Understanding the inheritance of mungbean yellow mosaic virus (MYMV) resistance in mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek)

Mungbean, Vigna radiata, third in the series of important pulse crops, still suffers from yield loss due to mungbean yellow mosaic disease caused by mungbean yellow mosaic virus (MYMV). Hence, studies on plant-microbe interaction are necessary for understanding the inheritance of resistance. This study concentrated on identification of linked molecular markers for MYMV resistance and to find the genetic inheritance of MYMV resistance in mungbean. A total of 413 germplasm entries in a MYMV hot spot area (Vamban) were subjected to natural field infection and 13 selected resistant lines were subjected to Agrobacterium infection using strains harboring partial genome of two different MYMV isolates, VA221 and VA239. Among the resistant lines, KMG189 showed strain-specific resistance to VA221 and had no symptoms during field trials. Ninety F₂ genotypes were developed from the cross made between KMG189 (MYMV-resistant) and VBN(Gg)2 (MYMV-susceptible), segregated in the Mendelian single cross ratio 3S:1R; susceptibility of all the F₁s to MYMV suggested that the MYMV resistance in mungbean is governed by a single recessive gene. Two SCAR markers CM9 and CM815 were developed through bulk segregant analysis, and the linkage analysis proved CM815 SCAR marker to be linked at 5.56 cM with MYMV resistance gene and SCAR CM9 had nil recombination percentage, suggesting it to be very closely linked to the MYMV resistance gene. SCAR marker CM9 was present in chromosome number 3 of mungbean suggesting novel loci for virus resistance in mungbean. The identified loci can be used for developing varieties resistant to MYMV in mungbean.     

Quantitative trait loci mapping of Cercospora leaf spot resistance in mungbean, <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek

Cercospora leaf spot (CLS) caused by the fungus Cercospora canescens Illis & Martin is a serious disease in mungbean (Vigna radiata (L.) Wilczek), and disease can reduce seed yield by up to 50%. We report here for the first time quantitative trait loci (QTL) mapping for CLS resistance in mungbean. The QTL analysis was conducted using F₂ (KPS1 × V4718) and BC₁F₁ [(KPS1 × V4718) × KPS1] populations developed from crosses between the CLS-resistant mungbean V4718 and CLS-susceptible cultivar Kamphaeng Saen 1 (KPS1). CLS resistance in F₂ populations was evaluated under field conditions during the wet seasons of 2008 and 2009, and resistance in BC₁F₁ was evaluated under field conditions during the wet season in 2008. Seven hundred and fifty-three simple sequence repeat (SSR) markers from various legumes were used to assess polymorphism between KPS1 and V4718. Subsequently, 69 polymorphic markers were analyzed in the F₂ and BC₁F₁ populations. The results of segregation analysis indicated that resistance to CLS is controlled by a single dominant gene, while composite interval mapping consistently identified one major QTL (qCLS) for CLS resistance on linkage group 3 in both F₂ and BC₁F₁ populations. qCLS was located between markers CEDG117 and VR393, and accounted for 65.5–80.53% of the disease score variation depending on seasons and populations. An allele from V4718 increased the resistance. The SSR markers flanking qCLS will facilitate transferral of the CLS resistance allele from V4718 into elite mungbean cultivars.     

Cloning and Drought Resistance Analysis of Soybean <i>GmHsps_p23-like</i> Gene

Soybean (Glycine max (L.) Merr.) is an important cultivated crop, which requires much water during its growth, and drought seriously affects soybean yields. Studies have shown that the expression of small heat shock proteins can enhance drought resistance, cold resistance and salt resistance of plants. In this experiment, soybean GmHsps_p23-like gene was successfully cloned by RT-PCR, the protein encoded by the GmHsps_p23-like gene was subjected to bioinformatics analysis, and the pCAMBIA3301-GmHsps_p23-like overexpression vector and pCBSG015-GmHsps_p23-like gene editing vector were constructed. Agrobacterium-mediated method was used to transform soybeans to obtain positive plants. RT-PCR detection, rehydration experiment and drought resistance physiological and biochemical index detection were performed on the T₂ generation positive transgenic soybean plants identified by PCR and Southern hybridization. The results showed that the overexpression vector plant GmHsps_p23-like gene expression increased. After rehydration, the transgenic overexpression plants returned to normal growth, and the damage to the plants was low. After drought stress, the SOD and POD activities and the PRO content of the transgenic overexpression plants increased, while the MDA content decreased. The reverse was true for soybean plants with genetically modified editing vectors. The drought resistance of the overexpressed soybeans under drought stress was higher than that of the control group, and had a stronger drought resistance. It showed that the expression of soybean GmHsps_p23-like gene can improve the drought resistance of soybean. The cloning and functional verification of soybean GmHsps_p23-like gene had not been reported yet. This is the first time that PCR technology has been used to amplify the soybean GmHsps_p23-like gene and construct an expression vector for this gene. This research has laid the foundation for transgenic technology to improve plant drought resistance and cultivate new drought-resistant transgenic soybean varieties.     

Cloning and Characterization of <i>EuGID1</i> in <i>Eucommia ulmoides</i> Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants, and regulated the growth and development of plants. In this study, a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver. The cDNA of EuGID1 was 1556 bp, and the open reading frame was 1029 bp, which encoded 343 amino acids. EuGID1 had the homology sequence with the hormone-sensitive lipase family. Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta. EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs. Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.     

Variation for Resistance to <i>Alternaria tenuissima</i> and Potential Structural Mechanism among Different Cultivars of <i>Chrysanthemum morifolium</i>

Black spot disease, caused by the necrotrophic fungus Alternaria tenuissima (Fr.) Wiltsh (A. tenuissima), is considered a highly destructive disease of Chrysanthemum (Chrysanthemum morifolium Ramat.). A set of 17 accessions of commercial chrysanthemum cultivars were evaluated for resistance to A. tenuissima by seedling artificial inoculation. It was found that the reaction of the accessions to artificial inoculation ranged from resistant to highly susceptible. Five varieties of chrysanthemum (‘Zhongshan Taogui’, ‘Jinba’, ‘Zhongshan Jinguan’, ‘Jinling Wanhuang’ and ‘Jinling Yangguang’) were resistant; two varieties of chrysanthemum (‘Zhongshan Xinggui’ and ‘Zhongshan Jinkui’) were moderately resistant; and others were susceptible to various degrees, four varieties of chrysanthemum (‘Zhongshan Zihe’, ‘Zhongshan Jiuhong’, ‘Zaoyihong’ and ‘Jinling Jiaohuang’) were highly susceptible, especially. Some leaf morphological features of two resistant and two highly susceptible cultivars were further researched. Trichome density, length, height, gland size and stomata density were found to be associated with plant passive resistance. Resistant varieties that were identified in present study will be promising germplasm for exploitation of breeding programmes aimed at developing A. tenuissima-resistant cultivars and increasing genetic diversity.     

Identification and Evaluation of Insect and Disease Resistance in Transgenic <i>Cry1Ab13-1</i> and <i>NPR1</i> Maize

PCR detection, quantitative real-time PCR (q-RTPCR), outdoor insect resistance, and disease resistance identification were carried out for the detection of genetic stability and disease resistance through generations (T₂, T₃, and T₄) in transgenic maize germplasms (S3002 and 349) containing the bivalent genes (insect resistance gene Cry1Ab13-1 and disease resistance gene NPR1) and their corresponding wild type. Results indicated that the target genes Cry1Ab13-1 and NPR1 were successfully transferred into both germplasms through tested generations; q-PCR confirmed the expression of Cry1Ab13-1 and NPR1 genes in roots, stems, and leaves of tested maize plants. In addition, S3002 and 349 bivalent gene-transformed lines exhibited resistance to large leaf spots and corn borer in the field evaluation compared to the wild type. Our study confirmed that Cry1Ab13-1 and NPR1 bivalent genes enhanced the resistance against maize borer and large leaf spot disease and can stably inherit. These findings could be exploited for improving other cultivated maize varieties.     

<Emphasis Type="Italic">Vr</Emphasis><Subscript><Emphasis Type="Italic">2</Emphasis></Subscript>: a new apple scab resistance gene

Reports from several European countries of the breakdown of the Vf resistance, the most frequently used source of resistance in breeding programs against apple scab, emphasize the urgency of diversifying the basis of apple scab resistance and pyramiding different apple scab resistances with the use of their associated molecular markers. GMAL 2473 is an apple scab resistant selection thought to carry the resistance gene Vr. We report the identification by BSA of three AFLP markers and one RAPD marker associated with the GMAL 2473 resistance gene. SSRs associated with the resistance gene were found by (1) identifying the linkage group carrying the apple scab resistance and (2) testing the SSRs previously mapped in the same region. One such SSR, CH02c02a, mapped on linkage group 2, co-segregates with the resistance gene. GMAL 2473 was tested with molecular markers associated with other apple scab resistance genes, and accessions carrying known apple scab resistance genes were tested with the SSR linked to the resistance gene found in GMAL 2473. The results indicate that GMAL 2473 does not carry Vr, and that a new apple scab resistance gene, named Vr ₂, has been identified.     

Identification of Resistance to Pathogenesis Related Protein <i>GmPR1L</i> in Tobacco <i>Botrytis cinerea</i> Infection

Soybean (Glycine max (Linn.) Merr.) annual leguminous crop is cultivated all over the world. The occurrence of diseases has a great impact on the yield and quality of soybean. In this study, based on the RNA-seq of soybean variety M18, a complete CDS (Coding sequence) GmPR1L of the pathogenesis-related protein 1 family was obtained, which has the ability to resist fungal diseases. The overexpression vector and interference expression vector were transferred into tobacco NC89, and the resistance of transgenic tobacco (Nicotiana tabacum L.) to Botrytis cinerea infection was identified. The results show that: Compared with the control, the activities of related defense enzymes SOD (Superoxide dismutase), POD (Peroxidase), PAL (L-phenylalanine ammonia-lyase) and PPO (Polyphenol oxidase) in the over-expressed transgenic tobacco OEA1 and OEA2 increased to different degrees, and increased significantly at different infection time points. The activities of defense enzymes in the interfering strains IEA1 and IEA2 were significantly lower than those in the control strains. The results of resistance level identification showed that the disease spot rate of OEA1 was significantly lower than that of the control line, and the disease spot rate of OEA2 was significantly lower than that of the control line. The plaque rate of the interfering expression line IEA1-IEA2 was significantly higher than that of the control line. It is preliminarily believed that the process related protein GmPR1L can improve the resistance of tobacco to B. cinerea.     

Association mapping of quantitative resistance for <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Stem canker caused by the fungus Leptosphaeria maculans is a major disease of Brassica napus. Quantitative resistance factors appear to be important components for effective and durable control of this pathogen. Quantitative trait loci (QTL) for stem canker resistance have previously been identified in the Darmor variety. However, before these QTL can be used in marker-assisted selection (MAS) to breed resistant varieties, they must be validated in a wide range of genetic backgrounds. We used an association mapping approach to confirm the markers located within the QTL previously identified in Darmor and establish their usefulness in MAS. For this, we characterized the molecular diversity of an oilseed rape collection of 128 lines showing a large spectrum of responses to infection by L. maculans, using 72 pairs of primers for simple sequence repeat and other markers. We used different association mapping models which either do or do not take into account the population structure and/or family relatedness. In all, 61 marker alleles were found to be associated with resistance to stem canker. Some of these markers were associated with previously identified QTL, which confirms their usefulness in MAS. Markers located in regions not harbouring previously identified QTL were also associated with resistance, suggesting that new QTL or allelic variants are present in the collection. All of these markers associated with stem canker resistance will help identify accessions carrying desirable alleles and facilitate QTL introgression.     

Conventional Breeding and Molecular Markers for Blast Disease Resistance in Rice (<i>Oryza sativa</i> L.)

Monogenic lines, which carried 23 genes for blast resistance were tested and used donors to transfer resistance genes by crossing method. The results under blast nursery revealed that 9 genes from 23 genes were susceptible to highly susceptible under the three locations (Sakha, Gemmeza, and Zarzoura in Egypt); Pia, Pik, Pik-p, Piz-t, Pita, Pi b, Pi, Pi 19 and Pi 20. While, the genes Pii, Pik-s, Pik-h, Pi z, Piz-5, Pi sh, Pi 3, Pi 1, Pi 5, Pi 7, Pi 9, Pi 12, Pikm and Pita-2 were highly resistant at the same locations. Clustering analysis confirmed the results, which divided into two groups; the first one included all the susceptible genes, while the second one included the resistance genes. In the greenhouse test, the reaction pattern of five races produced 100% resistance under artificial inoculation with eight genes showing complete resistance to all isolates. The completely resistant genes: Pii, Pik-s, Piz, Piz-5 (=bi2) (t), Pita (=Pi4) (t), Pita, Pi b and Pi1 as well as clustering analysis confirmed the results. In the F₁ crosses, the results showed all the 25 crosses were resistant for leaf blast disease under field conditions. While, the results in F₂ population showed seven crosses with segregation ratio of 15 (R):1 (S), two cross gave segregated ratio of 3 R:1 S and one gave 13:3. For the identi- fication of blast resistance genes in the parental lines, the marker K3959, linked to Pik-s gene and the variety IRBLKS-F5 carry this gene, which was from the monogenic line. The results showed that four genotypes; Sakha 105, Sakha 103, Sakha 106 and IRBLKS-F5 were carrying Pik-s gene, while was absent in the Sakha 101, Sakha 104, IRBL5-M, IRBL9-W, IRBLTACP1 and IRBL9-W(R) genotypes. As for Pi 5 gene, the results showed that it was present in Sakha 103 and Sakha 104 varieties and absent in the rest of the genotypes. In addition, Pita-Pita- 2 gene was found in the three Egyptian genotypes (Sakha 105, Sakha 101 and Sakha 104) plus IRBLTACP1 monogenetic. In F2 generation, six populations were used to study the inheritance of blast resistance and specific primers to confirm the ratio and identify the resistance genes. However, the ratios in molecular markers were the same of the ratio under field evaluation in the most population studies. These findings would facilitate in breeding programs for gene pyramiding and gene accumulation to produce durable resistance for blast using those genotypes.     

Linkage disequilibrium mapping of a<Emphasis Type="Italic"> Verticillium dahliae</Emphasis> resistance quantitative trait locus in tetraploid potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) through a candidate gene approach

We have used the linkage disequilibrium mapping method to test for an association between a candidate gene marker and resistance to Verticillium dahliae in tetraploid potato. A probe derived from the tomato Verticillium resistance gene (Ve1) identified homologous sequences (StVe1) in potato, which in a diploid population map to chromosome 9, in a position analogous to that of the tomato resistance gene. When a molecular marker closely linked (1.5 cM) to the homologues was used as a candidate gene marker on 137 tetraploid potato genotypes (mostly North American cultivars), the association between the marker and resistance was confirmed (P<0.001). The amount of phenotypic variation in resistance explained by the allele of the STM1051 marker was greater than 10% and 25% in two subpopulations that were inferred from coancestry data matrix. Cloning of homologues from the highly resistant potato cv. Reddale indicates that the resistance quantitative trait locus (QTL) comprises at least an eleven-member family, encoding plant-specific leucine-rich repeat proteins highly similar to the tomato Ve genes. The sequence analysis shows that all homologues are uninterrupted open reading frames and thus represent putative functional resistance genes. This is the first time that the linkage disequilibrium method has been used to find an association between a resistance gene and a candidate gene marker in tetraploid potato. We have shown that it is possible to map QTL directly on already available potato cultivars, without developing a new mapping population.Communicated by F. SalaminiAn erratum to this article can be found at     

Positional cloning of <Emphasis Type="Italic">ds1</Emphasis>, the target leaf spot resistance gene against <Emphasis Type="Italic">Bipolaris sorghicola</Emphasis> in sorghum

Target leaf spot is one of the major sorghum diseases in southern Japan and caused by a necrotrophic fungus, Bipolaris sorghicola. Sorghum resistance to target leaf spot is controlled by a single recessive gene (ds1). A high-density genetic map of the ds1 locus was constructed with simple sequence repeat markers using progeny from crosses between a sensitive variety, bmr-6, and a resistant one, SIL-05, which allowed the ds1 gene to be genetically located within a 26-kb region on the short arm of sorghum chromosome 5. The sorghum genome annotation database for BTx623, for which the whole genome sequence was recently published, indicated a candidate gene from the Leucine-Rich Repeat Receptor Kinase family in this region. The candidate protein kinase gene was expressed in susceptible plants but was not expressed or was severely reduced in resistant plants. The expression patterns of ds1 gene and the phenotype of target leaf spot resistance were clearly correlated. Genomic sequences of this region in parental varieties showed a deletion in the promoter region of SIL-05 that could cause reduction of gene expression. We also found two ds1 alleles for resistant phenotypes with a stop codon in the coding region. The results shown here strongly suggest that the loss of function or suppression of the ds1 protein kinase gene leads to resistance to target leaf spot in sorghum.     

Development of a New Cold-Tolerant Maize (<i>Zea mays</i> L.) Germplasm Using the <i>ICE1</i> Gene from <i>Arabidopsis thaliana</i>

To develop cold-tolerant maize germplasms and identify the activation of INDUCER OF CRT/DRE-BINDING FACTOR EXPRESSION (ICE1) expression in response to cold stress, RT-PCR was used to amplify the complete open reading frame sequence of the ICE1 gene and construct the plant expression vector pCAMBIA3301-ICE1-Bar. Immature maize embryos and calli were transformed with the recombinant vector using Agrobacterium tumefaciens-mediated transformations. From the regenerated plantlets, three T1 lines were screened and identified by PCR. A Southern blot analysis showed that a single copy of the ICE1 gene was integrated into the maize (Zea mays L.) genomes of the three T1 generations. Under low temperature-stress conditions (4°C), the relative conductivity levels decreased by 27.51%–31.44%, the proline concentrations increased by 12.50%–17.50%, the malondialdehyde concentrations decreased by 16.78%–18.37%, and the peroxidase activities increased by 19.60%–22.89% in the T1 lines compared with those of the control. A real-time quantitative PCR analysis showed that the ICE1 gene was ectopically expressed in the roots, stems, and leaves of the T1 lines. ICE1 positively regulates the expression of the CBF genes in response to cold stress. Thus, this study showed the successful transformation of maize with the ICE1 gene, resulting in the generation of a new maize germplasm that had increased tolerance to cold stress.     

Molecular mapping of pea powdery mildew resistance gene <Emphasis Type="Italic">er2</Emphasis> to pea linkage group III

Powdery mildew caused by Erysiphe pisi D.C. is one of the most serious diseases that inflict heavy losses to pea crop world-wide. Identification of resistance sources and their incorporation into susceptible cultivars remains the most effective method of controlling the disease. The present study investigated the resistance phenotype, inheritance, and genomic location of gene(s) controlling resistance to powdery mildew in pea genotype ‘JI2480’. The powdery mildew resistance in ‘JI2480’ appeared to be a spatial phenomenon showing expression only in leaf tissues. By segregation analysis of an F₂ progeny of cross ‘Lincoln/JI2480’, the leaf resistance of ‘JI2480’ was shown to be controlled by a single recessive gene, presumed to be er2. Through linkage analysis of 111 resistant F₂ progeny plants with simple sequence repeat (SSR) and random amplified polymorphic DNA (RAPD) markers adopted from the published linkage maps, the er2 gene was localized on pea linkage group III (LGIII). The assignment of er2 to LGIII, a position different from that reported for er1, has resolved the long standing controversy in the literature regarding the existence and genomic location of er2 gene. A RAPD marker OPX-17_1400, exhibiting cis phase linkage (2.6 cM) to er2 was successfully converted to a sequence characterized amplified region (SCAR) marker, ScX17_1400. The SCAR marker ScX17_1400 will ensure speedy and precise introgression of er2 into susceptible cultivars by permitting selection of er2 heterozygotes amongst BC _n F₁s without progeny tests and resistance screening.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

The inheritance of resistance to Verticillium wilt caused by race 1 isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the lettuce cultivar La Brillante

Verticillium wilt of lettuce caused by Verticillium dahliae can cause severe economic damage to lettuce producers. Complete resistance to race 1 isolates is available in Lactuca sativa cultivar (cv.) La Brillante and understanding the genetic basis of this resistance will aid development of new resistant cultivars. F₁ and F₂ families from crosses between La Brillante and three iceberg cultivars as well as a recombinant inbred line population derived from L. sativa cv. Salinas 88 × La Brillante were evaluated for disease incidence and disease severity in replicated greenhouse and field experiments. One hundred and six molecular markers were used to generate a genetic map from Salinas 88 × La Brillante and for detection of quantitative trait loci. Segregation was consistent with a single dominant gene of major effect which we are naming Verticillium resistance 1 (Vr1). The gene described large portions of the phenotypic variance (R ² = 0.49–0.68) and was mapped to linkage group 9 coincident with an expressed sequence tag marker (QGD8I16.yg.ab1) that has sequence similarity with the Ve gene that confers resistance to V. dahliae race 1 in tomato. The simple inheritance of resistance indicates that breeding procedures designed for single genes will be applicable for developing resistant cultivars. QGD8I16.yg.ab1 is a good candidate for functional analysis and development of markers suitable for marker-assisted selection.