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To meet the future demand of the global population, new varieties to adapt to regional climate changes such as salinity, drought, and submergence and new race emergence of pest disease and insects are expected to be developed for plant breeders. To facilitate efficient screening of germplasm and gene exploration on bi-parental cross populations and genome-wide association study (GWAS), the construction of a compact and genetically characterized germplasm platform is necessary in the National gene bank. In this study, genetic relationships of the representative core collections (CC) conserved in the National Seedbank of Myanmar were investigated using the genotyping-by-sequencing approach. It was found that the accessions were classified into the three clusters corresponding so-called japonica, indica, and aroma subpopulations. For efficient germplasm screening and gene exploration by GWAS on the indica landrace germplasm in Myanmar, a compact diversity panel designated “G” consisting of the 249 accessions was assembled, and genome-wide nucleotide variants were collected by the whole genome sequencing. The principal component analysis using genome-wide variants revealed that the selected accessions did not have an apparent population structure, but the first and second principal component scores correlated to the latitude and longitude, suggesting that latitude is the more causal factor of the geographical variation within the Myanmar landrace. When the reference genome of the leading variety in Myanmar was used, the mixed model genome-wide association analysis provided higher -log10(p) scores and detected seven peaks of apiculus pigmentation of spikelet. The five peak associations of the seven fall into the linkage disequllibrium blocks or neighbor blocks containing the isolated genes involved in anthocyanin pigmentation in rice. These results demonstrated that the combination of the landrace GWAS of the National seed banks and reference sequences genetically related to the landrace provide better research environments in characterization and gene exploration.

The HEUKCHEEM gene plays an important role in spine color formation. A white spine occurs due to two mutations in HEUKCHEEM and is closely related to the regional distribution of these mutants. Mapping analysis revealed that the HEUKCHEEM gene is co-segregated with the B locus in the regulation of black spine color development in cucumber fruit. HEUKCHEEM induced the expression of the genes involved in the anthocyanin biosynthetic pathway, leading to the accumulation of anthocyanins in black spines. The transiently over-expressed HEUKCHEEM in cucumber and tobacco plants enhanced the expression of anthocyanin biosynthesis-related genes, leading to anthocyanin accumulation. However, two mutations-insertion of the 6994 bp mutator-like transposable element (MULE) sequence into the second intron and one single-nucleotide polymorphism (SNP) of C to T in the second exon of HEUKCHEEM-were identified in white spines, leading to no accumulation of anthocyanin biosynthesis-related gene transcripts and anthocyanins. Furthermore, association analysis using 104 cucumber accessions with different geographical origins revealed that the types of mutations in HEUKCHEEM are strongly linked to geographical origins. The MULE insertion is found extensively in cucumbers with white spines in East Asia and Australia. However, cucumbers with white spines in other areas could be significantly influenced by a single SNP mutation. Our results provide fundamental information on spine color development in cucumber fruits and spine color-based cucumber breeding programs.

To evaluate and utilize potentially valuable quantitative trait loci or genes of wild relatives in the genetic background of domesticated crop species, chromosome segment substitution lines (CSSLs) are a valuable tool. CSSLs can be constructed through the exchange of chromosome segments of AA genome species of the genus Oryza with cultivated rice, Oryza sativa L. Here we report the development of three sets of CSSLs carrying segments of AA genome species closely related to Oryza sativa-O. glaberrima (IRGC 103777 from Mali), O. rufipogon (W1962 from China), and O. nivara (IRGC 105715 from Cambodia)-in the genetic background of ssp. japonica cultivar Taichung 65 through the use of 101 to 121 simple-sequence-repeat markers in whole-genome genotyping and marker-assisted selection. The materials are available via the National Bioresource Project (Rice) Oryzabase Web page.

QTL mapping and RT-PCR analyses identified the CsGy5G015660 as a strong powdery mildew resistance candidate gene and natural variation of CsGy5G015660 allele was observed using 115 core germplasm. Powdery mildew (PM) is among the most serious fungal diseases encountered in the cultivation of cucurbits. The development of PM-resistant inbred lines is thus of considerable significance for cucumber breeding programs. In this study, we applied bulked segregant analysis combined with QTL-seq to identify PM resistance loci using F2 population derived from a cross between two Korean cucumber inbred lines, PM-R (resistant) and PM-S (susceptible). Genome-wide SNP profiling using bulks of the two extreme phenotypes identified two QTLs on chromosomes 5 and 6, designated pm5.2 and pm6.1, respectively. The two PM resistance loci were validated using molecular marker-based classical QTL analysis: pm5.2 (30% R2 at LOD 11) and pm6.1 (11% R2 at LOD 3.2). Furthermore, reverse transcriptase-PCR analyses, using genes found to be polymorphic between PM-R and PM-S, were conducted to identify the candidate gene(s) responsible for PM resistance. We found that transcripts of the gene CsGy5G015660, encoding a putative leucine-rich repeat receptor-like serine/threonine-protein kinase (RPK2), showed specific accumulation in PM-R prior to the appearance of disease symptoms, and was accordingly considered a strong candidate gene for PM resistance. In addition, cleaved amplified polymorphic sequence markers from CsGy5G015660 were developed and used to screen 35 inbred lines. Natural variation in the CsGy5G015660 allele was also observed based on analysis of a core collection of 115 cucumber accessions. Our results provide new genetic insights for gaining a better understanding of the genetic basis of PM resistance in cucumber, and pave the way for further utilization in cucumber PM resistance breeding programs.

The Mildew resistance locus o (Mlo) is a plant-specific gene family that encodes a protein with seven-transmembrane (TM) domains that play an important role in plant resistance to powdery mildew (PM). PM caused by Podosphaera xanthii is a widespread plant disease and represents the major fungal threat for many Cucurbits. The recently reported Cucurbita maxima genome sequence data provides an opportunity to identify and characterize the MLO gene family in this species. A total of twenty genes designated CmaMLO1 to CmaMLO20 have been identified using an in silico cloning method with the MLO gene sequences of Cucumis sativus, Cucumis melo, Citrullus lanatus, and Cucurbita pepo as probes. These CmaMLOs were evenly distributed on 15 of the 20 C. maxima chromosomes without any obvious clustering. Multiple sequence alignment showed that common structural features of MLO gene family, such as TM domains, a calmodulin-binding domain, and 30 important amino acid residues for MLO function, were well conserved. Phylogenetic analysis of the CmaMLO genes and other plant species revealed seven different clades (I through VII); however, clade IV is specific to monocots (rice, barley and wheat). Expression analysis showed that four of the five CmaMLO genes that belonged to clade V were up-regulated in pumpkins infected with P. xanthii. Phylogenetic and expression analysis provided preliminary evidence that these five genes could be susceptibility genes that are important for PM resistance. This study is the first comprehensive report on MLO genes in C. maxima to our knowledge. These findings will facilitate the functional analysis of MLOs related to PM susceptibility and are valuable resources for the development of disease resistance in pumpkin.

Reduced seed shattering was a critical evolutionary step in crop domestication. Two cultivated rice species, Oryza sativa and Oryza glaberrima, were independently domesticated from the wild species Oryza rufipogon in Asia and Oryza barthii in Africa, respectively. A single nucleotide polymorphism (SNP) in the c gene, which encodes a trihelix transcription factor, causes nonshattering in O. sativa. However, the genetic mechanism of nonshattering in O. glaberrima is poorly understood. We conducted an association analysis for the coding sequences of SH3/SH4 in AA- genome rice species and the mutation suggested to cause nonshattering was demonstrated to do so using a positional-cloning approach in the O. sativa genetic background. We found that the loss of seed shattering in O. glaberrima was caused by an SNP resulting in a truncated SH3/SH4 protein. This mutation appears to be endemic and to have spread in the African gene pool by hybridization with some O. barthii accessions. We showed that interaction between the O. sativa and O. glaberrima domestication alleles of SH3 in heterozygotes induces a 'throwback' seed-shattering phenotype similar to that in the wild species. Identification of the causative SNP provides new insights into the molecular basis of seed shattering in crops and may facilitate investigation of the history of African rice domestication.

QTL mapping using NGS-assisted BSA was successfully applied to an F 2 population for downy mildew resistance in cucumber. QTLs detected by NGS-assisted BSA were confirmed by conventional QTL analysis. Downy mildew (DM), caused by Pseudoperonospora cubensis, is one of the most destructive foliar diseases in cucumber. QTL mapping is a fundamental approach for understanding the genetic inheritance of DM resistance in cucumber. Recently, many studies have reported that a combination of bulked segregant analysis (BSA) and next-generation sequencing (NGS) can be a rapid and cost-effective way of mapping QTLs. In this study, we applied NGS-assisted BSA to QTL mapping of DM resistance in cucumber and confirmed the results by conventional QTL analysis. By sequencing two DNA pools each consisting of ten individuals showing high resistance and susceptibility to DM from a F2 population, we identified single nucleotide polymorphisms (SNPs) between the two pools. We employed a statistical method for QTL mapping based on these SNPs. Five QTLs, dm2.2, dm4.1, dm5.1, dm5.2, and dm6.1, were detected and dm2.2 showed the largest effect on DM resistance. Conventional QTL analysis using the F2 confirmed dm2.2 (R 2 = 10.8–24 %) and dm5.2 (R 2 = 14–27.2 %) as major QTLs and dm4.1 (R 2 = 8 %) as two minor QTLs, but could not detect dm5.1 and dm6.1. A new QTL on chromosome 2, dm2.1 (R 2 = 28.2 %) was detected by the conventional QTL method using an F3 population. This study demonstrated the effectiveness of NGS-assisted BSA for mapping QTLs conferring DM resistance in cucumber and revealed the unique genetic inheritance of DM resistance in this population through two distinct major QTLs on chromosome 2 that mainly harbor DM resistance.

Hybrid incompatibility in F1hybrids or later generations is often observed as sterility or inviability. This incompatibility acts as postzygotic reproductive isolation, which results in the irreversible divergence of species. Here, we show that the reciprocal loss of duplicated genes encoding mitochondrial ribosomal protein L27 causes hybrid pollen sterility in F1hybrids of the cultivated riceOryza sativaand its wild relativeO.glumaepatula. Functional analysis revealed that this gene is essential for the later stage of pollen development, and distribution analysis suggests that the gene duplication occurred before the divergence of the AA genome species. On the basis of these results, we discuss the possible contribution of the “founder effect” in establishing this reproductive barrier.

F1 sterility, one of the most common post-zygotic reproductive barriers, is frequently observed in both interspecific and intraspecific crosses of rice. Elucidating the genetic and cytological mechanisms of F1 pollen sterility is important to exploit genetic resources and to understand the evolutionary dynamics of post-zygotic reproductive isolation in rice. Here, we report two F1 pollen sterility loci, designated S36 and S25, found in an interspecific cross between O. sativa ssp. japonica (Taichung 65) and O. nivara (IRGC105444), and an intraspecific cross between O. sativa ssp. japonica (Asominori) and ssp. indica (IR24). Genetic analyses revealed that both loci are located on distal end of the short arm of chromosome 12 and that allelic interaction at the heterozygous locus caused the sterility of male gametes carrying the japonica alleles in both cases. Comparison of map positions of S36 and S25 suggested that these two loci might be the same locus. Cytological investigation revealed that abnormality of sterile pollen grains caused by S36 occurred mainly at the late bicellular stage after initiation of starch accumulation. This study would provide the better understanding on the genetic nature and the cytological mechanism of F1 pollen sterility in rice.

Cucumber is a typical monoecious having separate male and female flowers on the same plant, and sex expression in cucumber is mainly determined by three major genes: F/f, M/m and A/a. Gynoecy plays an important role in cucumber hybrid breeding, and the use of gynoecious lines as maternal parents ensures high productivity. This study aimed to identify a co-dominant marker linked to Female (F) locus to distinguish homozygous and heterozygous gynoecious lines for efficient selection of gynoecy in cucumber breeding. Firstly, we analyzed a 50-kb genomic sequence of five gynoecious and five monoecious inbred lines to detect the polymorphism linked to F locus. A pair of specific primers, Cs-BCAT-F/Cs-BCAT-R, based on an In/Del polymorphism at 3′-UTR of branched-chain amino acid transaminase (BCAT) gene was designed to examine the polymorphism in gynoecious and monoecious parents, their F 1 hybrids and two F 2 segregating populations. The PCR fragments amplified with Cs-BCAT-F/Cs-BCAT-R co-segregated with sexual phenotypes in the F 2 populations, behaving as a co-dominant marker with two alleles: F (gynoecious) and f (monoecious). When we further verified the consistency of the co-dominant marker using additional 55 diverse inbred lines, it can explain ~90 % of accessions. Linkage analysis also showed that the gene which enhances the number of female flowers co-segregated with the co-dominant marker on the long arm of chromosome 6. This is the first report for the development of F locus-specific co-dominant marker which can distinguish perfectly homozygous and heterozygous gynoecious, and it could be used in marker-assisted selection in cucumber breeding.

Loss of function of duplicated genes plays an important role in the evolution of postzygotic reproductive isolation. The widespread occurrence of gene duplication followed by rapid loss of function of some of the duplicate gene copies suggests the independent evolution of loss-of-function alleles of duplicate genes in divergent lineages of speciation. Here, we found a novel loss-of-function allele of S27 in the Asian annual wild species Oryza nivara, designated S27-niv (s), that leads to F(1) pollen sterility in a cross between O. sativa and O. nivara. Genetic linkage analysis and complementation analysis demonstrated that S27-niv (s) lies at the same locus as the previously identified S27 locus and S27-niv (s) is a loss-of-function allele of S27. S27-niv (s) is composed of two tandem mitochondrial ribosomal protein L27 genes (mtRPL27a and mtRPL27b), both of which are inactive. The coding and promoter regions of S27-niv (s) showed a number of nucleotide differences from the functional S27-T65 (+) allele. The structure of S27-niv (s) is different from that of a previously identified null S27 allele, S27-glum (s), in the South American wild rice species O. glumaepatula, in which mtRPL27a and mtRPL27b are absent. These results show that the mechanisms for loss-of-function of S27-niv (s) and S27-glum (s) are different. Our results provide experimental evidence that different types of loss-of-function alleles are distributed in geographically and phylogenetically isolated species and represent a potential mechanism for postzygotic isolation in divergent species.

This paper presents texture feature extraction and selection methods for on-line pattern classification evaluation. Feature selection for texture analysis plays a vital role in the field of image recognition. Despite many approaches done previously, this research is entirely different from them since it comes from the fundamental ideas of feature selection for image retrieval. The proposed approach is capable of selecting the best features without recourse to classification and segmentation procedures. In this approach, probability density function estimation and a modified Bhattacharyya distance method are applied for clustering texture features of images in sequences and for comparing multi-distributed clusters with one another, respectively.