Development of SCAR markers linked to powdery mildew (<Emphasis Type="Italic">Uncinula necator</Emphasis>) resistance in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. and <Emphasis Type="Italic">Vitis</Emphasis> sp.)

Sequence-characterized amplified regions markers (SCARs) were developed from six randomly amplified polymorphic DNA (RAPD) markers linked to the major QTL region for powdery mildew (Uncinula necator) resistance in a test population derived from the cross of grapevine cultivars “Regent” (resistant) × “Lemberger”(susceptible). RAPD products were cloned and sequenced. Primer pairs with at least 21 nucleotides primer length were designed. All pairs were tested in the F1 progeny of “Regent” × “Lemberger”. The SCAR primers resulted in the amplification of specific bands of expected sizes and were tested in additional genetic resources of resistant and susceptible germplasm. All SCAR primer pairs resulted in the amplification of specific fragments. Two of the SCAR markers named ScORA7-760 and ScORN3-R produced amplification products predominantly in resistant individuals and were found to correlate to disease resistance. ScORA7-760, in particular, is suitable for marker-assisted selection for powdery mildew resistance and to facilitate pyramiding powdery mildew resistance genes from various sources.     

Revealing the genetic architecture of FHB resistance in hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) by QTL meta-analysis

Fusarium head blight (FHB) in wheat results in reduced yield and quality and in accumulation of mycotoxins. The objective of this study was to identify genomic regions in wheat involved in the control of FHB resistance applying a QTL meta-analysis approach by combining QTL of 30 mapping populations to propose independent meta-QTL (MQTL). A consensus map was created on which initial QTL were projected. Nineteen MQTL comprising 2–13 initial QTL with widely varying confidence intervals were found on 12 chromosomes. Some of them coincided with genomic regions previously identified (e.g. chromosomes 3BS, 6B), however, some MQTL were newly detected by this study. Separate analysis of populations with the same resistant parent showed a rather high consistency for the Chinese spring wheat donor ‘Sumai 3’, but little consistency for the Chinese donor ‘Wangshuibai’ and the Swiss donor ‘Arina’. According to our results breeders can in future (1) choose parents for crossing not comprising the same resistance loci or QTL intervals, (2) exploit new MQTL, and (3) select markers of some of these MQTL to be used in marker-assisted selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of sequence characterized DNA markers linked to leaf rust (<Emphasis Type="Italic">Hemileia vastatrix</Emphasis>) resistance in coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Coffee leaf rust due to Hemileia vastatrix is one of the most serious diseases in Arabica coffee (Coffea arabica). A resistance gene (S_H3) has been transferred from C. liberica into C. arabica. The present work aimed at developing sequence-characterized genetic markers for leaf rust resistance. Linkage between markers and leaf rust resistance was tested by analysing two segregating populations, one F₂ population of 101 individuals and one backcross (BC₂) population of 43 individuals, derived from a cross between a susceptible and a SH3-introgressed resistant genotype. A total of ten sequence-characterized genetic markers closely associated with the S_H3 leaf rust resistance gene were generated. These included simple sequence repeats (SSR) markers, sequence-characterised amplified regions (SCAR) markers resulting from the conversion of amplified fragment length polymorphism (AFLP) markers previously identified and SCAR markers derived from end-sequences of bacterial artificial chromosome (BAC) clones. Those BAC clones were identified by screening of C. arabica genomic BAC library using a cloned AFLP-marker as probe. The markers we developed are easy and inexpensive to run, requiring one PCR step followed by gel separation. While three markers were linked in repulsion with the S_H3 gene, seven markers were clustered in coupling around the S_H3 gene. Notably, two markers appeared to co-segregate perfectly with the S_H3 gene in the two plant populations analyzed. These markers are suitable for marker-assisted selection for leaf rust resistance and to facilitate pyramiding of the S_H3 gene with other leaf rust resistance genes.     

A large-scale<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10^-3 to 10^-5 compared with random integration by non-homologous end joining. We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 10³ stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10^-2 using the gene for diphtheria toxin A fragment as a negative marker. The established transformation procedure provides a basis for efficient gene targeting in rice.Abbreviations AS: Acetosyringone - 5-FU: 5-Fluorouracil - FW: Fresh weight - GT: Gene targeting - HR: Homologous recombination - NHEJ: Non-homologous end joining Communicated by H. Ebinuma     

Tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of hemp (<Emphasis Type="Italic">Cannabis sativa</Emphasis> L.)

Summary Hemp (Cannabis sativa L.) is cultivated in many parts of the world for ils fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM kinetin, 3% sucrose, and 8 gl⁻¹ agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 μM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus was selected on medium containing 1–2% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed. Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines ranged from one to four.     

Mapping of quantitative trait loci associated with ultraviolet-B resistance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The detection of quantitative trait loci (QTLs) associated with UV-B resistance in rice should allow their practical application in breeding for such a complex trait, and may lead to the identification of gene characteristics and functions. Considerable variation in UV-B resistance exists within cultivated rice (Oryza sativa L.), but its detailed genetic control mechanism has not been well elucidated. We detected putative QTLs associated with the resistance to enhanced UV-B radiation in rice, using 98 BC₁F₅ (backcross inbred lines; BILs) derived from a cross between Nipponbare (a resistant japonica rice variety) and Kasalath (a sensitive indica rice variety). We used 245 RFLP markers to construct a framework linkage map. BILs and both parents were grown under visible light with or without supplemental UV-B radiation in a growth chamber. In order to evaluate UV-B resistance, we used the relative fresh weight of aerial parts (RFW) and the relative chlorophyll content of leaf blades (RCC). The BIL population exhibited a wide range of variation in RFW and RCC. Using composite interval mapping with a LOD threshold of 2.9, three putative QTLs associated with both RFW and RCC were detected on chromosomes 1, 3 and 10. Nipponbare alleles at the QTLs on chromosome 1 and 10 increased the RFW and RCC, while the Kasalath allele at the QTL on chromosome 3 increased both traits. Furthermore, the existence of both QTLs on chromosomes 1 and 10 for UV-B resistance was confirmed using chromosome segment substitution lines. Plants with Kasalath alleles at the QTL on chromosome 10 were more sensitive to UV-B radiation than plants with them on chromosome 1. These results also provide the information not only for the improvement of UV-B resistance in rice though marker-associated selection, but also for the identification of UV-B resistance mechanisms by using near-isogenic lines.Communicated by D.J. Mackill     

Discovery,localization, and sequence characterization of molecular markers for the crown rust resistance genes <Emphasis Type="Italic">Pc38, Pc39</Emphasis>, and <Emphasis Type="Italic">Pc48</Emphasis> in cultivated oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.)

Molecular markers for the crown rust resistance genes Pc38, Pc39, and Pc48 in cultivated oat (Avena sativa L.) were identified using near-isogenic lines and bulked segregant analysis. Six markers for Pc48, the closest being 6 cM away, were found in a Pendek-39 × Pendek-48 (Pendek3948) population, but none was found in a Pendek-48 × Pendek-38 (Pendek4838) population. Three markers for Pc39 were found in the Pendek3948 population, one of which cosegregated with the gene. This same marker was found to be 6 cM away from the gene in an OT328 × Dumont (OT328Du) population. Nine markers for Pc38 were found in the Pendek4838 population, eight of which are within 2 cM of the gene. One other marker for Pc38 was found in the OT328Du population; however, comparative mapping suggests that the Pc38 region in OT328Du is in a different location than that in Pendek4838. A number of markers unlinked to the genes under study formed linkage groups in both the Pendek3948 and Pendek4838 populations. Four of these show homology or homoeology to each other and to the Pc39 region in Pendek3948. Two RFLP clones closely linked to Pc38 code for a putative leucine-rich repeat transmembrane protein kinase and a cre3 resistance gene analogue. This study provides information to support molecular breeding in oat, and contributes to ongoing research into genomic regions associated with fungal pathogen resistance.     

Knockout of a starch synthase gene <Emphasis Type="Italic">OsSSIIIa</Emphasis>/<Emphasis Type="Italic">Flo5</Emphasis> causes white-core floury endosperm in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.     

High-resolution genetic mapping at the <Emphasis Type="Italic">Bph15</Emphasis> locus for brown planthopper resistance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Resistance to the brown planthopper (BPH), Nilaparvata lugens Stål, a devastating sucking insect pest of rice, is an important breeding objective in rice improvement programs. Bph15, one of the 17 major BPH resistance genes so far identified in both cultivated and wild rice, has been identified in an introgression line, B5, and mapped on chromosome 4 flanked by restriction fragment length polymorphism markers C820 and S11182. In order to pave the way for positional cloning of this gene, we have developed a high-resolution genetic map of Bph15 by positioning 21 DNA markers in the target chromosomal region. Mapping was based on a PCR-based screening of 9,472 F₂ individuals derived from a cross between RI93, a selected recombinant inbred line of B5 bearing the resistance gene Bph15, and a susceptible variety, Taichung Native 1, in order to identify recombinant plants within the Bph15 region. Recombinant F₂ individuals with the Bph15 genotype were determined by phenotype evaluation. Analysis of recombination events in the Bph15 region delimited the gene locus to an interval between markers RG1 and RG2 that co-segregated with the M1 marker. A genomic library of B5 was screened using these markers, and bacterial artificial chromosome clones spanning the Bph15 chromosome region were obtained. An assay of the recombinants using the sub-clones of these clones in combination with sequence analysis delimited the Bph15 gene to a genomic segment of approximately 47 kb. This result should serve as the basis for eventual isolation of the Bph15 resistance gene.     

Transposition behavior of nonautonomous a <Emphasis Type="Italic">hAT</Emphasis> superfamily transposon <Emphasis Type="Italic">nDart</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Transposable elements (TEs) have a significant impact on the evolution of gene function and genome structures. An endogenous nonautonomous transposable element nDart was discovered in an albino mutant that had an insertion in the Mg-protoporphyrin IX methyltransferase gene in rice. In this study, we elucidated the transposition behavior of nDart, the frequency of nDart transposition and characterized the footprint of nDart. Novel independent nDart insertions in backcrossed progenies were detected by DNA blotting analysis. In addition, germinal excision of nDart occurred at very low frequency compared with that of somatic excision, 0–13.3%, in the nDart1-4(3-2) and nDart1-A loci by a locus-specific PCR strategy. A total of 253 clones from somatic excision at five nDart loci in 10 varieties were determined. nDart rarely caused deletions beyond target site duplication (TSD). The footprint of nDart contained few transversions of nucleotides flanking to both sides of the TSD. The predominant footprint of nDart was an 8-bp addition. Precise excision of nDart was detected at a rate of only 2.2%, which occurred at two loci among the five loci examined. Furthermore, the results in this study revealed that a highly conserved mechanism of transposition is involved between maize Ac/Ds and rice Dart/nDart, which are two-component transposon systems of the hAT superfamily transposons in plant species.     

Population structure analysis and association mapping of bacterial blight resistance in <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accessions

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a serious disease in rice production worldwide. To understand the genetic diversity of bacterial blight resistance a population consisting of 175 indica accessions from nine countries was collected and detected their association between SSR (Simple Sequence Repeat) markers and resistance to six bacterial races. The resistance phenotypes of various rice accessions were evaluated through artificial inoculation under controlled conditions in 2013 and 2014. Association analysis showed that 17 SSR markers were significantly associated with resistance to four bacterial races and the phenotypic variations explained (PVE) ranged from 7.43 to 15.05%. Among the 17 associated SSR markers, two SSR markers located in previously reported genes regions, and 15 SSR markers were newly identified in this study. These results validated a new approach to map resistance genes of rice to bacterial blight. These markers could be used for marker-assisted selection (MAS) in rice bacterial blight resistance breeding programs.     

Fine genetic mapping and physical delimitation of the lesion mimic gene <Emphasis Type="Italic">spotted leaf 5</Emphasis> (<Emphasis Type="Italic">spl5</Emphasis>) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Spotted leaf 5 (spl5), a lesion mimic mutant, was first identified in rice (Oryza sativa L.) japonica cv. Norin8 in 1978. This mutant exhibits spontaneous disease-like lesions in the absence of any pathogens and resistance to rice blast and bacterial blight; however, the target gene has not yet been isolated. In the present study, we employed a map-based cloning strategy to finely map the spl5 gene. In an initial mapping with 100 F₂ individuals (spl5/spl5) derived from a cross between the spl5 mutant and indica cv. 93-11, the spl5 gene was located in a 3.3-cM region on chromosome 7 using six simple sequence repeat (SSR) markers. In a high-resolution genetic mapping, two F₂ populations with 3,149 individuals (spl5/spl5) were derived from two crosses between spl5 mutant and two indica cvs. 93-11 and Zhefu802 and six sequence-tagged site (STS) markers were newly developed. Finally, the spl5 gene was mapped to a region of 0.048 cM between two markers SSR7 and RM7121. One BAC/PAC contig map covering these markers’ loci and the spl5 gene was constructed through Pairwise BLAST analysis. Our bioinformatics analysis shows that the spl5 gene is located in the 80-kb region between two markers SSR7 and RM7121 with a high average ratio of physical to genetic distance (1.67 Mb/cM) and eighteen candidate genes. The analysis of these candidate genes indicates that the spl5 gene represents a novel class of regulators controlling cell death and resistance response in plants.     

Genetic differentiation for nuclear,mitochondrial and chloroplast genomes in common wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.) and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The genetic differentiation of nuclear, mitochondrial (mt) and chloroplast (cp) genomes was investigated by Southern and PCR analysis using 75 varieties of cultivated rice ( Oryza sativa L.) and 118 strains of common wild rice (CWR, Oryza rufipogon Griff.) from ten countries of Asia. The distinguishing differences between the Indica and Japonica cultivars were detected both in the nuclear genome and the cytoplasmic genome, confirming that the Indica-Japonica differentiation is of major importance for the three different classes of genome in cultivated rice. This differentiation was also detected in common wild rice with some differences among the genome compartments and the various regions. For nuclear DNA variation, both Indica-like and Japonica-like types were observed in the Chinese CWR, with the latter more-frequent than the former. No Japonica-like type was found in South Asia, and only two strains of the Japonica-like type were detected in Southeast Asia, thus the Indica-like type is the major type among South and Southeast Asian CWR. For mtDNA, only a few strains of the Japonica-like type were detected in CWR. For cpDNA, the Japonica type was predominant among the CWR strains from China, Bangladesh and Burma, while the Indica type was predominant among the CWR strains from Thailand, Malaysia, Cambodia and Sri Lanka, and both types were found in similar frequencies among the Indian CWR. Altogether, however, the degree of Indica-Japonica differentiation in common wild rice was much-less important than that in cultivated rice. Cluster analyses for nuclear and mitochondrial DNA variation revealed that some CWR strains showed large genetic distances from cultivated rice and formed clusters distinct from cultivated rice. Coincidence in the genetic differentiation between the three different classes of genome was much higher in cultivated rice than in CWR. Among the 75 cultivars, about 3/4 entries were "homoeotype" showing congruent results for nuclear, mt and cpDNA regarding the Indica-Japonica differentiation. In CWR, the proportions of homoeotypes were 5.7%, 15% and 48.8% in China, South Asia and Southeast Asia, respectively. Based on the average genetic distance among all the strains of CWR and cultivated rice for nuclear and mitochondrial genomes, the variability of the nuclear genome was found to be higher than that of the mitochondrial genome. The global pattern based on all genomes shows much-more diversification in CWR than that in cultivated rice.     

Map-based cloning and functional analysis of the chromogen gene <Emphasis Type="Italic">C</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The chromogen gene C is critical for anthocyanin regulation in rice, and apiculus color is an important agronomic trait in selective breeding and variety purification. Mapbased cloning and in-depth functional analysis of the C gene will be useful for understanding the molecular mechanism of anthocyanin biosynthesis and for rice breeding. Japonica landrace Lijiangxintuanheigu (LTH) has red apiculi and purple stigmas. Genetic analysis showed that red apiculus and purple stigma in LTH co-segregated indicating control by a single dominant gene, or by two completely linked genes. Using 1,851 recessive individuals from two F₂ populations, the target gene OsC was delimited to a 70.8 kb interval on chromosome 6 that contains the rice homologue of the maize anthocyanin regulatory gene C1. When the entire OsC gene and its full-length cDNA cloned from LTH were transformed into japonica cultivar Kitaake with colorless apiculi and stigmas all positive transformants had red apiculi but non-colored stigmas, validating that OsC alone was responsible for the apiculus color and represented the functional C gene. OsC was constitutively expressed in all tissues examined, with strongest expression in leaf blades. These results set a foundation to clarify the regulatory mechanisms of OsC in the anthocyanin biosynthetic pathway.