lines show a differential reaction when inoculated with different Xap strains, indicating the presence of pathogenic races. In order to study the inheritance of resistance to common bacterial blight in common bean, a breeding line that showed a differential foliar reaction to Xap strains was selected and was crossed with a susceptible parent. The inheritance of resistance to one of the selected Xap races was determined by analysis of segregation patterns in the F₁, F₂, F₃ and F₄ generations from the cross between the resistant parent PR0313-58 and the susceptible parent ‘Rosada Nativa’. The F₁, F₂ and F₃ generations were tested under greenhouse conditions. Resistant and susceptible F_3:4 sister lines were tested in the field. The statistical analysis of all generations followed the model for a dominant resistance gene. The resistant phenotype was found to co-segregate with the SCAR SAP6 marker, located on LG 10. These results fit the hypothesis that resistance is controlled by a single dominant gene. The symbol proposed for the resistance gene is Xap-1 and for the bacterial race, XapV1.     

Map-based cloning of the fertility restoration locus <Emphasis Type="Italic">Rfm1</Emphasis> in cultivated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>)

Hybridization technology has proven valuable in enhancing yields in many crops, but was only recently adopted in the small grain cereals. Hybrid varieties in barley (Hordeum vulgare) rely on the cytoplasmic male sterility (CMS) system msm1 derived from Hordeum vulgare ssp. spontaneum. The major restorer gene described for the msm1 system is known as Rfm1 and maps to the top of chromosome 6H. To gain further insight into mechanisms underlying male fertility restoration in barley, we used a map-based cloning approach to identify the nuclear gene involved in the restoration mechanism of this hybridization system. Taking advantage of the available genomic resources in barley in combination with a custom-made non-gridded BAC library developed from a restorer line, we cloned and sequenced the Rfm1 restorer locus. The characterization and annotation of the nucleotide sequence for the Rfm1 restorer allele allowed for the identification of the candidate gene for Rfm1. The Rfm1 locus carries a tandem repeat of a gene encoding a pentatricopeptide repeat (PPR) protein. Surprisingly, Rfm1 belongs to the PLS-DYW subfamily of PPR genes known for their involvement in RNA editing in plants organelles, but that to date have not been identified as restorer genes.     

Improved salt tolerance and seed cotton yield in cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) by transformation with <Emphasis Type="Italic">betA</Emphasis> gene for glycinebetaine synthesis

Homozygous transgenic cotton (Gossypium hirsutum L.) plants that accumulated glycinebetaine (GB) in larger quantities were more tolerant to salt than wild-type (WT) plants. Four transgenic lines, namely 1, 3, 4, and 5, accumulated significantly higher levels of GB than WT plants did both before and after salt stress. At 175 and 275 mM NaCl, seeds of all the transgenic lines germinated earlier and recorded a higher final germination percentage, and the seedlings grew better, than those of the WT. Under salt stress, all the lines showed some characteristic features of salt tolerance, such as higher leaf relative water content (RWC), higher photosynthesis, better osmotic adjustment (OA), lower percentage of ion leakage, and lower peroxidation of the lipid membrane. Levels of endogenous GB in the transgenic plants were positively correlated with RWC and OA. The results indicate that GB in transgenic cotton plants not only maintains the integrity of cell membranes but also alleviates osmotic stress caused by high salinity. Lastly, the seed cotton yield of transgenic lines 4 and 5 was significantly higher than that of WT plants in saline soil. This research indicates that betA gene has the potential to improve crop’s salt tolerance in areas where salinity is limiting factors for agricultural productivity.     

Amenability of castor to an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation strategy using a <Emphasis Type="Italic">cry1AcF</Emphasis> gene for insect tolerance

Agrobacterium tumefaciens mediated in planta transformation protocol was developed for castor, Ricinus communis. Two-day-old seedlings were infected with Agrobacterium strain EHA105/pBinBt8 harboring cry1AcF and established in the greenhouse. Screening the T₁ generation seedlings on 300 mg L⁻¹ kanamycin identified the putative transformants. Molecular and expression analysis confirmed the transgenic nature and identified high-expressing plants. Western blot analysis confirmed the co-integration of the nptII gene in the selected transgenic plants. Bioassay against Spodoptera litura corroborated with high expression and identified five promising effective lines. Analysis of the T₂ generation plants proved the stability of the transgene indicating the feasibility of the method.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with a fungal phytase gene improves phosphorus acquisition

A phytase gene (phyA), isolated from Aspergillus ficuum (AF537344), was introduced into cotton (Gossypium hirsutum L.) by Agrobacterium-mediated transformation to increase the phosphorus (P) acquisition efficiency of cotton. Southern and Northern blot analyses showed that the phyA was successfully incorporated into the cotton genome and expressed in transgenic lines. After growing for 45 days with phytate (Po) as the only P source, the shoot and root dry weights of the transgenic plants all increased by nearly 2.0-fold relative to those of wild-type plants, but were similar to those of transgenic plants supplied with inorganic phosphorus. The phytase activities of root extracts prepared from transgenic plants were 2.4- to 3.6-fold higher than those from wild-type plants, and the extracellular phytase activities of transgenic plants were also 4.2- to 6.3-fold higher. Furthermore, the expressed phytase was secreted into the rhizospheres as demonstrated by enzyme activity staining. The transgenic plants accumulated much higher contents of total P (up to 2.1-fold after 30 days of growth) than the wild-type plants when supplied with Po. These findings clearly showed that cotton plant transformed with a fungal phytase gene was able to secret the enzyme from the root, which markedly improved the plant’s ability to utilize P from phytate. This may serve as a promising step toward the development of new cotton cultivars with improved phosphorus acquisition.     

Identification of genetic sources with attenuated <Emphasis Type="Italic">Tomato chlorosis virus</Emphasis>-induced symptoms in <Emphasis Type="Italic">Solanum</Emphasis> (section <Emphasis Type="Italic">Lycopersicon</Emphasis>) germplasm

The whitefly-transmitted Tomato chlorosis virus (ToCV) (genus Crinivirus) is associated with yield and quality losses in field and greenhouse-grown tomatoes (Solanum lycopersicum) in South America. Therefore, the search for sources of ToCV resistance/tolerance is a major breeding priority for this region. A germplasm of 33 Solanum (Lycopersicon) accessions (comprising cultivated and wild species) was evaluated for ToCV reaction in multi-year assays conducted under natural and experimental whitefly vector exposure in Uruguay and Brazil. Reaction to ToCV was assessed employing a symptom severity scale and systemic virus infection was evaluated via RT-PCR and/or molecular hybridization assays. A subgroup of accessions was also evaluated for whitefly reaction in two free-choice bioassays carried out in Uruguay (with Trialeurodes vaporariorum) and Brazil (with Bemisia tabaci Middle-East-Asia-Minor1—MEAM1?=?biotype B). The most stable sources of ToCV tolerance were identified in Solanum habrochaites PI 127827 (mild symptoms and low viral titers) and S. lycopersicum ‘LT05’ (mild symptoms but with high viral titers). These two accessions were efficiently colonized by both whitefly species, thus excluding the potential involvement of vector-resistance mechanisms. Other promising breeding sources were Solanum peruvianum (sensu lato) ‘CGO 6711’ (mild symptoms and low virus titers), Solanum chilense LA1967 (mild symptoms, but with high levels of B. tabaci MEAM1 oviposition) and Solanum pennellii LA0716 (intermediate symptoms and low level of B. tabaci MEAM1 oviposition). Additional studies are necessary to elucidate the genetic basis of the tolerance/resistance identified in this set of Solanum (Lycopersicon) accessions.     

Superior CMS (<Emphasis Type="Italic">Ogura</Emphasis>) lines with better combining ability improve yield and maturity in cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">botrytis</Emphasis>)

Three CMS lines, Ogu1A, Ogu2A and Ogu3A were selected among ten lines after BC₇ based on superior commercial, floral and seed setting traits. Introgression of sterile Ogura cytoplasm in cauliflower nuclear background reduced the flower size but did not affect commercial and seed setting traits drastically. Line × Tester analysis was done by taking these three CMS lines free from floral deformities as female parent with nine diverse lines of snowball cauliflower as tester. The parent Ogu2A exhibited highest GCA effect for curd yield (4.51) and harvest index (1.97) while Ogu1A exhibited highest GCA for earliness (−2.73). The parent, Ogu2A exhibited significant GCA for curd length (0.39) while, none of the CMS lines showed significant GCA for curd diameter and depth. Heterosis for curd yield was highest in the hybrid, Ogu2A × Kt-22 (63.5%) followed by Ogu1A × WF (36.9%) and Ogu1A × Kt-15 was the best hybrid for earliness followed by Ogu3A × Kt-22 with heterosis of −14.4% and −11.7%. However, the number of heterotic hybrids for yield and earliness was low indicating narrow genetic base of the snowball cauliflower.     

Search in <Emphasis Type="Italic">Solanum</Emphasis> (section <Emphasis Type="Italic">Lycopersicon</Emphasis>) germplasm for sources of broad-spectrum resistance to four <Emphasis Type="Italic">Tospovirus</Emphasis> species

The genus Tospovirus was considered as monotypic with Tomato spotted wilt virus (TSWV) being the only assigned species. However, extensive studies with worldwide isolates revealed that this genus comprises a number of species with distinct virulence profiles. The Neotropical South America is one center of Tospovirus diversity with many endemic species. Groundnut ringspot virus (GRSV), TSWV, Tomato chlorotic spot virus (TCSV), and Chrysanthemum stem necrosis virus (CSNV) are the predominant tomato-infecting species in Brazil. Sources of resistance were found in Solanum (section Lycopersicon) mainly against TSWV isolates from distinct continents, but there is an overall lack of information about resistance to other viral species. One-hundred and five Solanum (section Lycopersicon: Solanaceae) accessions were initially evaluated for their reaction against a GRSV isolate by analysis of symptom expression and systemic virus accumulation using DAS-ELISA. A subgroup comprising the most resistant accessions was re-evaluated in a second assay with TSWV, TCSV, and GRSV isolates and in a third assay with a CSNV isolate. Seven S. peruvianum accessions displayed a broad-spectrum resistance to all viral species with all plants being free of symptoms and systemic infection. Sources of resistance were also found in tomato cultivars with the Sw-5 gene and also in accessions of S. pimpinellifolium, S. chilense, S. arcanum, S. habrochaites, S. corneliomuelleri, and S. lycopersicum. The introgression/incorporation of these genetic factors into cultivated tomato varieties might allow the development of genetic materials with broad-spectrum resistance, as well as with improved levels of phenotypic expression.     

Resistance screening of <Emphasis Type="Italic">Coffea</Emphasis> spp. accessions for <Emphasis Type="Italic">Pratylenchus coffeae</Emphasis> and <Emphasis Type="Italic">Radopholus arabocoffeae</Emphasis> in Vietnam

Coffee varieties with resistance for the plant-parasitic nematodes Pratylenchus coffeae and Radopholus arabocoffeae are limited in Vietnam. A selection of imported varieties and high yield varieties of Arabica coffee in Vietnam were evaluated for resistance to both plant-parasitic nematode species in Northern Vietnam. The same experiments were carried out with hybrid arabica coffee, three selected clones of Coffea canephora and one clone of Coffea excelsa in the Western Highland of Vietnam. The screened coffee accessions from Ethiopia (KH1, KH13, KH20, KH21, KH29, and KH31) were susceptible and good host for P. coffeae. Also accessions 90P4 (Portugal) and Oro azteca (Mexico) had a reproduction factor Rf > 1. Pluma Hidalgo (Mexico), 90/6 (Vietnam), 90P3 (Portugal), 90P2 (Vietnam), Variedad (Mexico), 90T (Portugal), and Garnica (Mexico) were poor hosts (Rf < 1) but not tolerant to P. coffeae, expressed by a reduction of root weight compared to untreated control plants. Most of the coffee accessions tested in Northern Vietnam were intolerant to R. arabocoffeae, except 90T which showed no reduction of root weight, even at high initial nematode densities (4,000/pot). Good hosts for R. arabocoffeae were Variedad, KH1, KH21, KH29, KH20, KH31, and KH13 with Rf > 1. Pluma Hidalgo, 90/6, 90P3, 90P2, 90T, Oro azteca, and Garnica were poor hosts (Rf < 1). In the Western Highland experiment, all arabica coffee accessions were susceptible for P. coffeae with Rf ranging from 1.41 to 1.59. Tolerance to P. coffeae was found in C. liberica var. Dewevrei, Hong34 and Nhuantren. Coffea excelsa, Hong34, Nhuantren, and H1C19 were tolerant to R. arabocoffeae at the highest inoculation density (4,000 nematodes/pot). The most susceptible accessions were Nhuantren and K55. Resistance (Rf < 1) to R. arabocoffeae was found in C. liberica var. Dewevrei and Hong34. This article reports on the first screening for resistance and tolerance to P. coffeae and R. arabocoffeae in coffee accessions in Vietnam and shows promising results for enhanced coffee-breeding.     

Production and characterization of inter-sectional hybrids between <Emphasis Type="Italic">Kalanchoe spathulata</Emphasis> and <Emphasis Type="Italic">K. laxiflora</Emphasis> ( = <Emphasis Type="Italic">Bryophyllum crenatum</Emphasis>)

The genus Kalanchoe is currently divided into section Kalanchoe and section Bryophyllum, and there has been no successful report on the production of inter-sectional hybrids. Therefore, reciprocal crosses were made between Kalanchoe spathulata (sect. Kalanchoe) and K. laxiflora (sect. Bryophyllum) in order to obtain basic information on the reproductive barriers between these two sections. The seeds were aseptically germinated in vitro and the plants were grown in greenhouse till flowering. When K. spathulata was used as a maternal donor, 39 out of 80 plants showed intermediate characteristics between K. spathulata and K. laxiflora. In contrast, no plants were obtained in the reverse crosses. Hybridity of these plants was confirmed by flow cytometric analysis, chromosome numbers and RAPD analysis. Bulbil formation on the leaf margin as one of the conspicuous characteristics of K. laxiflora was not observed in the hybrids. Some of the hybrid lines showed some pollen fertility, but failed to yield viable seeds by self-pollination or backcross-pollination. Successful production of the inter-sectional hybrid between the two species suggests that they are not so distantly related as considered previously.     

<Emphasis Type="Italic">Rf4</Emphasis> has minor effects on the fertility restoration of wild abortive-type cytoplasmic male sterile <Emphasis Type="Italic">japonica</Emphasis> (<Emphasis Type="Italic">Oryza sativa</Emphasis>) lines

Wild abortive (WA)-type cytoplasmic male sterility (CMS) has been exclusively used for breeding three-line hybrid indica rice, but it has not been applied for generating japonica hybrids because of the difficulties related to breeding japonica restorer lines. Determining whether the major restorer-of-fertility (Rf) gene used for indica hybrids can efficiently restore the fertility of WA-type japonica CMS lines may be useful for breeding WA-type japonica restorer lines. In this study, japonica restorer lines for Chinsurah Boro II (BT)-type CMS exhibited varying abilities to restore the fertility of ‘WA-LiuqianxinA’, which is a WA-type japonica CMS line. Additionally, Rf genes for WA-type CMS were identified in the BT-type japonica restorers. Meanwhile, ‘C9083’, which is a BT-type japonica restorer, exhibited a limited ability to restore the fertility of WA-type japonica CMS lines, and a genetic analysis revealed that the fertility restoration was controlled by one locus. The Rf gene was mapped to an approximately 370-kb physical region and was identified as Rf4. Furthermore, Rf gene dosage effects and the temperature influenced the fertility restoration of WA-type japonica CMS lines. This study is the first to confirm that Rf4 has only minor effects on the fertility restoration of WA-type japonica CMS lines. These results may be relevant for the development of WA-type japonica hybrids.     

Development and application of a molecular marker for TMV resistance based on <Emphasis Type="Italic">N</Emphasis> gene in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

Tobacco mosaic virus (TMV) caused serious loss in yield and quality of tobacco every year. It is a long-term goal to improve the tobacco resistance against TMV by tobacco breeding. N gene was the firstly reported TMV-resistant gene, which showed resistance against all Tobamoviruses except the Ob stain and belonged to the toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat class of plant resistance (R) genes. At present, N gene had already been widely used in tobacco conventional breeding, but there is rare available molecular maker used in marker-assisted selection of TMV resistance. In this study, we designed a pair of primers that specific amplify N gene fragment based on the sequence of N gene intron III, named N-marker. Then, we identified TMV resistance by two selecting methods, PCR with N-marker and inoculated with the TMV-C strain. Results from the two method showed that (1) 13 varieties among 67 tobacco varieties displayed hypersensitive reaction when inoculated with the TMV-C strain, also contained N gene fragments screened by PCR with N-marker; (2) 105 strains of 200 BC1 strains showed resistance against TMV when inoculated with TMV-C strain, meanwhile, 103 of the 105 strains contained N gene fragment verified by PCR with N-marker. Therefore, the N-marker is reliable for high throughput screening of germplasm resources and tobacco breeding materials in selection of N-mediated TMV resistance. Our study not only developed a molecular marker for tobacco breeding, but also identified new germplasm resources that are resistant to TMV.     

Asparagine synthetase genes (<Emphasis Type="Italic">AsnS1</Emphasis> and <Emphasis Type="Italic">AsnS2</Emphasis>) in durum wheat: structural analysis and expression under nitrogen stress

Wheat is one of the most widely grown cereal crops based on the amount of calories it provides in the human diet. Durum wheat (Triticum turgidum ssp. durum) is largely used for production of pasta and other products. In order to use genetic knowledge to improve the understanding of N-use efficiency, we carried out, for the first time in durum wheat, the isolation and the characterization of four members of the asparagine synthetase (AsnS) gene family. Phylogenetic inference clustered the Ttu-AsnS1 (1.1 and 1.2) and Ttu-AsnS2 (2.1 and 2.2) genes in AsnS gene class I, which is present in monocots and dicots. Class I genes underwent a subsequent duplication leading to the formation of two subgroups. Plants of Svevo cultivar were grown under N-stress conditions and expression of the four AsnS genes was investigated at three developmental stages (seedling, booting, and late milk development), crucial for N absorption, assimilation and remobilization. AsnS1 genes were down-regulated in N-stressed roots, stems and leaves during seedling growth and booting, but seemed to play a role in N remobilization in flag leaves during grain filling. AsnS2 genes were scarcely expressed in roots, stems, and leaves. In N-stressed spikes there was no differential expression in any of the genes. The genes were mapped in silico using a durum wheat SNP map, assigning Ttu-AsnS1 genes to chromosome 5 and Ttu-AsnS2 to chromosome 3. These findings provide a better understanding of the role of ASN genes in response to N stress in durum wheat.     

Molecular markers linked to <Emphasis Type="Italic">Bn</Emphasis>;<Emphasis Type="Italic">rf</Emphasis>: a recessive epistatic inhibitor gene of recessive genic male sterility in <Emphasis Type="Italic">Brassica napus </Emphasis>L.

7–7365AB is a recessive genic male sterile (RGMS) two-type line, which can be applied in a three-line system with the interim-maintainer, 7–7365C. Fertility of this system is controlled by two duplicate dominant epistatic genes (Bn;Ms3 and Bn;Ms4) and one recessive epistatic inhibitor gene (Bn;rf). Therefore an individual with the genotype of Bn;ms3ms3ms4ms4Rf_ exhibits male sterility, whereas, plant with Bn;ms3ms3ms4ms4rfrf shows fertility because homozygosity at the Bn;rf locus (Bn;rfrf) can inhibit the expression of two recessive male sterile genes in homozygous Bn;ms3ms3ms4ms4 plant. A cross of 7–7365A (Bn;ms3ms3ms4ms4RfRf) and 7–7365C (Bn;ms3ms3ms4ms4rfrf) can generate a complete male sterile population served as a mother line with restorer in alternative strips for the multiplication of hybrid seeds. In the present study, molecular mapping of the Bn;Rf gene was performed in a BC₁ population from the cross between 7–7365A and 7–7365C. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) technique was used to identify molecular markers linked to the gene of interest. From a survey of 768 primer combinations, seven AFLP markers were identified. The closest marker, XM5, was co-segregated with the Bn;Rf locus and successfully converted into a sequence characterized amplified region (SCAR) marker, designated as XSC5. Two flanking markers, XM3 and XM2, were 0.6 cM and 2.6 cM away from the target gene, respectively. XM1 was subsequently mapped on linkage group N7 using a doubled-haploid (DH) mapping population derived from the cross Tapidor × Ningyou7, available at IMSORB, UK. To further confirm the location of the Bn;Rf gene, additional simple sequence repeat (SSR) markers in linkage group N7 from the reference maps were screened in the BC₁ population. Two SSR markers, CB10594 and BRMS018, showed polymorphisms in our mapping population. The molecular markers found in the present study will facilitate the selection of interim-maintainer.