Identification of novel tan spot resistance QTLs using an SSR-based linkage map of tetraploid wheat

Durum wheat (Triticum turgidum L. subsp. durum, 2n = 4x = 28, AABB) is an important cereal used for making pasta products. Compared with bread wheat, durum wheat receives less attention in genetic and genomic studies. In this research, a tetraploid wheat doubled haploid (DH) population derived from the cross between the durum wheat cultivar ‘Lebsock’ and the T. turgidum subsp. carthlicum (2n = 4x = 28, AABB) accession PI 94749 was developed. The population consisted of 146 lines and was used to construct linkage maps of all 14 chromosomes. The maps consisted of 280 SSR markers and spanned 2,034.1 cM with an average density of one marker per 7.2 cM. The DH population and the whole genome linkage maps were then used to identify QTLs associated with tan spot resistance. The DH population was inoculated separately with two Ptr ToxA-producing isolates (Pti2 and 86-124) representing races 1 and 2, respectively, of Pyrenophora tritici-repentis, and five resistance QTLs were detected on chromosome arms 3AS, 3BL, 5AL and 7BL. Together, the QTLs explained a total of 46 and 41% of the phenotypic variation for reaction to Pti2 and 86-124, respectively. The Tsn1-Ptr ToxA interaction was not a significant factor in tan spot development in this population, and none of the QTLs corresponded to previously identified loci known to confer insensitivity to host-selective toxins (HSTs) produced by P. tritici-repentis. This result, together with those of other similar studies, indicates that the wheat–P. tritici-repentis pathosystem involves more factors than currently published host-toxin interactions. The DH population and genetic maps reported here will be useful for genetic dissection of important agronomic traits as well as the identification and development of markers for marker-assisted selection (MAS).     

Construction of an intraspecific integrated linkage map of pepper using molecular markers and doubled-haploid progenies.

An integrated molecular linkage map of pepper (Capsicum annuum L.), including mainly RFLP and RAPD markers, has been constructed by alignment of three intraspecific linkage maps generated by segregating doubled-haploid progenies. A total of 85 markers covered approximately 820 cM in 14 linkage groups. Four linkage groups were assigned to 4 chromosomes. Two new genes of agronomic interest were located: L controlling hypersensitive resistance to TMV and up controlling the erect habit of the fruits. The C gene controlling the fruit pungency was more precisely located. This map is estimated to represent from 36 to 59% of the total pepper genome. An examination of segregation data has revealed several genomic regions with aberrant segregation ratios often favouring the agronomic big-fruited parents, particularly in crosses involving the exotic parent CM334, suggesting that these genome regions are subjected to selection during the process of doubled-haploid production. The suitability of doubled-haploid progenies for mapping projects and the differences observed between this intraspecific integrated map with earlier published interspecific pepper maps are discussed.     

An SSR-based linkage map of Capsicum annuum

There are five cultivated species of pepper, of which Capsicum annuum is the most widely cultivated as a vegetable or spice and the main experimental material of most pepper breeding programs. However, the number of simple sequence-repeat (SSR) markers known for C. annuum is limited. To develop SSR markers for Capsicum species, we constructed four SSR-enriched libraries from the genomic DNA of C.␣annuum, sequenced 1873 clones, and isolated 626 unique SSR clones. A higher percentage of these SSR markers were taken from dinucleotide motif libraries than from trinucleotide motif libraries. Primer pairs for the 626 SSR clones were synthesized and tested for polymorphisms; 594 amplified products were detected with the expected size. However, only 153 products were polymorphic between the parents of our mapping population. Using 106 highly reproducible pairs from the primer pairs, we constructed a linkage map of C. annuum in an intraspecific doubled haploid population (n=117) that contains nine previously reported SSRs as well as AFLP, CAPS, and RAPD markers and the trait of fruit pungency. The map contains 374 markers, including 106 new SSR markers distributed across all 13 linkage groups, and covers 1042 cM. The polymorphism information content (PIC) of these new SSR markers was calculated using 14 lines of Capsicum species. The average number of alleles per locus was 2.9 and the average PIC value was 0.46, even within C. annuum. The SSR markers developed in this study will be useful for mapping and marker-assisted selection in pepper breeding, and the linkage map provides a reference genetic map for Capsicum species.     

Molecular linkage map for an intraspecific recombinant inbred population of durum wheat (Triticum turgidum L. var. durum)

Durum wheat (Triticum turgidum L. var. durum) is an economically and nutritionally important cereal crop in the Mediterranean region. To further our understanding of durum genome organization we constructed a durum linkage map using restriction fragment length polymorphisms (RFLPs), simple sequence repeats (SSRs) known as Gatersleben wheat microsatellites (GWMs), amplified fragment length polymorphisms (AFLPs), and seed storage proteins (SSPs: gliadins and glutenins). A population of 110 F₉ recombinant inbred lines (RILs) was derived from an intraspecific cross between two durum cultivars, Jennah Khetifa and Cham 1. The two parents exhibit contrasting traits for resistance to biotic and abiotic stresses and for grain quality. In total, 306 markers have been placed on the linkage map – 138 RFLPs, 26 SSRs, 134 AFLPs, five SSPs, and three known genes (one pyruvate decarboxylase and two lipoxygenases). The map is 3598 cM long, with an average distance between markers of 11.8 cM, and 12.1% of the markers deviated significantly from the expected Mendelian ratio 1:1. The molecular markers were evenly distributed between the A and B genomes. The chromosome with the most markers is 1B (41 markers), followed by 3B and 7B, with 25 markers each. The chromosomes with the fewest markers are 2A (11 markers), 5A (12 markers), and 4B (15 markers). In general, there is a good agreement between the map obtained and the Triticeae linkage consensus maps. This intraspecific map provides a useful tool for marker-assisted selection and map-based breeding for resistance to biotic and abiotic stresses and for improvement of grain quality. Received: 14 February 2000 / Accepted: 28 April 2000     

Construction of a genetic linkage map of the model legume Lotus japonicus using an intraspecific F2 population.

Among leguminous plants, the model legume Lotus japonicus (Regel) Larsen has many biological and genetic advantages. We have developed a genetic linkage map of L. japonicus based on amplified fragment length polymorphism (AFLP), simple sequence repeat polymorphism (SSRP) and derived cleaved amplified polymorphic sequence (dCAPS). The F2 mapping population used was derived from a cross between two L. japonicus accessions Gifu B-129 and Miyakojima MG-20. These parental accessions showed remarkable cytological differences, particularly with respect to size and morphology of chromosomes 1 and 2. Using fluorescence in situ hybridization (FISH) with BAC clones from Gifu B-129 and TAC (Transformation-competent Artificial Chromosome) clones from Miyakojima MG-20, a reciprocal translocation was found to be responsible for the cytological differences between chromosomes 1 and 2. The borders of the translocations were identified by FISH and by alignment toward the L. filicaulis x L. japonicus Gifu B-129 linkage map. The markers from the main translocated region were located on linkage groups 1 and 2 of the two accessions, Gifu B-129 and Miyakojima MG-20, respectively. The framework of the linkage map was constructed based on codominant markers, and then dominant markers were integrated separately in each linkage group of the parents. The resulting linkage groups correspond to the six pairs of chromosomes of L. japonicus and consist of 287 markers with 487.3 cM length in Gifu B-129 and 277 markers with 481.6 cM length in Miyakojima MG-20. The map and marker information is available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Towards an integrated linkage map of common bean 2. Development of an RFLP-based linkage map

Summary A restriction fragment length polymorphism (RFLP)-based linkage map for common bean (Phaseolus vulgaris L.) covering 827 centiMorgans (cM) was developed based on a F₂ mapping population derived from a cross between BAT93 and Jalo EEP558. The parental genotypes were chosen because they exhibited differences in evolutionary origin, allozymes, phaseolin type, and for several agronomic traits. The segregation of 152 markers was analyzed, including 115 RFLP loci, 7 isozyme loci, 8 random amplified polymorphic DNA (RAPD) marker loci, and 19 loci corresponding to 15 clones of known genes, 1 virus resistance gene, 1 flower color gene, and 1 seed color pattern gene. Using MAPMAKER and LINKAGE-1, we were able to assign 143 markers to 15 linkage groups, whereas 9 markers remained unassigned. The average interval between markers was 6.5 cM; only one interval was larger than 30 cM. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios (121 or 31) and mapped into four clusters. Probes of known genes belonged to three categories: seed proteins, pathogen response genes, and Rhizobium response genes. Within each category, sequences homologous to the various probes were unlinked. The I gene for bean common mosaic virus resistance is the first disease resistance gene to be located on the common bean genetic linkage map.     

Development of a new SSR-based linkage map in apricot and analysis of synteny with existing Prunus maps

Linkage maps of the apricot accessions ‘Lito’ and ‘BO 81604311’ were constructed using a total of 185 simple sequence repeat (SSR) markers sampled from those isolated in peach, almond, apricot and cherry; 74 were derived from a new apricot genomic library enriched for AG/CT microsatellite repeats (UDAp series), and in total, 98 had never been mapped in Prunus before. Eight linkage groups putatively corresponding to the eight haploid apricot chromosomes were identified for each parent. The two maps were 504 and 620 cM long, respectively, with 96 anchor markers showing a complete co-linearity between the two genomes. As few as three gaps larger than 15 cM were present in ‘Lito’ and six in the male parent; the maps align well with all the available SSR-based Prunus maps through the many common anchor loci. Only occasionally inverted positions between adjacent markers were found, and this can be explained by the small size of cross populations analysed in these Prunus maps and in those reported in literature. The newly developed apricot SSRs will help saturating the existing Prunus maps and will extend the choice of markers in the development of genetic maps for new breeding populations.     

A high-density rice genetic linkage map with 2275 markers using a single F2 population.

A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using > 130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.     

Towards an integrated linkage map of common bean

Summary Two genomic libraries were established to provide markers to develop an integrated map combining molecular markers and genes for qualitative and quantitative morpho-agronomic traits in common bean. Contrasting characteristics were observed for the two libraries. While 89% of the PstI clones were classified as single-copy sequences, only 21% of the EcoRIBamHI clones belonged in that category. Clones of these two libraries were hybridized against genomic DNA of nine genotypes chosen according to their divergent evolutionary origin and contrasting agronomic traits. Eight restriction enzymes were used in this study. PstI clones revealed 80–90% polymorphism between the Andean and Middle American gene pools and 50–60% polymorphism within these gene pools. However, under the same conditions only 30% of the EcoRI-BamHI clones showed polymorphism between the Middle American and Andean gene pools. Hybridization with PstI clones to EcoRI-, EcoRV-, or HindIII-digested genomic DNA resulted in a cumulative frequency of polymorphism of approximately 80%. Hybridizations to BamHI-, HaeIII-, HinfI-, PstI-, and XbaI-digested genomic DNA detected no additional polymorphisms not revealed by the former three enzymes. In the PstI library, a positive correlation was observed between the average size of hybridizing restriction fragments and the frequency of polymorphism detected by each restriction enzyme. This relationship is consistent with the higher proportion of insertion/deletion events compared with the frequency of nucleotide substitutions observed in that library.     

Genetic linkage map in sour cherry using RFLP markers

 Restriction fragment length polymorphism (RFLP) linkage maps of two tetraploid sour cherry (Prunus cerasus L., 2n=4x=32) cultivars, Rheinische Schattenmorelle (RS) and Erdi Botermo (EB), were constructed from 86 progeny from the cross RS×EB. The RS linkage map consists of 126 single-dose restriction fragment (SDRF, Wu et al. 1992) markers assigned to 19 linkage groups covering 461.6 cM. The EB linkage map has 95 SDRF markers assigned to 16 linkage groups covering 279.2 cM. Fifty three markers mapped in both parents were used as bridges between both maps and 13 sets of homologous linkage groups were identified. Homoeologous relationships among the sour cherry linkage groups could not be determined because only 15 probes identified duplicate loci. Fifty nine of the markers on the linkage maps were detected with probes used in other Prunus genetic linkage maps. Four of the sour cherry linkage groups may be homologous with four of the eight genetic linkage groups identified in peach and almond. Twenty one fragments expected to segregate in a 1 : 1 ratio segregated in a 2 : 1 ratio. Three of these fragments were used in the final map construction because they all mapped to the same linkage group. Six fragments exhibited segregation consistent with the expectations of intergenomic pairing and/or recombination. Received: 1 April 1998 / Accepted: 9 June 1998     

Enrichment of an intraspecific genetic map of upland cotton by developing markers using parental RAD sequencing

RAD sequencing was performed using DH962 and Jimian5 as upland cotton mapping parents. Sequencing data for DH962 and Jimian5 were assembled into the genome sequences of ≈55.27 and ≈57.06 Mb, respectively. Analysing genome sequences of the two parents, 1,323 SSR, 3,838 insertion/deletion (InDel), and 9,366 single-nucleotide polymorphism (SNP) primer pairs were developed. All of the SSRs, 121 InDels, 441 SNPs, and other 6,747 primer pairs were screened in the two parents, and a total of 535 new polymorphic loci were identified. A genetic map including 1,013 loci was constructed using these results and 506 loci previously published for this population. Twenty-seven new QTLs for yield and fibre quality were identified, indicating that the efficiency of QTL detection was greatly improved by the increase in map density. Comparative genomics showed there to be considerable homology and collinearity between the A_T and A₂ genomes and between the D_T and D₅ genomes, although there were a few exchanges and introgressions among the chromosomes of the A₂ genome. Here, the development of markers using parental RAD sequencing was effective, and a high-density intraspecific genetic map was constructed. This map can be used for molecular marker-assisted selection in cotton.     

Integrating the porcine physical and linkage map using cosmid-derived markers

An essential part in the development of informative linkage maps is to include genetic markers that have been anchored by physical mapping. Here a set of 18 porcine cosmid-derived genetic markers are reported that have been mapped by linkge analysis, and that also have been physically localized by fluorescence in situ hybridization (FISH). Three different strategies were used to establish polymorphic markers from the cosmid clones. Firstly, dinucleotide microsatellite loci were derived by sequencing cosmid subclones containing (CA), repeats. Secondly, variable SINE 3′ poly(A) tracts (SINEVA) were identified by direct SINE-PCR amplification of cosmid clones. Thirdly, the cosmids were used in Southern blot hybridization to detect restriction fragment length polymorphisms (RFLPs). Compared with the most recent consensus compilation of the porcine gene map, the present assignment of markers to chromosomes Zp, 3, 4, 10, 12q, and 16 represents the first loci mapped to these chromosomes, for which linkage as well as in situ data are now available.     

Genetics of screwworm: new genetic markers and preliminary linkage map

Eight new genetic markers for Cochliomyia hominivorax (Diptera: Calliphoridae), the screwworm, are characterized. The markers include three eye mutants, lemon-eye (le), cherry-eye (ch), and red-eye (re); one wing mutant, curly-wing (cw); and four allozyme markers, amylase (Amy-1), glycerol-3-phosphate dehydrogenase (Gpd), phosphoglucomutase (Pgm), and octanol dehydrogenase (Odh). The markers are associated into four linkage groups. Radiation-induced translocations were used to correlate the linkage groups with their respective chromosomes. A preliminary genetic linkage map with these and three previously characterized loci is presented.     

Development of simple sequence repeat (SSR) markers and construction of an SSR-based linkage map in Italian ryegrass (Lolium multiflorum Lam.)

In order to develop simple sequence repeat (SSR) markers in Italian ryegrass, we constructed a genomic library enriched for (CA)n-containing SSR repeats. A total of 1,544 clones were sequenced, of which 1,044 (67.6%) contained SSR motifs, and 395 unique clones were chosen for primer design. Three hundred and fifty-seven of these clones amplified products of the expected size in both parents of a two-way pseudo-testcross F₁ mapping population, and 260 primer pairs detected genetic polymorphism in the F₁ population. Genetic loci detected by a total of 218 primer pairs were assigned to locations on seven linkage groups, representing the seven chromosomes of the haploid Italian ryegrass karyotype. The SSR markers covered 887.8 cM of the female map and 795.8 cM of the male map. The average distance between two flanking SSR markers was 3.2 cM. The SSR markers developed in this study will be useful in cultivar discrimination, linkage analysis, and marker-assisted selection of Italian ryegrass and closely related species.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

SSR-based genetic linkage map construction in pistachio using an interspecific F<Subscript>1</Subscript> population and QTL analysis for leaf and shoot traits

Pistachio is one of the most commercially important nut trees in the world. To characterize the genetic controls of horticultural traits and facilitate marker-assisted breeding in pistachio, we constructed an SSR-based linkage map using an interspecific F₁ population derived from a cross between the cultivar “Siirt” (Pistacia vera L.) and the monoecious Pa-18 genotype of Pistacia atlantica Desf. This population was also used for the first QTL analysis in pistachio on leaf and shoot characters. In total, 1312 SSR primers were screened, and 388 loci were successfully integrated into parental linkage maps. The Siirt maternal map contained 306 markers, while the “Pa-18” paternal map included 285 markers along the 15 linkage groups. The Siirt map spanned 1410.4 cM, with an average marker distance of 4.6 cM; the Pa-18 map covered 1362.5 cM with an average marker distance of 4.8 cM. Phenotypic data were collected during the growing seasons of 2015 and 2016 for four traits: leaf length (LL), leaf width (LW), leaf length/leaf width ratio (LWR), number of leaflet pairs (NLL), and young shoot color (YSC). A total of 17 QTLs were identified in the parental maps. Four QTLs for LL and LW were located on LG2 and LG4, while four QTLs for LWR ratio on LG13 and LG14, two QTLs for NLL and two QTLs for YSC were on LG7 and LG9, respectively, with similar positions in both parental maps. The SSR markers, linkage maps, and QTLs reported here will provide a valuable resource for future molecular and genetic studies in pistachio.     

A genetic linkage map of durum wheat

 A genetic linkage map of tetraploid wheat [Triticum turgidum (L.) Thell.] was constructed using segregation data from a population of 65 recombinant inbred lines (RILs) derived from a cross between the durum wheat cultivar Messapia and accession MG4343 of T. turgidum (L.) Thell. ssp dicoccoides (Korn.) Thell. A total of 259 loci were analysed, including 244 restriction fragment length polymorphisms (RFLPs), one PCR (polymerase chain reaction) marker (a sequence coding for a LMW (low-molecular-weight) glutenin subunit gene located at the Glu-B3 locus), seven biochemical (six seed-storage protein loci and one isozyme locus) and seven morphological markers. A total of 213 loci were mapped at a LOD≥3 on all 14 chromosomes of the A and B genomes. The total length of the map is 1352 cM and the average distance between adjacent markers is 6.3 cM. Forty six loci could not be mapped at a LOD≥3. A fraction (18.6%) of the markers deviated significantly from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on chromosomes 1B, 3AL, 4AL, 6AL and 7AL. The durum wheat map was compared with the published maps of bread wheat using several common RFLP markers and general features are discussed. The markers detected the known structural rearrangements involving chromosomes 4A, 5A and 7B as well as the translocation between 2B-6B, but not the deletion on 2BS. This map provides a useful tool for analysing and breeding economically important quantitative traits and for marker-assisted selection, as well as for studies of genome organisation in small grain cereal species. Received: 5 January 1998 / Accepted: 31 March 1998     

Distribution of DArT, AFLP, and SSR markers in a genetic linkage map of a doubled-haploid hexaploid wheat population.

A genetic linkage mapping study was conducted in 93 doubled-haploid lines derived from a cross between Triticum aestivum L. em. Thell 'Arina' and a Norwegian spring wheat breeding line, NK93604, using diversity arrays technology (DArT), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR) markers. The objective of this study was to understand the distribution, redundancy, and segregation distortion of DArT markers in comparison with AFLP and SSR markers. The map contains a total of 624 markers with 189 DArTs, 165 AFLPs and 270 SSRs, and spans 2595.5 cM. All 3 marker types showed significant (p < 0.01) segregation distortion, but it was higher for AFLPs (24.2%) and SSRs (22.6%) than for DArTs (13.8%). The overall segregation distortion was 20.4%. DArTs showed the highest frequency of clustering (27.0%) at < 0.5 cM intervals between consecutive markers, which is 3 and 15 times higher than SSRs (8.9%) and AFLPs (1.8%), respectively. This high proportion of clustering of DArT markers may be indicative of gene-rich regions and (or) the result of inclusion of redundant clones in the genomic representations, which was supported by the presence of very high correlation coefficients (r > 0.98) and multicollinearity among the clustered markers. The present study is the first to compare the utility of DArT with AFLP and SSR markers, and the present map has been successfully used to identify novel QTLs for resistance to Fusarium head blight and powdery mildew and for anther extrusion, leaf segment incubation, and latency.     

Genetic linkage map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers

 The potential of PCR-based markers for construction of a genetic linkage map in Einkorn wheat was investigated. From a comparison of polymorphisms between two Einkorn wheats, Triticum monococcum (Mn) and T. boeoticum (Bt), we obtained 49 polymorphic bands produced by 33 primers for inter-simple sequence repeat (ISSR) and 36 polymorphic bands shown by 25 combinations of random amplified polymorphic DNA (RAPD) primers for mapping in 66 individuals in the F₂ population. Although 44 ISSR fragments and 29 RAPD fragments statistically showed a 3 : 1 segregation ratio in the F₂ population, only 9 markers each of the ISSR and RAPD bands were able to be mapped on the RFLP linkage map of Einkorn wheat. ISSR markers were distributed throughout the chromosomes. The mapped positions of the ISSR markers seemed to be similar to those obtained by the RFLP markers. On the other hand, 4 of the 9 RAPD markers could map the RFLP marker-poor region on the short arm of 3A^m, suggesting a potential to map novel regions containing repetitive sequences. Comparisons of the genetic linkage map of Einkorn wheat to the linkage map and cytological map of common wheat revealed that the marker orders between the two maps of Einkorn wheat and common wheat coincided except for 4A, which harbors chromosome rearrangements specific for polyploid wheats, indicating a conservatism between the two genomes. Recombinations in Einkorn wheat chromosomes took place more frequently around the centromere and less at the distal part of chromosomes in comparison to those in common wheat. Nevertheless, recombinations even in Einkorn wheat chromosomes were strongly suppressed around the centromere. In fact, the markers located within 1 cM of the centromere were located almost in the central part of the chromosome arm. Received: 7 June 1997 / Accepted: 17 June 1997     

A genetic linkage map of azuki bean constructed with molecular and morphological markers using an interspecific population (Vigna angularis x V. nakashimae)

A genetic linkage map of azuki bean (Vigna angularis) was constructed with molecular and morphological markers using an F₂ population of an interspecific cross between azuki bean and its wild relative, V. nakashimae. In total, 132 markers (108 RAPD, 19 RFLP and five morphological markers) were mapped in 14 linkage groups covering 1250 cM; ten remained unlinked. The clusters of markers showing distorted segregation were found in linkage groups 2, 8 and 12. By comparing the azuki linkage map with those of mungbean and cowpea, using 20 RFLP common markers, some sets of the markers were found to belong to the same linkage groups of the respective maps, indicating that these linkage blocks are conserved among the three Vigna species. This map provides a tool for markerassisted selection and for studies of genome organization in Vigna species.