QTL-seq analysis of powdery mildew resistance in a Korean cucumber inbred line.

KEY MESSAGE: 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.

Two types of mutations in the HEUKCHEEM gene functioning in cucumber spine color development can be used as signatures for cucumber domestication.

MAIN CONCLUSION: 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.

Identification of quantitative trait loci governing subgynoecy in cucumber.

KEY MESSAGE: QTL-seq analysis identified three major QTLs conferring subgynoecy in cucumbers. Furthermore, sequence and expression analyses predicted candidate genes controlling subgynoecy. The cucumber (Cucumis sativus L.) is a typical monoecious having individual male and female flowers, and sex differentiation is an important developmental process that directly affects its fruit yield. Subgynoecy represents a sex form with a high degree of femaleness and would have alternative use as gynoecy under limited resource conditions. Recently, many studies have been reported that QTL-seq, which integrates the advantages of bulked segregant analysis and high-throughput whole-genome resequencing, can be a rapid and cost-effective way of mapping QTLs. Segregation analysis in the F2 and BC1 populations derived from a cross between subgynoecious LOSUAS and monoecious BMB suggested the quantitative nature of subgynoecy in cucumbers. Both genome-wide SNP profiling of subgynoecious and monoecious bulks constructed from F2 and BC1 plants consistently identified three significant genomic regions, one on chromosome 3 (sg3.1) and another two on short and long arms of chromosome 1 (sg1.1 and sg1.2). Classical QTL analysis using the F2 confirmed sg3.1 (R2 = 42%), sg1.1 (R2 = 29%) and sg1.2 (R2 = 18%) as major QTLs. These results revealed the unique genetic inheritance of subgynoecious line LOSUAS through two distinct major QTLs, sg3.1 and sg1.1, which mainly increase degree of femaleness, while another QTL, sg1.2, contributes to decrease it. This study demonstrated that QTL-seq allows rapid and powerful detection of QTLs using preliminary generation mapping populations such as F2 or BC1 population and further that the identified QTLs could be useful for molecular breeding of cucumber lines with high yield potential.

Development of introgression lines of AA genome Oryza species, O. glaberrima, O. rufipogon, and O. nivara, in the genetic background of O. sativa L. cv. Taichung 65.

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.

A single base change explains the independent origin of and selection for the nonshattering gene in African rice domestication.

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 for downy mildew resistance in cucumber via bulked segregant analysis using next-generation sequencing and conventional methods.

KEY MESSAGE: 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.

Mitochondrial gene in the nuclear genome induces reproductive barrier in rice.

Hybrid incompatibility in F(1) hybrids 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 F(1) hybrids of the cultivated rice Oryza sativa and its wild relative O. 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.

Independent evolution of a new allele of F1 pollen sterility gene S27 encoding mitochondrial ribosomal protein L27 in Oryza nivara.

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.