Roland Schafleitner

The okra (Abelmoschus esculentus) transcriptome as a source for gene sequence information and molecular markers for diversity analysis.

A combined leaf and pod transcriptome of okra (Abelmoschus esculentus (L.) Moench) has been produced by RNA sequencing and short read assembly. More than 150,000 unigenes were obtained, comprising some 46 million base pairs of sequence information. More than 55% of the unigenes were annotated through sequence comparison with databases. The okra transcriptome sequences were mined for simple sequence repeat (SSR) markers. From 935 non-redundant SSR motifs identified in the unigene set, 199 were chosen for testing in a germplasm set, resulting in 161 polymorphic SSR markers. From this set, 19 markers were selected for a diversity analysis on 65 okra accessions comprising three different species, revealing 58 different genotypes and resulted in clustering of the accessions according to species and geographic origin. The okra gene sequence information and the marker resource are made available to the research community for functional genomics and breeding research.

The AVRDC – The World Vegetable Center mungbean (Vigna radiata) core and mini core collections

BackgroundLarge ex situ germplasm collections generally harbor a wide range of crop diversity. AVRDC – The World Vegetable Center is holding in trust the world’s second largest mungbean (Vigna radiata) germplasm collection with more than 6,700 accessions. Screening large collections for traits of interest is laborious and expensive. To enhance the access of breeders to the diversity of the crop, mungbean core and mini core collections have been established.ResultsThe core collection of 1,481 entries has been built by random selection of 20% of the accessions after geographical stratification and subsequent cluster analysis of eight phenotypic descriptors in the whole collection. Summary statistics, especially the low differences of means, equal variance of the traits in both the whole and core collection and the visual inspection of quantile-quantile plots comparing the variation of phenotypic traits present in both collections indicated that the core collection well represented the pattern of diversity of the whole collection. The core collection was genotyped with 20 simple sequence repeat markers and a mini core set of 289 accessions was selected, which depicted the allele and genotype diversity of the core collection.ConclusionsThe mungbean core and mini core collections plus their phenotypic and genotypic data are available for distribution to breeders. It is expected that these collections will enhance the access to biodiverse mungbean germplasm for breeding.

Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome

We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911). In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum), two monocots (Oryza sativa and Zea mays), two gymnosperms (Pinus taeda and Ginkgo biloba) and one moss (Physcomitrella patens). Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.

Phylogeographical Structure in Mitochondrial DNA of Legume Pod Borer (Maruca vitrata) Population in Tropical Asia and Sub-Saharan Africa.

This study was undertaken to assess the genetic diversity and host plant races of M. vitrata population in South and Southeast Asia and sub-Saharan Africa. The cytochrome c oxidase subunit 1 (cox1) gene was used to understand the phylogenetic relationship of geographically different M. vitrata population, but previous studies did not include population from Southeast Asia, the probable center of origin for Maruca, and from east Africa. Extensive sampling was done from different host plant species in target countries. Reference populations from Oceania and Latin America were used. An amplicon of 658 bp was produced by polymerase chain reaction, and 64 haplotypes were identified in 686 M. vitrata individuals. Phylogenetic analysis showed no difference among the M. vitrata population from different host plants. However, the results suggested that M. vitrata has formed two putative subspecies (which cannot be differentiated based on morphological characters) in Asia and sub-Saharan Africa, as indicated by the high pairwise FST values (0.44–0.85). The extremely high FST values (≥0.93) of Maruca population in Latin America and Oceania compared to Asian and African population seem to indicate a different species. On the continental or larger geographical region basis, the genetic differentiation is significantly correlated with the geographical distance. In addition, two putative species of Maruca, including M. vitrata occur in Australia, Indonesia and Papua New Guinea. The negative Tajima’s D and Fu’s FS values showed the recent demographic expansion of Maruca population. The haplotype network and Automatic Barcode Gap Discovery analyses confirmed the results of phylogenetic analysis. Thus, this study confirmed the presence of three putative Maruca species, including one in Latin America, one in Oceania (including Indonesia) and M. vitrata in Asia, Africa and Oceania. Hence, the genetic differences in Maruca population should be carefully considered while designing the pest management strategies in different regions.

Transcriptomic and Proteomic Research To Explore Bruchid-Resistant Genes in Mungbean Isogenic Lines.

Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for human nutrition in Asia. Bruchids (Callosobruchus spp.) currently cause heavy damage as pests of grain legumes during storage. We used omics-related technologies to study the mechanisms of bruchid resistance in seeds of the nearly isogenic lines VC1973A (bruchid-susceptible) and VC6089A (bruchid-resistant). A total of 399 differentially expressed genes (DEGs) were identified between the two lines by transcriptome sequencing. Among these DEGs, 251 exhibited high expression levels and 148 expressed low expression levels in seeds of VC6089A. Forty-five differential proteins (DPs) were identified by isobaric tags for relative and absolute quantification (iTRAQ); 21 DPs had higher abundances in VC6089A, and 24 DPs had higher abundances in VC1973A. According to transcriptome and proteome data, only three DEGs/DPs, including resistant-specific protein (g39185), gag/pol polyprotein (g34458), and aspartic proteinase (g5551), were identified and located on chromosomes 5, 1, and 7, respectively. Both g39185 and g34458 genes encode a protein containing a BURP domain. In previous research on bruchid molecular markers, the g39185 gene located close to the molecular markers of major bruchid-resistant locus may be a bruchid-resistant gene.

Cucurbit Powdery Mildew-resistant Bitter Gourd Breeding Lines Reveal Four Races of Podosphaera xanthii in Asia

Bitter gourd (Momordica charantia L.) is a commercially and nutritionally important market vegetable in Asia cultivated mainly by smallholder farmers. Cucurbit powdery mildew (CPM) caused by Podosphaera xanthii (Px) is a nearly ubiquitous and serious fungal disease of bitter gourd. Five bitter gourd breeding lines (THMC 113, THMC 143, THMC 153, THMC 167, and THMC 170) were selected at theWorld Vegetable Center for resistance to a local isolate of Px in Kamphaeng Saen, Thailand.We evaluated the resistance potential of these five inbred lines against local isolates of Px at 12 locations in five Asian countries. Plants were inoculated with the respective local Px isolate 15 and 30 days after transplanting and additional Px-infected plants of the inoculated control were interplanted throughout each test. Plants were rated 60 days after transplanting for CPM reaction using a 0 (no evidence of infection) to 5 (>75% infection evident on individual leaves) disease severity scale. THMC 153 and THMC 167 were resistant to the local race of Px in all locations, whereas THMC 143 was observed resistant in all test locations except one in China. THMC 113 was resistant in each location except one in India. THMC 170 was susceptible in three locations in India. The multilocation tests revealed four unique Px races on bitter gourd in different Asian countries and sources of resistance for breeding CPM-resistant bitter gourd cultivars. Six strains of Px isolated from other cucurbits (Cucumis and Cucurbita) and representing five melon CPM races were unable to infect the susceptibleM. charantia accession THMC 144 and the five resistant breeding lines, indicating pathotype differences between them and an isolate of M. charantia origin typed as race 1 on melon. THMC 143 and THMC 167, which originated from India, exhibited good yield potential in trials conducted in Thailand, Myanmar, Vietnam, and Bangladesh. HORTSCIENCE VOL. 53(3) MARCH 2018 337 Bitter gourd (M. charantia L.) is an important cucurbitaceous market vegetable in Asia, where more than 340,000 ha are devoted to its cultivation annually (McCreight et al., 2013). Its cultivation is gaining popularity in some African countries such as Ghana, Zambia, Congo, and Madagascar for local consumption or for export to Europe and the Middle East to cater the demand of emigrant Asian communities. It is also cultivated to a lesser extent in the southern United States and Australia (Northern Territory, Queensland, New South Wales, and Victoria), where popular Asian hybrid cultivars are cultivated for consumption mainly by ethnic communities from Asia (Morgan and Midmore, 2002). Bitter gourd fruit is a rich source of betacarotene, vitamin C, folic acid, magnesium, phosphorus, and potassium (Dhillon et al., 2017; Yuwai et al., 1991). The health and pharmacological properties of bitter gourd have been well documented (Tan et al., 2016). Currently, 422 million people worldwide have diabetes (World Health Organization, 2016) and Type 2 diabetes accounts for around 90% (379 million). Bitter gourd fruit is used in folk medicine to manage Type 2 diabetes (Abascal and Yarnell, 2005; Grover and Yadav, 2004; Lans, 2006). Cucurbit powdery mildew (CPM) caused by Px is a serious fungal foliar disease of cucurbit production in open fields and greenhouses. Disease outbreak brings reduction in plant growth, premature foliage loss, and reduction in yield and fruit quality (Keinath and DuBose, 2004). CPM on bitter gourd is currently controlled by fungicides, although fungicide resistance has developed in some areas (Lebeda et al., 2010; McGrath, 2006). The pathogen is highly variable in virulence and represented by many pathotypes (Lebeda et al., 2011) and races (Lebeda et al., 2016). The use of disease-resistant varieties is an economical and safe approach for disease management. Accessions resistant to CPM have been identified in melon (Cucumis melo; Dhillon et al., 2012), watermelon (Citrullus lanatus; Thomas et al., 2005), cucumber (Cucumis sativus; Block and Reitsma, 2005), squash (Cucurbita pepo; Lebeda and K rístkov a, 1996), pumpkin (Cucurbita moschata; Wessel-Beaver, 1993), and bottle gourd (Lagenaria siceraria; Kousik et al., 2008). Resistance to CPM is, however, often race-specific and not durable (Lebeda et al., 2008, 2016). Commercial cultivars of bitter gourd resistant to CPM are not currently available. We developed five inbred lines resistant to CPM after screening 150 accessions of a global collection of bitter gourd in the World Vegetable Center genebank against the local CPM population at Kamphaeng Saen (Thailand). A single resistant plant was identified in each of five segregating populations derived from five genebank accessions that originated from India, Thailand, Taiwan, and Belize. Multiple cycles of inbreeding and selection led to the development of the five CPM-resistant inbred lines. We evaluated four of these inbred lines in 2011 against local isolates of Px in Thailand, Taiwan, and the United States (South Carolina, Florida, California) (Dhillon et al., 2015). We report here the reactions of the five bitter gourd CPM-resistant inbred lines against local isolates of Px at 12 locations in five Asian countries (China, India, Thailand, Vietnam, and Philippines) in 2013 and 2014. In addition, we sought to relate Asian CPM– bitter gourd interactions to the more developed body of knowledge of CPM–melon interactions, first by challenging these lines with European and the Mediterranean CPM isolates, and second by challenging a set of melon CPM race differentials with a singlespore strain isolated from a local isolate of Px on bitter gourd from Kamphaeng Saen, Thailand. The latter test also challenged representatives of cucumber, summer squash, and watermelon, cucurbit species on which few CPM races have been identified, with the exception of watermelon where four CPM races have been defined (Davis et al., 2007; Kousik et al., 2011; Mercier et al., 2014; Zhang et al., 2011). Furthermore, we evaluated horticultural fruit characters of the CPM-resistant bitter gourd breeding lines in the field test at Kamphaeng Saen, Thailand in 2014, and assessed the yield potential of two of the lines in Thailand, Myanmar, Vietnam, and Bangladesh in 2016, to assess their horticultural value as sources of CPM resistance. Materials and Methods Germplasm and field test sites. Five bitter gourd CPM-resistant inbred lines and a susceptible bitter gourd check line were evaluated against the respective local Px isolates at 12 locations in five countries in 2013 and 2014 (Table 1). Each field test was planted in a randomized complete block design with three replications of five plants per plot. Entries were planted on raised, 1.6-m wide beds covered with black plastic mulch. Plots were 5-m long on a single bed; each consisted of five transplants spaced 1-m apart. Plants were trellised on the plastic net erected on vertical bamboo poles. Field test. At each location, a spore suspension was prepared by detaching heavily sporulating leaves of susceptible THMC 144 and washing them with a spray of 100 mL of water and filtering through a double layer of cheesecloth. The suspension was diluted to a concentration of 4 · 10 conidia/mL of water as determined by a hemocytometer. This was freshly prepared as required for each inoculation. Seedlings were inoculated 15 and 30 d after transplanting, at the threeleaf stage of growth, at each location. The spore suspension was sprayed over the plants until runoff, by using a pressurized sprayer. THMC 144 plants with abundantly sporulating CPMwere used as spreader plants, placed between rows as additional sources of powdery mildew inoculum. Disease severity was rated on leaves of individual plants 30 d after the second inoculation using a 0–5 visual rating scale, where 0 = no symptom; 1 = 1% to 10%; 2 = 11% to 25%; 3 = 26% to 50%; 4 = 51% to 75%; and 5 = >75% of leaf surface covered by mycelium. Plant ratings of 0 and 1 were considered resistant. The susceptible check had a mean rating of 5.0 at all test locations. The 0–5 scale was converted to percentage usingmidpoints: 0 = 0%, 1= 5.5%, 2= 18%, 3= 38%, 4 = 63%, and 5 = 97% and the data were subjected to analysis of variance (ANOVA) using SAS general linear model (GLM) procedure (SAS Institute, Cary, NC). Mean separation was performed using Fisher’s least significant differences (LSD) at P# 0.05. Growth chamber tests. There were two growth chamber tests. The first evaluated the five breeding lines against European and Mediterranean CPM isolates, whereas the second evaluated various cucurbits with a single-spore CPM strain from Kamphaeng Saen, Thailand. Five plants, each of the five bitter gourd CPM-resistant inbred lines, and a susceptible check (Table 2) were inoculated at two-leaf stage, similarly as explained previously, with six CPM strains isolated from cucurbits in Europe and the Mediterranean area and typed for race on melon: Sm3 (race 1), S87-7 (race 2F), 00Sm39 (race 3), 98Sm65 (race 5), and 04Sm2 and 08Sm9 (race 3.5). A singlespore CPM strain isolated from M. charantia grown in an open field in Kamphaeng Saen, Thailand, was inoculated on five plants each of ‘Marketer’ cucumber, ‘Diamant’ summer squash, ‘Sugar Baby’ watermelon, and melon CPM race differentials [‘V edrantais’, ‘PMR 45’, ‘PMR 5’, WMR 29, PI 124112, 90625 (PI 313970), and AR Hale’s Best Jumbo]. Plants were raised in a glasshouse until the second leaf stage and then incubated after inoculation in a growth chamber (16 h day 26 C/8 h night 20 C) at GAFL, INRA, Montfavet, France. Disease severity was rated on leaves of individual plants after 10–14 d of inoculation, using 0–3 visual rating scale, where 0 = no visible Received for publication 2 Oct. 2017. Accepted for publication 11 Jan. 2018. Funding for this research was provided by the Federal Ministry for Economic Cooperation and Development, Germany (BMZ), Japan Ministry of Agriculture, Forestry and Fish

Molecular Markers Associated to Two Non-allelic Genic Male Sterility Genes in Peppers (Capsicum annuum L.)

Male sterility is of high importance in hybrid seed production of hot and sweet peppers. Genic (or nuclear) male sterility (GMS) is a simply inherited (usually monogenic recessive) and highly stable trait. However, one major disadvantage of using GMS is 1:1 segregation of male sterile to male fertile plants in every subsequent generation. Molecular markers tightly linked to genic male sterility (ms) genes would facilitate an efficient and rapid transfer of ms genes into different genetic backgrounds through marker-assisted backcrossing. The two non-allelic genic male sterility genes ms3 and msw in hot and sweet pepper backgrounds, respectively, are monogenic recessive. Genotyping by sequencing (GBS) in an F2 population segregating for ms3 gene in hot pepper and in an F6 inbred near-isogenic line (NIL) population segregating for msw gene in sweet pepper yielded 9,713 and 7,453 single nucleotide polymorphism markers, respectively. Four candidate SNPs co-segregating with ms3 gene and one co-segregating with msw gene were identified by bulk segregant analysis and physically mapped to chromosomes 1 and 5, respectively. In hot pepper, two markers [HPGMS2 (CAPS) and HPGMS3 (dCAPS)] located 3.83 cM away from the ms3 gene and in sweet pepper the dCAPS marker SPGMS1 co-segregated (completely linked) with the msw gene were developed. These markers will increase the efficacy of the male sterility genes for pepper breeding, as they can be useful in developing the genic male sterile lines in parental inbred lines of commercial hybrids through marker-assisted backcrossing, hybrid seed production, and genetic purity testing of hybrid seeds.

PCR-based assays for validation of single nucleotide polymorphism markers in rice and mungbean.

BackgroundSingle nucleotide polymorphism (SNP) markers are the method of choice for genetic analyses including diversity and quantitative trait loci (QTL) studies. Marker validation is essential for QTL studies, but the cost and workload are considerable when large numbers of markers need to be verified. Marker systems with low development costs would be most suitable for this task.ResultsWe have tested allele specific polymerase chain reaction (PCR), tetra markers and a genotyping tool based on the single strand specific nuclease CEL-I to verify randomly selected SNP markers identified previously either with a SNP array or by genotyping by sequencing in rice and mungbean, respectively. The genotyping capacity of allele-specific PCR and tetra markers was affected by the sequence context surrounding the SNP; SNPs located in repeated sequences and in GC-rich stretches could not be correctly identified. In contrast, CEL-I digestion of mixed fragments produced from test and reference DNA reliably pinpointed the correct genotypes, yet scoring of the genotypes became complicated when multiple SNPs were present in the PCR fragments. A cost analysis showed that as long the sample number remains small, CEL-I genotyping is more cost-effective than tetra markers.ConclusionsCEL-I genotyping performed better in terms of genotyping accuracy and costs than tetra markers. The method is highly useful for validating SNPs in small to medium size germplasm panels.