A linkage map of cultivated cucumber (Cucumis sativus L.) with 248 microsatellite marker loci and seven genes for horticulturally important traits

A genetic map was developed with microsatellite (simple sequence repeat, SSR) markers and 148 recombinant inbred lines (RILs) derived from a cross between two cultivated cucumber (Cucumis sativus L.) inbred lines 9110Gt and 9930, which was also segregating for seven horticulturally important traits including bitterfree foliage (bi), gynoecious sex expression (F), uniform immature fruit color (u), glossy fruit skin (d), heavy netting of mature fruit (H), no fruit ribbing (fr), and virescent leaf (v-1). Linkage analysis placed 248 microsatellite loci into seven linkage groups spanning 711.9?cM with a mean marker interval of 2.8?cM. Based on shared markers with an early cucumber genetic map, the 7 linkage groups could be assigned to seven cucumber chromosomes. The four fruit epidermal feature-related genes, u, d, H and fr were found to be tightly linked loci in Chromosome 5, and the other three (F, bi and v-1) were placed in different locations of Chromosome 6. It was the first time to map the four genes H, fr, bi and v-1 with molecular markers. In addition, this is the first report of the inheritance of fruit ribbing in cucumber, which was controlled by a single, dominant gene designated as Fr. Mapping information from this study opens the way for marker-assisted selection and map-based cloning of these horticulturally important genes in cucumber.     

Identification and fine mapping of molecular markers closely linked to fruit spines size <Emphasis Type="Italic">ss</Emphasis> gene in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Fruit spine size is one of the importantly external quality traits effected the economic value of cucumber fruit. Morphological–cytological observation of the fruit spine size phenotype indicated that large spine formation arises from an increasing of spiny pedestal cell number caused by cell division, and best periods to accurately score fruit spine size trait was 4th day before flowering to 7th day after flowering according the continuous observation. Genetic analysis showed that a single dominant gene determined the fruit spine size trait in cucumber. BC₁ population (189 individuals) of two inbred lines (large spine PI197088 and small spine SA0422) was used for primary mapping of the SS/ss locus with 7 markers covering an interval of 37.1 cM. An F₂ segregating population of 1032 individuals constructed from the same two parents (PI197088 and SA0422) was used to fine mapping of the SS/ss locus. Six new markers linked to the gene were successfully screened for construction of a fine linkage map, in which the SS/ss locus was located in the region flanked by marker SE1 (3 recombinants) and SSR43 (2 recombinants) with a 189 kb physical distance. Markers from this study will be valuable for candidate gene cloning and marker-assisted selection for cucumber breeding.     

Stability of seed oil quality traits in high and mid-oleic acid sunflower hybrids

Powdery mildew caused by Podosphaera xanthii is an important disease of melon, and race 2F is the predominant race in most areas of China. Resistance to P. xanthii race 2F in melon K7-1 was controlled by a dominant gene, designated Pm-2F, in a 106-member population of recombinant inbred lines derived from K7-1× susceptible K7-2. Using bulked segregant analysis with molecular markers, we have identified two polymorphic simple sequence repeats (SSR) to determine that Pm-2F is located on linkage group II. Comparative genomic analyses using mapped SSR markers and the cucumber genome sequence showed that the melon chromosomal region carrying Pm-2F is homologous to a 288,223 bp genomic region on cucumber chromosome (chr) 1. The SSR markers on chr 1 of cucumber, SSR02734, SSR02733 and CS27 were found linked with Pm-2F. Comparative mapping showed that two SSR markers (SSR02734 and CMBR8) flanked the Pm-2F locus and two nucleotide binding site-leucine-rich repeat resistance genes were identified in the collinear region of cucumber. A cleaved amplified polymorphic sequence (CAPS) marker was developed from the sequence of resistance genes and it delimits the genomic region carrying Pm-2F to 0.8 cM. The evaluation of 165 melon accessions and 13 race differential lines showed that the newly developed CAPS (CAPS-Dde I) marker can be used as a universal marker for effective marker assisted selection in melon powdery mildew resistance breeding. The putative resistance gene cluster provides a potential target site for further fine mapping and cloning of Pm-2F.     

Comparison and development of EST–SSRs from two 454 sequencing libraries of Gossypium barbadense

EST–SSRs of Gossypium barbadense are mainly developed using traditional Sanger sequencing. However, due to the high cost and low throughput of Sanger sequencing, it is necessary to use high throughput sequencing technology for the development of more ESTs to more effectively analyze the structure and function of this species. In this study, a G. barbadense acc. 3–79 unnormalized fiber cDNA library (219.63 Mb) and a G. barbadense cv. Hai7124 normalized root cDNA library (204.61 Mb) were obtained by 454 sequencing. EST–SSRs were identified from the two libraries, and only 7,255 SSRs were obtained from the unnormalized library, with an average frequency of 1/31.00 kb. In contrast, 16,087 SSRs were obtained from the normalized library, with an average frequency of 1/13.02 kb. The frequencies of dinucleotides and tetranucleotides in the two libraries were very different. Comparing the two libraries, we found that a normalized cDNA library is more efficient for mining SSRs. Integrating the two libraries allowed the development of 1,129 EST–SSR markers, and 311 polymorphic loci were integrated into our interspecific BC₁ genetic linkage map. The mapping results showed that the distribution of EST–SSRs on sub-genomes and chromosomes was uneven; however, the distribution of the mapped G. barbadense EST–SSRs on homologous chromosomes was similar, with the exception of Chr05 versus Chr19 and Chr12 versus Chr26. This study provided new EST–SSR markers that will facilitate studies on cotton genetics and breeding.     

Correlated inheritance of fruit neck with fruit length and linkage relations with 10 other characteristics of cucumber

Summary The inheritance of cucumber fruit neck size and its linkage relationships with the fruit length and 10 other characteristics was investigated. Frequency distributions and the means of generations indicate intermediate inheritance for neck size and fruit lenght. Correlation and regression analysis suggest strong linkage between fruit neck size and fruit length. Chi square analysis of the F₂ and BC generations indicate independent assortment of the fruit neck size and the following characters: bitterness, female sex expression, spine color, spine size, warted fruit, uniform color of immature fruit, mature fruit color, dull fruit skin, epidermal structure, and powdery mildew resistance.     

Genetic mapping and QTL analysis of fruit and flower related traits in cucumber (Cucumis sativus L.) using recombinant inbred lines

A set of 224 recombinant inbred lines (RILs) derived from a narrow cross between two fresh eaten types (S94 (Northern China type) × S06 (Northern European type)) (Cucumis sativus L.) was used to construct a genetic linkage map. With the RILs a 257-point genetic map was constructed including 206 SRAPs, 22 SSRs, 25 SCARs, 1 STS, and three economically important morphological markers (small spines (ss), uniform immature fruit color (u), dull fruit skin (D)). The seven linkage groups covered 1005.9 cM with a mean marker interval of 3.9 cM. The ss locus was linked to D and u, and they were all on Linkage group 6. The RIL map contained a total of 51 sequence-specific markers, which made possible the comparison of molecular linkage maps developed in different laboratories. Using the F_6:7 derived families, a total of 78 QTLs were detected with relatively high LOD scores (2.9–84.4) for nine fruit-related traits (fruit weight, length, and diameter, fruit flesh thickness, seed-cavity diameter, fruit-stalk length, fruit pedicel length, length/diameter and length/stalk ratio) and three flower-related traits (first flower node, first female flower node and female flower ratios). Several sequence-anchor markers (CSWCT25, CS30, CMBR41, CS08 etc.) were closely linked with some QTLs for fruit weight, fruit length, fruit flesh thickness and sex expression, which can be used for the future marker-assisted selection to improve the fruit traits in cucumber breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. X. J. Yuan and J. S. Pan contributed equally to this investigation.     

Molecular mapping and candidate gene analysis for fruit epidermal structure in cucumber

Firmness of skin is an important quality of processing cucumbers, which is a feature of the palisade epidermis in these types compared to flat epidermis in fresh cucumbers. This study was conducted to map the Pe (palisade epidermis) and analyse the candidate gene. Populations derived from the cross of two inbred lines, NCG122 with fruit flat epidermis and NCG121 with fruit palisade epidermis, were used to identify the inheritance of palisade epidermis and to map the gene involved in its development. The results showed that the palisade epidermis trait is controlled by a single gene, Pe, that is dominant over flat epidermis. Seven simple sequence repeat (SSR) and five insertion deletion (Indel) markers were identified to be linked to the Pe gene. It was mapped to cucumber chromosome 5 (Chr.5) between SSR14611 and Indelpe12 with genetic distances of 0.3 cM and 0.2 cM, respectively. The physical distance of the genomic region harbouring the gene was 227.5 kb with 26 predicted candidate genes. The accuracy of marker‐assisted selection using the molecular markers, Indelpe12 and SSR14611, was 68% and 88%, respectively.     

Inheritance and molecular tagging of MYMIV resistance gene in blackgram (Vigna mungo L. Hepper)

Yellow Mosaic disease (YMD) is one of the most destructive diseases of blackgram (Vigna mungo) causing heavy yield losses every year. Mungbean Yellow Mosaic India Virus (MYMIV) is one of the YMD causing begomoviruses prevalent in the major blackgram growing area (northern and central part) of India. Inheritance of MYMIV resistance gene was studied in blackgram using F₁, F₂ and F_2:3 derived from cross DPU 88-31(resistant)× AKU 9904 (susceptible). The results of genetic analysis showed that a single dominant gene controls the MYMIV resistance in blackgram genotype DPU 88-31. The F₂ population from the same cross was also used to tag and map the MYMIV resistance gene using SSR markers. Out of 361 markers, 31 were found polymorphic between the parents. However, marker CEDG 180 was found to be linked with resistance gene following the bulked segregant analysis. This marker was mapped in the F₂ mapping population of 168 individuals at a map distance of 12.9 cm. The validation of this marker in nine resistant and seven susceptible genotypes has suggested its use in marker assisted breeding for developing MYMIV resistant genotypes in blackgram.     

Development and validation of molecular markers closely linked to the wheat stripe rust resistance gene YrC591 for marker-assisted selection

Stripe rust resistance gene YrC591, present in wheat cultivar C591, is effective against currently important Puccinia striiformis Westend. f. sp. tritici isolates in China. An F_2:3 population (127 lines) was developed by crossing C591 with susceptible cultivar Taichung 29. Thirty four simple sequence repeat (SSR) and 155 sequence tagged site (STS) markers located on chromosome 7BL were used to perform bulk segregant analysis. Eight SSR markers, cfa2040, wmc273, wmc166, gwm984, barc32 wmc276, barc182 and gwm146, and 6 STS markers, mag1714, mag1757, mag1811, BE425120, BE471173 and BG607810, were polymorphic between the parents and contrasting resistant and susceptible DNA pools. F_2:3 lines were genotyped with these polymorphic markers. Linkage analysis indicated that YrC591 was flanked by Xmag1714 and Xbarc182 with genetic distances of 1.2 and 0.4 cM, respectively. In addition, validation of the SSR markers cfa2040, wmc273 and barc32, and STS markers mag1714 and BE425120 was carried out using wheat lines with C591 as a parent, indicating that these markers should be effective in tracing this gene in marker-assisted selection.     

Backcross introgression of plastomic factors controlling chilling tolerance into elite cucumber (Cucumis sativus L.) germplasm: early generation recovery of recurrent parent phenotype

Environmental stresses such as chilling temperatures can decrease germination, emergence, flower and fruit development, marketable yield, and postharvest fruit storage longevity in cucumber (Cucumis sativus L.). While response to chilling injury in cucumber is controlled by simple plastidic (maternal) and nuclear (paternal) factors, no chilling tolerant U.S. processing varieties are commercially available. Furthermore, even though three single nucleotide polymorphic sites have been identified as plastid components associated with chilling tolerance in cucumber, it is not known how these factors interact with nuclear factors controlling economically important traits. Therefore, an experiment was designed to evaluate the rate of recovery of the chilling susceptible (cytoplasm) genotype during introgression backcrossing (IB), where it was used as a recurrent parent after the initial mating to a line possessing chilling tolerant cytoplasm (donor parent). Phenotypic yield and quality trait data were collected on processing type backcross progeny (BC_1–5 and BC₂S₃) derived from an initial ‘Chipper’ (tolerant) × line M 29 (susceptible) mating, and rate of progression to the recurrent parent was determined by simple sequence repeat marker and morphological trait analyses. Substantial degrees of the recurrent parent phenotype and nuclear genome were recovered by the BC₂ generation (P = 0.001), with nearly complete recovery of recurrent parental traits and its nuclear genome occurring by the BC₃. General combining ability (GCA) of derived BC₂S₃ lines was significant for yield, yield/plant, length (L), diameter (D), and L:D ratios. The BC₂S₃ line GCA and rate of progression towards the recurrent parent for economically important traits suggests that elite chilling tolerant cucumber germplasm can be developed rapidly through IB and marker genotyping.     

Genetic mapping of gummy stem blight (Didymella bryoniae) resistance genes in Cucumis sativus-hystrix introgression lines

Gummy stem blight (GSB, Didymella bryoniae (Auersw.) Rehm) is a devastating disease occurring worldwide in cucumber (Cucumis sativus L.) production and causing considerable yield loss. No commercially available cultivars are resistant to GSB. By screening 52 introgression lines (ILs) derived from the cross of C. hystrix × C. sativus and eight cucumber cultivar/lines through a whole plant assay, three ILs (HH1-8-1-2, HH1-8-5, HH1-8-1-16) were identified as GSB resistant lines. Six common introgression regions in these three ILs were on Chromosomes 1, 4, and 6. To further map the resistance in the ILs, three mapping populations (2009F₂, 2009F₂′ and 2010F₂) from a cross between resistant IL HH1-8-1-2 and susceptible 8419 were constructed and used for QTL mapping with SSR markers. Two quantitative trait loci (QTLs) were identified; one on Chromosome 4 and the other on Chromosome 6. The interval for Chromosome 4 QTL is 12 cM spanning 3.569 Mbp, and the interval for Chromosome 6 QTL is 11 cM covering 1.299 Mbp. The mapped QTLs provide a foundation for map-based cloning of the genes and establishing an understanding of the associated mechanisms underlying GSB resistance in cucumber.     

Genetic mapping of QTLs for horticulture traits in a F2-3 population of bitter gourd (Momordica charantia L.)

An extensive genetic linkage map was constructed for bitter gourd (Momordica charantia L.) via the study of F₂ progenies derived from two cultivated inbred lines (gynoecia Z-1-4 and 189-4-1). The map included 194 loci on 11 chromosomes consisting of 26 EST-SSR loci, 28 SSR loci, 124 AFLP loci, and 16 SRAP loci. This map covered 1005.9 cM with 12 linkage groups. A total of 43 quantitative trait loci (QTLs), with a single QTL associated with 5.1–33.1 % phenotypic variance, were identified on nine chromosomes for 13 horticulture traits by analyzing the F_2-3 families and the genetic linkage map. The 13 horticulture traits which were investigated in three environments included female flower ratios (FFR), first female flower node (FFFN), fruit length, fruit diameter, flesh thickness, fruit shape, fruit pedicel length, fruit length pedicel ratios, fruit weight (FW), fruit numbers per plant (FPP), yield per plant (YPP), stem diameter (SD), and internodes length (IL). One QTL cluster region was detected on Lg-5 which contained the most important QTLs for YPP, FPP, FFFN, FFR, and FW with high contributions to phenotypic variance (5.8–25.4 %).     

QTL mapping for the tocopherols at milk stage of kernel development in sweet corn

Tocopherols have several beneficial effects in plants, and are indispensable micronutrients for humans. Sweet corn is a major source of tocopherols in high concentrations. In this investigation, tocopherol compounds in sweet corn were analyzed by high performance liquid chromatography. To detect quantitative trait loci (QTL) controlling accumulation of tocopherols at the milk stage in sweet corn, a F₂ population consisting of 229 F_2:3 lines was created from the cross between a high-total tocopherols line (A6) and a low-total tocopherols line (A57). A genetic map was constructed using 136 polymorphic molecular markers including one gene-targeted marker based on the tocopherol biosynthesis pathway (HPPD). Eleven putative QTLs for tocopherol content and composition were detected by composite interval mapping and located on Chr. 1, Chr. 2, Chr. 5, Chr. 6 and Chr. 10. Phenotypic variance explained by each QTL ranged from 4.74 to 41.16 %. Eight mapped QTLs were co-localized, suggesting that the same QTL affected the amounts of more than one tocopherol compound. One candidate gene-targeted marker (HPPD) showed co-localization with the major QTL for γ-tocopherol and total tocopherols. Only one interval (umc1177–bnlg1429) on chromosome one exhibited a QTL for α, δ, γ, and total tocopherols with high LOD and R² values. The primary conclusion of this work is that two major QTLs located on Chr. 1 and Chr. 5 can be used for improvement of sweet corn nutrition quality by marker-assisted selection.     

Molecular mapping of QTLs for resistance to Fusarium wilt (race 1) and Ascochyta blight in chickpea (Cicer arietinum L.)

Fusarium wilt (FW) and Ascochyta blight (AB) are two important diseases of chickpea which cause 100 % yield losses under favorable conditions. With an objective to validate and/or to identify novel quantitative trait loci (QTLs) for resistance to race 1 of FW caused by Fusarium oxysporum f. sp. ciceris and AB caused by Ascochyta rabiei in chickpea, two new mapping populations (F_2:3) namely ‘C 214’ (FW susceptible) × ‘WR 315’ (FW resistant) and ‘C 214’ (AB susceptible) × ‘ILC 3279’ (AB resistant) were developed. After screening 371 SSR markers on parental lines and genotyping the mapping populations with polymorphic markers, two new genetic maps comprising 57 (C 214 × WR 315) and 58 (C 214 × ILC 3279) loci were developed. Analysis of genotyping data together with phenotyping data collected on mapping population for resistance to FW in field conditions identified two novel QTLs which explained 10.4–18.8 % of phenotypic variation. Similarly, analysis of phenotyping data for resistance to seedling resistance and adult plant resistance for AB under controlled and field conditions together with genotyping data identified a total of 6 QTLs explaining up to 31.9 % of phenotypic variation. One major QTL, explaining 31.9 % phenotypic variation for AB resistance was identified in both field and controlled conditions and was also reported from different resistant lines in many earlier studies. This major QTL for AB resistance and two novel QTLs identified for FW resistance are the most promising QTLs for molecular breeding separately or pyramiding for resistance to FW and AB for chickpea improvement.     

Assessment of wheat variety stability using SSR markers

In International Union for the Protection of New Varieties of Plants member countries, assessment of wheat variety stability is a statutory requirement before new varieties are registered and plant breeders’ rights are granted. However, it is impossible to test the stability of varieties accurately and quickly, or to assess the stability of a large number of varieties in a short time based on the morphological traits defined by the national standard. The objective of this study was to establish method and procedure of wheat variety stability assessment using SSR markers as a replacement for morphological observations. In preliminary study, the methods to identify non-homozygous SSR loci and calculate the homozygous SSR loci ratio (SSR–HLR) of wheat varieties were established. On this basis, the genotypes at 347 molecular markers loci of 20 advanced lines demonstrated that SSR–HLRs were 84.7–94.8 % in the F₄ lines, 96.1–99.4 % in the F₅ lines, and ≥98 % in the F₆ lines, respectively. Eighty of 347 markers with good polymorphism, stable PCR amplification, and high resolving power of genotyping were recommended to detect SSR–HLR for 633 wheat regional trial varieties. Comparing morphological observation, the varieties with SSR–HLR >95 % were deemed stable; the varieties with SSR–HLR <91 % were deemed unstable; the varieties with SSR–HLR ranging from 91 to 95 % were required field identification for stability assessment. Based on the relationship between the homozygous SSR loci ratio and wheat variety stability, procedures for the assessment of wheat variety stability using SSR markers were established.     

Molecular mapping of the blast resistance gene Pi49 in the durably resistant rice cultivar Mowanggu

Rice blast, caused by the fungus Magnaporthe oryzae, is the most devastating fungal disease of rice. Mowanggu, a local japonica cultivar in Yunnan Province, China, confers broad-spectrum resistance to this pathogen. To identify the resistance gene(s) in Mowanggu, we obtained an F₂ population and 280 F₈ recombinant inbred lines (RILs) from a cross between Mowanggu and CO39, a highly susceptible indica cultivar. A linkage map with 145 simple sequence repeat (SSR) and single feature polymorphism markers over 12 chromosomes was constructed using the 280 RILs. The resistance evaluation of the F₂ and F₈ populations in both the growth chamber and in a natural rice blast nursery showed that a single dominant gene controls blast resistance in Mowanggu. Moreover, nine quantitative trait loci, which were responsible for different partial resistance components, were mapped on chromosomes 2, 3, 6, 8, 9, and 12, making contributions to the phenotypic variation ranging from 3.03 to 6.18 %. The dominant resistance gene, designated Pi49, was mapped on chromosome 11 with genetic distance of 1.01 and 1.89 cM from SSR markers K10 and K134, respectively. The physical distance between K10 and K134 is about 181 kb in the Nipponbare genome. The Pi49 gene accounted for the major phenotypic variation of disease severity in the growth chamber (where plants were inoculated with single blast isolates) and also accounted for most of the phenotypic variance of disease severity, lesion number, diseased leaf area, and lesion size in the blast nursery. Our study not only identified tightly linked markers for introgression of Pi49 into elite rice cultivars via marker-aided selection but also provides a starting point for map-based cloning of the new resistance gene.     

Genome-wide InDel marker system for application in rice breeding and mapping studies

In order to widen the genetic basis for local rice breeding programs, a set of 506 PCR-based novel insertion/deletion (InDel) markers were designed. The markers have size differences larger than 30 base-pairs and were generated by published genome sequences of rice (Oryza sativa L.) indica and japonica subspecies. Among the 506 InDel markers, 133 markers are polymorphic between the selected parents Taiken 2 and Taichung Sen 10, which are elite Taiwanese japonica and indica varieties, respectively. The InDel to amplicon size ratios, based on the japonica template, were in the range of 8.8–45.6 %, with an average of 21.6 %. As a result, the PCR product can be efficiently separated and easily scored by running 30 min of non-submerging electrophoresis on 2–3 % agarose gels. Commonly observed non-parental bands originated from heteroduplex are considered beneficial in distinguishing homo- and hetero-zygotes. A linkage map constructed solely on these markers arranged in 12 linkage groups and extended over 1,413.9 cM with an average distance of 9.8 cM between markers, based on a segregating population of 286 F₂ individuals derived from a cross between Taiken 2 and Taichung Sen 10. The result indicated that InDel markers with moderate size differences were abundant and uniformly distributed across the whole genome of rice. Due to their simplicity in design and robustness in genotyping, these InDel markers have been routinely used in our quantitative trait loci mapping studies and marker-assisted selection programs for rice. We suggest that InDel markers with moderate size differences could be a valuable alternative in addition to the widely used SSR and SNP markers for rice breeding, and particularly suitable for field laboratories at breeding stations and/or laboratories with limited resources.     

Construction of an integrated genetic map based on maternal and paternal lineages of tea (Camellia sinensis)

Genetic study on important traits of tea is difficult because of its self-incompatibility in nature. Moreover, development of a new variety usually needs more than 20 years, since it takes many years from seedling to matured plants for trait investigation. Genetic map is an essential tool for genetic study and breeding. In this study, we have developed an integrated genetic map of tea (Camellia sinensis) using a segregating F1 population derived from a cross between two commercial cultivars (‘TTES 19’ and ‘TTES 8’). A total of 574 polymorphic markers (including SSR, CAPS, STS, AFLP, ISSR and RAPD), 69 markers with highly significant levels of segregation distortion (P < 0.001) (12.0 %) were excluded from further analyses. Of the 505 mapped markers, there were 265 paternal markers (52.5 %), 163 maternal markers (32.3 %), 65 doubly heterozygous dominant markers (12.9 %), and 12 co-dominant markers (2.4 %). The co-dominant markers and doubly heterozygous dominant markers were used as bridge loci for the integration of the paternal and maternal maps. The integrated map comprised 367 linked markers, including 36 SSR, 3 CAPS, 1 STS, 250 AFLP, 13 ISSR and 64 RAPD that were assigned to 18 linkage groups. The linkage groups represented a total map length of 4482.9 cM with a map density of 12.2 cM. This genetic map has the highest genetic coverage so far, which could be applied to comparative mapping, QTL mapping and marker assisted selection in the future.