Genetic mapping and QTL analysis of horticultural traits in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) using recombinant inbred lines

A set of 171 recombinant inbred lines (RIL) were developed from a narrow cross in cucumber (Cucumis sativus L.; 2n = 2x = 14) using the determinate (de), gynoecious (F), standard-sized leaf line G421 and the indeterminate, monoecious, little-leaf (ll) line H-19. A 131-point genetic map was constructed using these RILs and 216 F₂ individuals to include 14 SSRs, 24 SCARs, 27 AFLPs, 62 RAPDs, 1 SNP, and three economically important morphological [F (gynoecy), de (determinate habit), ll (little leaf)] markers. Seven linkage groups spanned 706 cM with a mean marker interval of 5.6 cM. The location of F and de was defined by genetic linkage and quantitative trait locus (QTL) analysis to be associated with SSR loci CSWCT28 and CSWCTT14 at 5.0 cM and 0.8 cM, respectively. RIL-based QTL analysis of the number of lateral branches in three environments revealed four location-independent factors that cumulatively explained 42% of the observed phenotypic variation. QTLs conditioning lateral branching (mlb1.1), fruit length/diameter ratio (ldr1.2) and sex expression (sex1.2) were associated with de. Sex expression was influenced by three genomic regions corresponding to F and de both on linkage Group 1, and a third locus (sex6.1) on linkage Group 6. QTLs conditioning the number of fruit per plant (fpl1.2), the number of lateral branches (mlb1.4) and fruit length/diameter ratio (ldr1.3) were associated with ll. The potential value of these marker-trait associations (i.e., yield components) for plant improvement is portended by the relatively high LOD scores (2.6 to 13.0) and associated R² values (1.5% to 32.4%) that are affiliated with comparatively few genetic factors (perhaps 3 to 10).Communicated by H.C. BeckerMention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable     

Molecular mapping of a male fertility restorer locus of Brassica oleracea using expressed sequence tag-based single nucleotide polymorphism markers and analysis of a syntenic region in Arabidopsis thaliana for identification of genes encoding pentatricopeptide repeat proteins

An F₂ population was developed from a cross between a mur-cytoplasmic male sterile broccoli line and a restorer Chinese kale line. Phenotypic analysis of F₂ plants indicated that the pollen fertility is controlled by two genes and segregated in a duplicate gene interaction mode with a ratio of 15:1. A total of 236 single nucleotide polymorphism (SNP) markers were developed utilizing 1,448 primers designed for production of expressed sequence tag (EST)-SNP markers of Raphanus sativus and analyzed by the dot-blot technique in 205 F₂ individuals. A linkage map was constructed with a total of 142 markers and these markers were assigned to nine linkage groups together with simple sequence repeat markers mapped previously on the published linkage maps of Brassica oleracea. The linkage map spanned 909 cM with an average marker distance of 6.4 cM. A fertility restorer locus (Rfm1) was mapped on LG1, corresponding to chromosome 3, along with a flower color locus at a distance of 25 cM. SNP markers flanking the Rfm1 locus were BoCL2642s at a distance of 2.5 cM on one side and BoCL2901s at a distance of 7.5 cM on the other side. All the SNP markers showed homology with Arabidopsis thaliana and Brassica rapa genome sequences. Three pentatricopeptide repeat genes of the P-subfamily, particularly expressed in buds of the restorer line, were identified and these genes could be potential candidate fertility restorer genes.     

A SNP haplotype associated with a gene resistant to Xanthomonas axonopodis pv. malvacearum in upland cotton (Gossypium hirsutum L.)

An F_4:5 population of 285 families with each tracing back to a different F₂ plant, derived from a cotton bacterial blight resistant line ‘DeltaOpal’ and a susceptible line ‘DP388’, was artificially inoculated with bacterial blight race 18 (Xanthomonas axonopodis pv. malvacearum) to assay their resistance or susceptibility to the disease. The segregation in the F_4:5 population indicates that the resistance was conditioned by a single dominant gene designated B _12. Simple sequence repeat (SSR) markers identified as putatively linked to the resistance gene by bulked segregant analysis were confirmed on the entire F_4:5 population. Three SSR markers, CIR246, BNL3545 and BNL3644 on chromosome 14, were found closely linked to B ₁₂ . The association between CIR246 and B ₁₂ was validated among 354 plants of 16 diverse varieties. Based on Monsanto SSR/single nucleotide polymorphism (SNP) consensus map, SNP markers closely linked to CIR246 were used to screen ‘DeltaOpal’ and ‘DP388’ for polymorphism. The polymorphic SNP markers were run on the F_4:5 population and the four SNP markers spanning 3.4 cM were found to flank the resistance gene on chromosome 14. The linkage between B ₁₂ and the 4-SNP marker haplotype was validated using 18 elite cotton lines. This 4-SNP marker haplotype can be used for marker assisted selection for bacterial blight resistance breeding programs or for screening germplasm collections for this locus rapidly.     

Contents and recovery of gibberellins in monoecious and gynoecious cucumber plants

Diffusates from seedlings and root exudates from 6-week-old plants of a monoecious line of cucumber, Cucumis sativus L., contained considerably higher levels of gibberellin-(GA-) like substances than did those from plants of an isogenic gynoecious line. Most of the GA-like activity was found in a chromatogram region typical of GA₁ and GA₃; some activity, particularly in root exudates, appeared also at an R_F similar to that of GA₄ and GA₇.

When seedlings were treated with ³H-labeled GA₁, more radioactivity was found in the diffusates from monoecious seedlings than from gynoecious ones. The same was true of biological activity in root diffusates from older plants which had been treated with gibberellin A₄₊₇.

In conjunction with evidence present in literature, these results support the idea that endogenous GAs play a part in the regulation of sex expression in cucumber, relatively high levels favoring the formation of staminate flowers.

     

Mapping of <Emphasis Type="Italic">Sihc1</Emphasis>, which controls hull color,using a high-density genetic map based on restriction site-associated DNA sequencing in foxtail millet [<Emphasis Type="Italic">Setaria italica</Emphasis> (L.) P. Beauv.]

Hull color (HC) in foxtail millet appears to be a major indicator of nutritional quality. To attempt to identify and characterize the gene or quantitative trait locus (QTL) related to HC and to provide a basis for quality and yield breeding of foxtail millet, a restriction site-associated DNA sequencing (RAD-seq) analysis was employed to reveal genome-wide single nucleotide polymorphisms (SNPs) and to genotype the F₂ progeny from a cross between two cultivars: Heizhigu with green HC and Changnong 35 with yellow HC. A high-density linkage map spanning 1542.87 cM was constructed using 1694 bin markers, consisting of 46,256 SNP markers, and a QTL locus controlling HC was identified and temporarily named as Sihc1 (Setaria italica hull color). Results showed that Sihc1 was located in the interval between markers M33926512 and M34281352 on the end of chromosome 6, and explained 80.26% of the phenotypic variation with a logarithm of odds ratio of 93.46. The additive and dominant effects of Sihc1 were 1.0025 and ?0.9991, respectively. Sihc1 was narrowed to a 354.84 kb physical region, inclusive of 30 open reading frames, and further analysis suggested that Seita.6G226500, Seita.6G226800, Seita.6G228300, and Seita.6G228600 might be the key candidates of Sihc1.     

The M locus and ethylene-controlled sex determination in andromonoecious cucumber plants.

Sex determination in cucumber (Cucumis sativus L.) plants is genetically controlled by the F and M loci. These loci interact to produce three different sexual phenotypes: gynoecious (M-F-), monoecious (M-ff), and andromonoecious (mmff). Gynoecious cucumber plants produce more ethylene than do monoecious plants. We found that the levels of ethylene production and the accumulation of CS-ACS2 mRNA in andromonoecious cucumber plants did not differ from those in monoecious plants and were lower than the levels measured in gynoecious plants. Ethylene inhibited stamen development in gynoecious cucumbers but not in andromonoecious ones. Furthermore, ethylene caused substantial increases in the accumulation of CS-ETR2, CS-ERS, and CS-ACS2 mRNA in monoecious and gynoecious cucumber plants, but not in andromonoecious one. In addition, the inhibitory effect of ethylene on hypocotyl elongation in andromonoecious cucumber plants was less than that in monoecious and gynoecious plants. These results suggest that ethylene responses in andromonoecious cucumber plants are reduced from those in monoecious and gynoecious plants. This is the first evidence that ethylene signals may influence the product of the M locus and thus inhibit stamen development in cucumber. The andromonoecious line provides novel material for studying the function of the M locus during sex determination in flowering cucumbers.     

Sexual differentiation in cucumber: The effects of abscisic acid and other growth regulators on various sex genotypes

AbA, ethephon and gibberellin were applied to cucumber plantsof monoecious, gynoecious, andromoneocious and hermaphroditeinbred lines, as well as to F₁ (gynoecious?monoecious) plants.Exogenous AbA enhanced the male tendency in monoecious cucumberplants and the female tendency in gynoecious plants, irrespectiveof light regime. Exogenous ethephon treatments increased thefemale tendency in monoecious plants, and decreased it in gynoeciousones. These effects were influenced by day length. ExogenousAbA counteracted the effect of gibberellin (A₄₊₇) treatmentin gynoecious plants, but had no such effect in monoecious ones. In addition to its differential effect on sexual differentiation,AbA stimulated flower development in gynoecious plants and inhibitedit in monoecious plants. These responses to AbA are discussedin the light of previously reported effects of plant growthregulators on various sex types of cucumber. The present resultsare being integrated into an updated working hypothesis on sexcontrol in cucumbers. (Received August 30, 1976; )     

Rapid location of Glomerella leaf spot resistance gene locus in apple by whole genome re-sequencing

Glomerella leaf spot (GLS) is a new fungal disease of apple that damages apple leaves mainly during the summer in China. For efficient GLS-resistant apple breeding by marker-assisted selection (MAS) and a better understanding of the molecular mechanisms of the resistance, it is important to find molecular markers that are tightly linked to GLS resistance genes and construct fine mapping. However, the development and selection of DNA markers are time-consuming and labor-intensive processes. Next-generation sequencing technology provides a powerful tool to overcome this limitation and is faster and more efficient in establishing the association of GLS resistance with molecular markers or searching for candidate genes. In this study, we report a method for rapid location of a GLS resistance gene locus (R _gls ) in apple by whole genome re-sequencing technology coupled with bulked segregant analysis (BSA). A total of 3,399,950 single nucleotide polymorphisms (SNPs) were identified. Through the genome-wide comparison of SNP profiles between the resistant and the susceptible bulks constructed from F₁ individuals derived from a cross between “Golden Delicious” and “Fuji,” the R _gls locus was identified on apple chromosome 15 between 2 and 5 Mb. In this region, eight SNP markers were validated using high resolution melting (HRM), and the fine genetic mapping of the eight markers was constructed. The R _gls locus was sandwiched by two flanking markers SNP4208 and SNP4257, with the recombination frequency of 0.97% (2/207). The marker SNP4236 co-segregated with R _gls . The physical size of the R _gls locus was estimated to be 49 kb. In this genetic interval, nine genes were predicted. Our study provides an effective method for rapid identification of genomic regions and development of the diagnostic markers for MAS. This strategy is potentially useful for other agronomic traits or plant species.     

Identification of a nuclear-recessive gene locus for male sterility on A2 chromosome using the Brassica 60 K SNP array in non-heading Chinese cabbage

WS24-3 is a newly bred recessive genic male sterility line of the non-heading Chinese cabbage (Brassica rapa ssp. chinensis). Here, an F₂ population was produced from the cross between WS24-3 and a male-fertile breeding line (WS135). The Illumina Brassica 60 K single nucleotide polymorphism (SNP) array was used for SNPs detecting between sterile and fertile bulks from the F₂ population, and 62 SNPs were identified. BLAST analysis of the 62 SNPs revealed that the A2 chromosome of Brassica rapa genome contained 22 SNPs, whereas the other chromosomes did not contain more than 6 SNPs each. These data indicated that the potential target gene locus, named Bra2Ms, might be located on A2. Based on 10 of the 22 SNPs, allele-specific-polymerase chain reaction (AS-PCR) primers and single sequence repeat (SSR) primers were designed, 5 AS-PCR primers and 9 SSR primers showed difference between the bulks in electrophoretic determination. Analysis of these markers in F₂ population revealed that Bra2Ms was genetically delimited to a region of 1.2 cM. We also detected two co-segregated markers SSRa2-951 and SSRa2-960 in this region. The markers identified in our study might facilitate the transfer of recessive genic male sterility alleles to other favorable genetic backgrounds. Furthermore, these markers will support a map-based clone of Bra2Ms.     

Some Morphogenic Differences between Monoecious and Gynoecious Cucumber Seedlings as Related to Ethylene Production

Exposure of gibberellic acid-treated seedlings of a monoecious cucumber cultivar `Chipper' (Cucumis sativus L.) to ethylene caused thickening of the hypocotyl, inhibited longitudinal growth, and had no effect on fresh weight. Downward curvature of cotyledons was increased by the presence of ethylene. A gynoecious breeding line, `Gy 3,' had thicker hypocotyls and displayed its cotyledons in a more downward position than `Chipper'. Excised hypocotyls of the gynoecious seedlings produced three times as much ethylene as did the monoecious Chipper hypocotyls. Thus, ethylene may play a role in the regulation of cucumber seedling morphology.     

A major QTL associated with Fusarium oxysporum race 1 resistance identified in genetic populations derived from closely related watermelon lines using selective genotyping and genotyping-by-sequencing for SNP discovery

Key message

A major quantitative trait locus (QTL) for Fusarium oxysporum Fr. f. sp. niveum race 1 resistance was identified by employing a “selective genotyping” approach together with genotyping-by-sequencing technology to identify QTLs and single nucleotide polymorphisms associated with the resistance among closely related watermelon genotypes.

Abstract

Fusarium wilt is a major disease of watermelon caused by the soil-borne fungus Fusarium oxysporum Schlechtend.:Fr. f. sp. niveum (E.F. Sm.) W.C. Snyder & H.N. Hans (Fon). In this study, a genetic population of 168 F₃ families (24 plants in each family) exhibited continuous distribution for Fon race 1 response. Using a “selective genotyping” approach, DNA was isolated from 91 F₂ plants whose F₃ progeny exhibited the highest resistance (30 F₂ plants) versus highest susceptibility (32 F₂ plants), or moderate resistance to Fon race 1 (29 F₂ plants). Genotyping-by-sequencing (GBS) technology was used on these 91 selected F₂ samples to produce 266 single nucleotide polymorphism (SNP) markers, representing the 11 chromosomes of watermelon. A major quantitative trait locus (QTL) associated with resistance to Fon race 1 was identified with a peak logarithm of odds (LOD) of 33.31 and 1-LOD confidence interval from 2.3 to 8.4 cM on chromosome 1 of the watermelon genetic map. This QTL was designated “Fo-1.1” and is positioned in a genomic region where several putative pathogenesis-related or putative disease-resistant gene sequences were identified. Additional independent, but minor QTLs were identified on chromosome 1 (LOD 4.16), chromosome 3 (LOD 4.36), chromosome 4 (LOD 4.52), chromosome 9 (LOD 6.8), and chromosome 10 (LOD 5.03 and 4.26). Following the identification of a major QTL for resistance using the “selective genotyping” approach, all 168 plants of the F ₂ population were genotyped using the SNP nearest the peak LOD, confirming the association of this SNP marker with Fon race 1 resistance. The results in this study should be useful for further elucidating the mechanism of resistance to Fusarium wilt and in the development of molecular markers for use in breeding programs of watermelon.     

Development and linkage mapping of novel sex-linked markers for marker-assisted cultivar breeding in pistachio (<Emphasis Type="Italic">Pistacia vera</Emphasis> L.)

The dioecious character of Pistacia vera L (the pistachio tree) limits its breeding capacity. Thus, early stage selection of males can save time, labor, and land. This study aimed to develop sex-linked single nucleotide polymorphism (SNP) markers, together with expressed sequence tag-derived simple sequence repeats (EST-SSRs), to determine position of the sex locus in pistachio by constructing a linkage map of its sex chromosome for the first time. Nine novel sex-linked SNP markers were successfully identified by SNaPshot minisequencing analysis of 25 SNP loci from 17 restriction site-associated DNA (RAD) reads in 309 individuals. All nine markers were heterozygous in females and homozygous in males supporting a ZW/ZZ sex determination system in pistachio. A total of 105 segregating SSRs and sex-linked markers were used to identify the sex chromosome and the position of the sex locus through analysis of a Siirt × Ba?yolu F₁ population with 122 progenies. Of these 105 markers, four common and four paternal SSRs were mapped onto the sex chromosome, along with the phenotypic sex locus and sex-linked markers. The resulting consensus map had a total length of 65.19 cM. The sex locus and sex-linked SNP markers were located in the center of the chromosome at a distance of 31.86 and 31.92 cM, respectively. This study presents valuable information about the sex chromosome and sex locus position as well as novel polymorphic EST-SSRs and nine sex-linked SNP markers in pistachio.     

The level of phytohormones in monoecious and gynoecious cucumbers as affected by photoperiod and ethephon

The endogenous levels of auxin, gibberellin, and inhibitors were followed in monoecious and gynoecious cucumber (Cucumis sativus L.) plants, and in plants treated with the ethylene-releasing compound Ethephon (2-chloroethyl phosphonic acid). Higher auxin inhibitor and lower gibberellin levels were associated with female tendency. The endogenous level of gibberellin and auxin decreased in Ethephon-treated plants. Application of Ethephon induced a rise in abscisic acid. Root application of abscisic acid promoted female tendency of gynoecious cucumbers grown under conditions which increase maleness. High CO₂ levels, which are known to antagonize ethylene, increased maleness of gynoecious cucumbers. The possibility of interrelationship between gibberellin, auxin, ethylene, and abscisic acid on sex expression are discussed.     

Mapping of the <Emphasis Type="Italic">multifoliate pinna</Emphasis> (<Emphasis Type="Italic">mfp</Emphasis>) leaf-blade morphology mutation in grain pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

The multifoliate pinna (mfp) mutation alters the leaf-blade architecture of pea, such that simple tendril pinnae of distal domain are replaced by compound pinna blades of tendrilled leaflets in mfp homozygotes. The MFP locus was mapped with reference to DNA markers using F₂ and F_2:5 RIL as mapping populations. Among 205 RAPD, 27 ISSR and 35 SSR markers that demonstrated polymorphism between the parents of mapping populations, three RAPD markers were found linked to the MFP locus by bulk segregant analyses on mfp/mfp and MFP/MFP bulks assembled from the F_2:5 population. The segregational analysis of mfp and 267 DNA markers on 96 F₂ plants allowed placement of 26 DNA markers with reference to MFP on a linkage group. The existence of common markers on reference genetic maps and MFP linkage group developed here showed that MFP is located on linkage group IV of the consensus genetic map of pea.     

Reassessment of QTLs for Late Blight Resistance in the Tomato Accession L3708 Using a Restriction Site Associated DNA (RAD) Linkage Map and Highly Aggressive Isolates of Phytophthora infestans

Tomato late blight caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary is a major threat to tomato production in cool and wet environments. Intensified outbreaks of late blight have been observed globally from the 1980s, and are associated with migration of new and more aggressive populations of P. infestans in the field. The objective of this study was to reassess late blight resistance in the wild tomato accession L3708 (Solanum pimpinellifolium L.) against pathogens of different aggressiveness. An F_2:3 genetic mapping population was developed using L3708 as the paternal parent. Two isolates of P. infestans, Pi39A and Pi733, were used for inoculation. Pi733 is a highly aggressive genotype that defeats three known late blight resistance genes, Ph-1, Ph-2, and Ph-5t in tomato. In contrast, Pi39A is a less aggressive genotype that defeats only Ph-1. Restriction site Associated DNA Sequencing (RAD-Seq) technology was used to massively sequence 90 bp nucleotides adjacent to both sides of PstI restriction enzyme cutting sites in the genome for all individuals in the genetic mapping population. The RAD-seq data were used to construct a genetic linkage map containing 440 single nucleotide polymorphism markers. Quantitative trait locus (QTL) analysis identified a new disease-resistant QTL specific to Pi733 on chromosome 2. The Ph-3 gene located on chromosome 9 could be detected whichever isolates were used. This study demonstrated the feasibility and efficiency of RAD-Seq technology for conducting a QTL mapping experiment using an F_2:3 mapping population, which allowed the identification of a new late blight resistant QTL in tomato.     

Rapid identification of an adult plant stripe rust resistance gene in hexaploid wheat by high-throughput SNP array genotyping of pooled extremes

Key message

High-throughput SNP array analysis of pooled extreme phenotypes in a segregating population by KASP marker genotyping permitted rapid, cost-effective location of a stripe rust resistance QTL in wheat.

Abstract

German wheat cultivar “Friedrichswerther” has exhibited high levels of adult plant resistance (APR) to stripe rust in field environments for many years. F_2:3 lines and F₆ recombinant inbred line (RILs) populations derived from a cross between Friedrichswerther and susceptible landrace Mingxian 169 were evaluated in the field in 2013, 2016 and 2017. Illumina 90K iSelect SNP arrays were used to genotype bulked extreme pools and parents; 286 of 1135 polymorphic SNPs were identified on chromosome 6B. Kompetitive Allele-Specific PCR (KASP) markers were used to verify the chromosome region associated with the resistance locus. A linkage map was constructed with 18 KASP-SNP markers, and a major effect QTL was identified within a 1.4 cM interval flanked by KASP markers IWB71602 and IWB55937 in the region 6BL3-0-0.36. The QTL, named QYr.nwafu-6BL, was stable across environments, and explained average 54.4 and 47.8% of the total phenotypic variation in F_2:3 lines and F₆ RILs, respectively. On the basis of marker genotypes, pedigree analysis and relative genetic distance QYr.nwafu-6BL is likely to be a new APR QTL. Combined high-throughput SNP array genotyping of pooled extremes and validation by KASP assays lowers sequencing costs compared to genome-wide association studies with SNP arrays, and more importantly, permits rapid isolation of major effect QTL in hexaploid wheat as well as improving accuracy of mapping in the QTL region. QYr.nwafu-6BL with flanking KASP markers developed and verified in a subset of 236 diverse lines can be used in marker-assisted selection to improve stripe rust resistance in breeding programs.