Next‐generation sequencing for identification of candidate genes for Fusarium wilt and sterility mosaic disease in pigeonpea (Cajanus cajan)

To map resistance genes for Fusarium wilt (FW) and sterility mosaic disease (SMD) in pigeonpea, sequencing‐based bulked segregant analysis (Seq‐BSA) was used. Resistant (R) and susceptible (S) bulks from the extreme recombinant inbred lines of ICPL 20096 × ICPL 332 were sequenced. Subsequently, SNP index was calculated between R‐ and S‐bulks with the help of draft genome sequence and reference‐guided assembly of ICPL 20096 (resistant parent). Seq‐BSA has provided seven candidate SNPs for FW and SMD resistance in pigeonpea. In parallel, four additional genotypes were re‐sequenced and their combined analysis with R‐ and S‐bulks has provided a total of 8362 nonsynonymous (ns) SNPs. Of 8362 nsSNPs, 60 were found within the 2‐Mb flanking regions of seven candidate SNPs identified through Seq‐BSA. Haplotype analysis narrowed down to eight nsSNPs in seven genes. These eight nsSNPs were further validated by re‐sequencing 11 genotypes that are resistant and susceptible to FW and SMD. This analysis revealed association of four candidate nsSNPs in four genes with FW resistance and four candidate nsSNPs in three genes with SMD resistance. Further, In silico protein analysis and expression profiling identified two most promising candidate genes namely C.cajan_01839 for SMD resistance and C.cajan_03203 for FW resistance. Identified candidate genomic regions/SNPs will be useful for genomics‐assisted breeding in pigeonpea.     

Breeding and selection of Buxus for resistance to Calonectria pseudonaviculata

Box blight is a widespread disease of Buxus caused by the pathogen Calonectria pseudonaviculata. It is responsible for significant losses in nurseries, gardens and wild boxwood populations. Our goal was to maximize the efficiency of a breeding programme towards increased disease resistance. The use of artificial inoculation of young F1 seedlings with C. pseudonaviculata spores under greenhouse conditions appeared to be a reliable tool for early selection of interesting prebreeding material. Overall, the four hybrid populations screened showed a segregating behaviour between their parents when determining the percentage of diseased leaves and lesion diameter. Genotypes were also found with an increased tolerance as compared to the parental species. Approximately 50% of the seedlings had the same score for both parameters after artificial inoculation in the greenhouse and in the field. Of the seedlings that showed severe symptoms in the greenhouse, <15% showed no disease symptoms in the field. Therefore, for larger breeding programmes, we propose a two‐step selection procedure: first artificial inoculation at seedling level to eliminate all genotypes with severe symptoms and then evaluation of the remaining seedlings in the field. Using this strategy, we were able to select several genotypes in our four hybrid populations with improved resistance to C. pseudonaviculata.     

A Mechanistic Probe into the Dual Inhibition of T. cruzi Glucokinase and Hexokinase in Chagas Disease Treatment – A Stone Killing Two Birds?

Glucokinase (GLK) and Hexokinase (HK) have been characterized as essential targets in Trypanosoma cruzi (Tc)-mediated infection. A recent study reported the propensity of the concomitant inhibition of TcGLK and TcHK by compounds GLK2-003 and GLK2-004, thereby presenting an efficient approach in Chagas disease treatment. We investigated this possibility using atomic and molecular scaling methods. Sequence alignment of TcGLK and TcHK revealed that both proteins shared approximately 33.3 % homology in their glucose/inhibitor binding sites. The total binding free energies of GLK2-003 and GLK2-004 were favorable in both proteins. PRO92 and THR185 were pivotal to the binding and stabilization of the ligands in TcGLK, likewise their conserved counterparts, PRO163 and THR237 in TcHK. Both compounds also induced a similar pattern of perturbations in both TcGLK and TcHK secondary structure. Findings from this study therefore provide insights into the underlying mechanisms of dual inhibition exhibited by the compounds. These results can pave way to discover and optimize novel dual Tc inhibitors with favorable pharmacokinetics properties eventuating in the mitigation of Chagas disease.     

Common Bean Reaction to Fusarium oxysporum f. sp. Phaseoli, the Cause of Severe Vascular Wilt in Central Africa

During the September‐December season of 1990, severe symptoms of Fusarium wilt were for the first time observed on a popular climbing bean (Phaseolus vulgaris L.) cultivar. G 2333. introduced within the previous 5 years. Seventy‐three bean genotypes were screened for resistance lo the disease, using artificial inoculation. The effect of inoculation density on the reaction of four selected genotypes was also investigated. Of the 29 climbing bean genotypes evaluated, 19 were resistant, including 11 of the 15 pre‐release or released cultivars. Of the 44 bush bean cultivars evaluated, 28 were resistant, five were intermediate and 11 were susceptible. All susceptible cultivars showed vascular discoloration. In both susceptible and resistant genotypes, the fungus spread almost equally from the entry points in inoculated roots to the base of the plants, but colonization and vertical spread within the vascular system were markedly less in resistant than in susceptible cultivars. At 20 and 30 cm above soil level, the fungus was only recovered from susceptible cultivars. Increasing inoculum density from 10² to 10⁷ conidia/ml did not affect the resistance of cultivars RWR 950 and G 685 but. in the susceptible cultivars G 2333 and MLB‐48‐89 A. it resulted in early appearance, high incidence and severity of the disease.     

Effect of yellow rust on yield components of barley cultivars with race-specific and slow rusting resistance to yellow rust

Yellow rust caused by Puccinia striiformis f. sp. hordei is an important disease of barley (Hordeum vulgare L.) in some parts of the world. We compared the effectiveness of different types of resistance in field plots at Ardabil Agricultural Research Station (Iran) during 2010–2011. Yield components along with slow rusting parameters including final rust severity (FRS), apparent infection rate (r), relative area under disease progress curve (rAUDPC) and coefficient of infection (CI) were evaluated for 25 barley cultivars. In all, two barley cultivars with race-specific resistance, 19 cultivars with different levels of slow rusting resistance and four susceptible cultivars were included in two experiments with and without fungicide protection under high disease pressure. Barley cultivars with slow rusting resistance displayed a range of severity responses indicating phenotypic diversity. Mean thousand kernels weight (TKW) losses for susceptible, race-specific and slow rusting genotypes were 31, 3 and 12%, respectively, and mean kernels per spike (KPS) losses for susceptible, race-specific and slow rusting genotypes were 19, 0.2 and 8%, respectively. Correlation coefficient of mean TKW and KPS losses with epidemiological parameters; rAUDPC, r, CI and FRS were highly significant. Slow rusting cultivars with low values of different parameters as well as genotypes with low yield component losses despite moderate disease levels were identified. Such genotypes can be used for breeding barely genotypes with high levels of resistance and negligible yield losses.     

山羊GOLA-DQA2基因多态性与血液免疫性状的相关分析

文章采用PCR-RFLP技术对莱芜黑山羊、波尔山羊、鲁波山羊3个山羊种群的GOLA-DQA2基因外显子2进行遗传多态性研究, 并对其血液免疫指标的效应进行分析。结果表明, 3个山羊种群共检测到4种基因型, 由3个等位基因控制; GOLA-DQA2基因外显子2的第77、79、80和169位的碱基表现出多态性; 多数血液免疫指标品种效应是主要效应; 鲁波山羊中, AB基因型的红细胞计数、白细胞计数分别显著高于BB基因型、BC基因型(P<0.05); AB基因型的红细胞压积显著高于BB、BC 基因型(P<0.05); BC基因型的噬中性粒细胞比率显著高于BB基因型(P<0.05); 波尔山羊和莱芜黑山羊中, 基因型与血液免疫指标之间也有一定的相关性, 但没有达到显著水平。由上述结果初步推测, 在鲁波山羊中, AB、BC基因型是影响红细胞计数、白细胞总数、噬中性粒细胞比率等血液免疫指标的重要因素。研究结果揭示GOLA-DQA2基因与血液免疫指标之间有一定的相关性, 可为山羊的抗病育种提供一定的参考。     

Valuable New Resistances Ensure Improved Management of Sclerotinia Stem Rot (Sclerotinia sclerotiorum) in Horticultural and Oilseed Brassica Species

Field resistances against Sclerotinia rot (SR) (Sclerotinia sclerotiorum) were determined in 52 Chinese genotypes of Brassica oleracea var. capitata, 14 Indian Brassica juncea genotypes carrying wild weedy Brassicaceae introgression(s) and four carrying B‐genome introgression, 22 Australian commercial Brassica napus varieties, and 12 B. napus and B. juncea genotypes of known resistance. All plants were individually inoculated by securing an agar disc from a culture of S. sclerotiorum growing on a glucose‐rich medium to the stem above the second internode with Parafilm tape. Mean stem lesion length across tested genotypes ranged from <1 to >68 mm. While there was considerable diversity within the germplasm sets from each country, overall, 65% of the B. oleracea var. capitata genotypes from China showed the highest levels of stem resistance, a level comparable with the highest resistance ever recorded for oilseed B. napus or B. juncea from China or Australia. One Indian B. juncea line carrying weedy introgression displayed a significant level of both stem and leaf resistance. However, the vast majority of commercial Australian oilseed B. napus varieties fell within the most susceptible 40% of genotypes tested for stem disease. There was no correlation between expressions of stem versus leaf resistance, suggesting their independent inheritance. A few Chinese B. oleracea var. capitata genotypes that expressed combined extremely high‐level stem (≤1 mm length) and leaf (≤0.5 mean number of infections/plant) resistance will be particularly significant for developing new SR‐resistant horticultural and oilseed Brassica varieties.     

Correlative evidence for peroxidase involvement in disease resistance against <Emphasis Type="Italic">Alternaria</Emphasis> leaf blight of tomato

Eighteen tomato genotypes, with varying degree of response to Alternaria leaf blight disease (ALBD) were used to assess the possible involvement of protease and peroxidase activities in disease response. Pre-infectional protease activity varied noticeably in tested genotypes. Highest pre-infectional protease activity was observed in susceptible genotype CLN-2123. Post-infectional protease activity level was generally lower when compared with pre-infectional level in all genotypes with exception of unchanged level in Tibrido. There was no correlation between post-infectional protease activity and percent disease index (%DI). In contrast, pre- and post-infectional leaf peroxidase activities showed a significant (p < 0.01) negative correlation with %DI. Genotypes with higher pre-infectional peroxidase activity performed better on exposure to Alternaria alternata infection and accumulate enhanced peroxidase activity. Tibrido accumulated highest peroxidase activity while level was lowest in 1621P, which showed highest ALBD incidence. Moreover, genotypes with better resistance to A. alternata infection maintained higher post-infectional peroxidase activity. In resistant (Tibrido) and all moderately resistant genotypes, leaf peroxidase activity raised after inoculation when compared with the pre-inoculation level. I summary, higher pre- and post-infectional peroxidase activity was found to be associated with Alternaria leaf blight resistance. The peroxidase activity can be used as a biochemical tool in marker-assisted screening of tomato germplasm for Alternaria leaf blight resistance.     

荷斯坦牛Nramp1基因遗传多态性及其与乳房炎相关性的研究

利用PCR-SSCP技术检测了344头中国荷斯坦牛Nramp1基因exon 11的基因多态性, 并分析了其不同基因型与乳房炎及产奶量性状的关系。结果表明: 实验群体发现3种基因型AA、AB、BB, 其中A等位基因为优势等位基因, 等位基因频率为0.767, 而B等位基因频率则为0.233。经χ2适合性检验, 群体处于Hardy-Weinberg平衡状态(P>0.05)。测序结果显示: 扩增片段分别在200 bp(C/G)和254 bp(T/G)存在碱基突变, 并导致了氨基酸改变, 分别为丙氨酸替换为脯氨酸(Ala356Pro)、亮氨基酸替换为蛋氨酸(Leu374Met)。通过构建最小二乘线性模型, 进行Nramp1基因多态性与产奶量、体细胞评分(SCS)的相关性分析表明, AA型个体的SCS最小二乘均值显著低于BB﹑AB型(P<0.05), 而AA型﹑AB个体的产奶量最小二乘均值显著高于BB型(P<0.01, P<0.05), AA基因型可作为乳房炎抗性的优良基因型。因此, 可将Nramp1作为奶牛乳房炎候选基因应用于分子标记辅助选择育种。     

Characterisation of Ascochyta rabiei isolates and evaluation of genotypic stability in chickpea

Ascochyta rabiei isolates were characterised for their variability using a set of host differentials following cloth chamber screening technique. Sixty chickpea genotypes were evaluated against the characterised 10 individual pathotypes separately to identify genotypes with stable resistance during 2007–2008. Twenty four genotypes showed resistance to all the pathotypes; whereas 18 genotypes were resistant to moderately resistant to these pathotypes. The above genotypes can be considered good sources of stable resistance and recommended as donors or for direct cultivation in north western plain zone of India.     

Tolerance and overcompensation to infection by Phytophthora infestans in the wild perennial climber Solanum dulcamara

Studies of infection by Phytophthora infestans—the causal agent of potato late blight—in wild species can provide novel insights into plant defense responses, and indicate how wild plants might be influenced by recurrent epidemics in agricultural fields. In the present study, our aim was to investigate if different clones of Solanum dulcamara (a relative of potato) collected in the wild differ in resistance and tolerance to infection by a common European isolate of P. infestans. We performed infection experiments with six S. dulcamara genotypes (clones) both in the laboratory and in the field and measured the degree of infection and plant performance traits. In the laboratory, the six evaluated genotypes varied from resistant to susceptible, as measured by degree of infection 20 days post infection. Two of the four genotypes susceptible to infection showed a quadratic (concave downward) relationship between the degree of infection and shoot length, with maximum shoot length at intermediate values of infection. This result suggests overcompensation, that is, an increase in growth in infected individuals. The number of leaves decreased with increasing degree of infection, but at different rates in the four susceptible genotypes, indicating genetic variation for tolerance. In the field, the inoculated genotypes did not show any disease symptoms, but plant biomass at the end of the growing season was higher for inoculated plants than for controls, in‐line with the overcompensation detected in the laboratory. We conclude that in S. dulcamara there are indications of genetic variation for both resistance and tolerance to P. infestans infection. Moreover, some genotypes displayed overcompensation. Learning about plant tolerance and overcompensation to infection by pathogens can help broaden our understanding of plant defense in natural populations and help develop more sustainable plant protection strategies for economically important crop diseases.     

How does salinity influence habitat selection and growth in juvenile American eels Anguilla rostrata?

The influence of salinity on habitat selection and growth in juvenile American eels Anguilla rostrata captured in four rivers across eastern Canada was assessed in controlled experiments in 2011 and 2012. Glass eels were first categorized according to their salinity preferences towards fresh (FW), salt (SW) or brackish water (BW) and the growth rate of each group of elvers was subsequently monitored in controlled FW and BW environments for 7 months. Most glass eels (78–89%) did not make a choice, i.e. they remained in BW. Salinity preferences were not influenced by body condition, although a possible role of pigmentation could not be ruled out. Glass eels that did make a choice displayed a similar preference for FW (60–75%) regardless of their geographic origin but glass eels from the St Lawrence Estuary displayed a significantly higher locomotor activity than those from other regions. Neither the salinity preferences showed by glass eels in the first experiment nor the rearing salinities appeared to have much influence on growth during the experiments. Elvers from Nova Scotia, however, reached a significantly higher mass than those from the St Lawrence Estuary thus supporting the hypothesis of genetically (or epigenetically) based differences for growth between A. rostrata from different origins. These results provide important ecological knowledge for the sustained exploitation and conservation of this threatened species.     

至少6个突变基因型冬虫夏草菌在冬虫夏草子座中表达的动态变化

本研究检测了在冬虫夏草成熟过程中突变基因型冬虫夏草菌在子座中表达的动态变化。根据冬虫夏草菌基因内转录间隔区(ITS)序列中大量散在的点突变,设计了8个单核苷酸多态性(SNP)延伸引物,采用SNP质谱基因分型法测定各个SNP位点的单核苷酸延伸反应,观察冬虫夏草子座中转换突变和颠换突变基因型菌在冬虫夏草成熟过程中表达的动态变化。其中5个SNP延伸引物(067721-211,067721-240,067721-477,067721-531和067721-581)位于rDNA ITS1和ITS2段,用于区分GC和AT偏倚基因型,但不区分2个AT偏倚基因型。另外3个延伸引物(067740-324,067740-328和067740-360)位于rDNA 5.8S段,用于区分2个AT基因型。采用PCR+EcoRⅠ限制性酶切法检验2个GC偏倚基因型冬虫夏草菌在冬虫夏草成熟过程中表达的动态变化。结果表明:冬虫夏草子座rDNA ITS1-5.8S-ITS2片段的8个SNP位点的质谱图谱显示冬虫夏草子座中存在至少5个冬虫夏草菌转换突变和颠换突变等位基因。它们的表达在冬虫夏草成熟过程中呈现动态变化。067721-211和067721-477位点的AT偏倚型等位基因的峰高明显高于GC偏倚型;随着冬虫夏草的成熟,这2个位点的AT型等位基因峰高大幅度下降。SNP质谱图不仅显示转换突变的等位基因,颠换突变等位基因也有很高的检出率,它们的峰高在一些位点甚至超过GC和AT基因型等位基因;随着冬虫夏草的成熟,颠换突变等位基因的检出率和峰高趋于下降。在区分2个AT偏倚基因型的研究中,AB067744和AB067740 2个基因型的等位基因同时存在于未成熟冬虫夏草子座中。随着冬虫夏草的成熟,AB067744基因型等位基因的峰高明显降低,而AB067740基因型等位基因的峰高明显升高。PCR产物EcoRⅠ酶切试验发现子座中存在2组GC偏倚型菌,具有完全不同的成熟模式。综上,冬虫夏草子座中存在至少6个突变基因型冬虫夏草菌,其表达随着冬虫夏草的成熟呈现动态变化。这些突变基因型菌表达的动态变化对于冬虫夏草子座的萌发和成熟具有重要菌物学意义。     

Functional markers based molecular characterization and cloning of resistance gene analogs encoding NBS-LRR disease resistance proteins in finger millet (<Emphasis Type="Italic">Eleusine coracana)</Emphasis>

Magnaporthe grisea, the blast fungus is one of the main pathological threats to finger millet crop worldwide. A systematic search for the blast resistance gene analogs was carried out, using functional molecular markers. Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encodes nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from Eleusine coracana. Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from resistant genotypes of finger millet using PCR based approach with primers designed from conserved regions of NBS domain. Attempts were made to identify molecular markers linked to the resistance gene and to differentiate the resistant bulk from the susceptible bulk. A total of 9 NBS-LRR and 11 EST-SSR markers generated 75.6 and 73.5% polymorphism respectively amongst 73 finger millet genotypes. NBS-5, NBS-9, NBS-3 and EST-SSR-04 markers showed a clear polymorphism which differentiated resistant genotypes from susceptible genotypes. By comparing the banding pattern of different resistant and susceptible genotypes, five DNA amplifications of NBS and EST-SSR primers (NBS-05_504, NBS-09₇₁₁, NBS-07₆₈₈, NBS-03₅₀₉ and EST-SSR-04₂₄₁) were identified as markers for the blast resistance in resistant genotypes. Principal coordinate plot and UPGMA analysis formed similar groups of the genotypes and placed most of the resistant genotypes together showing a high level of genetic relatedness and the susceptible genotypes were placed in different groups on the basis of differential disease score. Our results provided a clue for the cloning of finger millet blast resistance gene analogs which not only facilitate the process of plant breeding but also molecular characterization of blast resistance gene analogs from Eleusine coracana.     

Identification and molecular mapping of a Fusarium wilt resistant gene in upland cotton

Fusarium wilt (FW) is one of the most economically damaging cotton diseases worldwide, causing yellowing, wilting, defoliation, vascular tissue damage and ultimately death. Identification of molecular markers linked to FW genes is vital to incorporate resistance into elite cotton cultivars. An intraspecific F₂ in Gossypium hirsutum L. was developed by crossing with a highly resistant cultivar Zhongmiansuo 35 (ZMS35) and a susceptible cultivar Junmian 1 to screen simple sequence repeats (SSRs) closely linked to the FW resistance gene. FW was identified in F_2:3 families by evaluating seedling leaf symptoms and vascular tissue damage at plant maturity under natural field infection conditions over 2 years. The results showed that FW resistance segregated in a 3:1 ratio as a simple monogenic trait in F_2:3 families. Molecular mapping identified a FW resistance gene closely linked with the SSR marker JESPR304₋₂₈₀ in chromosome D3(c17). We proposed to name this gene FW ^R . A composite interval mapping method detected four QTLs for FW resistance in Chr.A7(c7), D1(c15), D9(c23) and D3, respectively. Among them, one major QTL (LOD > 20) was tagged near marker JESPR304 within an interval of 0.06–0.2 cM, and explained over 52.5–60.9% of the total phenotypic variance. The data confirmed the existence of a major gene in Chr.D3. This is the first report of molecular mapping of a major gene contributing FW resistance in cotton. The present research therefore provides an opportunity to understand the genetic control of resistance to FW and conduct molecular marker-assisted selection breeding to develop FW resistant cultivars.     

The first set of EST resource for gene discovery and marker development in pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis>L.)

Background  

Pigeonpea (Cajanus cajan (L.) Millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses [Fusarium wilt (FW), sterility mosaic disease (SMD), etc.] are serious challenges for sustainable crop production. Modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. Availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. With an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of FW- and SMD- responsive expressed sequence tags (ESTs).     

Drought Tolerance in Mung Bean is Associated with the Genotypic Divergence,Regulation of Proline,Photosynthetic Pigment and Water Relation

Drought is one of the critical conditions for the growth and productivity of many crops including mung bean (Vigna radiata L. Wilczek). Screening of genotypes for variations is one of the suitable strategies for evaluating crop adaptability and global food security. In this context, the study investigated the physiological and biochemical responses of four drought tolerant (BARI Mung-8, BMX-08010-2, BMX-010015, BMX-08009-7), and four drought sensitive (BARI Mung-1, BARI Mung-3, BU Mung-4, BMX-05001) mung bean genotypes under wellwatered (WW) and water deficit (WD) conditions. The WW treatment maintained sufficient soil moisture (22% ± 0.5%, i.e., 30% deficit of available water) by regularly supplying water. Whereas, the WD treatment was maintained throughout the growing period, and water was applied when the wilting symptom appeared. The drought tolerant (DT) genotypes BARI Mung-8, BMX-08010-2, BMX-010015, BMX-08009-7 showed a high level of proline accumulation (2.52–5.99 mg g⁻¹ FW), photosynthetic pigment (total chlorophyll 2.96–3.27 mg g⁻¹ FW at flowering stage, and 1.62–2.38 mg g⁻¹ FW at pod developing stage), plant water relation attributes including relative water content (RWC) (82%–84%), water retention capacity (WRC) (12–14) as well as lower water saturation deficit (WSD) (19%–23%), and water uptake capacity (WUC) (2.58–2.89) under WD condition, which provided consequently higher relative seed yield. These indicate that the tolerant genotypes gained better physiobiochemical attributes and adaptability in response to drought conditions. Furthermore, the genotype BMX- 08010-2 showed superiority in terms of those physio-biochemical traits, susceptibility index (SSI) and stress tolerance index (STI) to other genotypes. Based on the physiological and biochemical responses, the BMX-08010-2 was found to be a suitable genotype for sustaining yield under drought stress, and subsequently, it could be recommended for crop improvement through hybridization programs. In addition, the identified traits can be used as markers to identify tolerant genotypes for drought-prone areas.