Chromosome rearrangements during domestication of cucumber as revealed by high-density genetic mapping and draft genome assembly

Cucumber, Cucumis sativus L. is the only taxon with 2n = 2x = 14 chromosomes in the genus Cucumis. It consists of two cross‐compatible botanical varieties: the cultivated C. sativus var. sativus and the wild C. sativus var. hardwickii. There is no consensus on the evolutionary relationship between the two taxa. Whole‐genome sequencing of the cucumber genome provides a new opportunity to advance our understanding of chromosome evolution and the domestication history of cucumber. In this study, a high‐density genetic map for cultivated cucumber was developed that contained 735 marker loci in seven linkage groups spanning 707.8 cM. Integration of genetic and physical maps resulted in a chromosome‐level draft genome assembly comprising 193 Mbp, or 53% of the 367 Mbp cucumber genome. Strategically selected markers from the genetic map and draft genome assembly were employed to screen for fosmid clones for use as probes in comparative fluorescence in situ hybridization analysis of pachytene chromosomes to investigate genetic differentiation between wild and cultivated cucumbers. Significant differences in the amount and distribution of heterochromatins, as well as chromosomal rearrangements, were uncovered between the two taxa. In particular, six inversions, five paracentric and one pericentric, were revealed in chromosomes 4, 5 and 7. Comparison of the order of fosmid loci on chromosome 7 of cultivated and wild cucumbers, and the syntenic melon chromosome I suggested that the paracentric inversion in this chromosome occurred during domestication of cucumber. The results support the sub‐species status of these two cucumber taxa, and suggest that C. sativus var. hardwickii is the progenitor of cultivated cucumber.     

Use of VeraCode 384-plex assays for watermelon diversity analysis and integrated genetic map of watermelon with single nucleotide polymorphisms and simple sequence repeats

Watermelon (Citrullus lanatus var. lanatus) is one of the most important vegetable crops in the world. Molecular markers have become the tools of choice for resolving watermelon taxonomic relationships and evolution. Increased numbers of single nucleotide polymorphism (SNP) markers together with simple sequence repeat (SSR) markers would be useful for phylogenetic analyses of germplasm accessions and for linkage mapping for marker-assisted breeding with quantitative trait loci and single genes. We aimed to construct a genetic map based on SNPs (generated by Illumina Veracode multiplex assays for genotyping) and SSR markers and evaluate relationships inferred from SNP genotypes between 130 watermelon accessions collected throughout the world. We incorporated 282 markers (232 SNPs and 50 SSRs) into the linkage map. The genetic map consisted of 11 linkage groups spanning 924.72 cM with an average distance of 3.28 cM between markers. Because all of the SNP-containing sequences were assembled with the whole-genome sequence draft for watermelon, chromosome numbers could be readily assigned for all the linkage groups. We found that 134 SNPs were polymorphic in 130 watermelon accessions chosen for diversity studies. The current 384-plex SNP set is a powerful tool for characterizing genetic relatedness and for developing medium-resolution genetic maps.     

Development and characterization of microsatellite markers in Cucumis

This study provides a set of useful SSR markers and describes their development, characterization and application for diversity studies.Sixty one Cucumis SSR markers were developed, most of them (46) from melon (Cucumis melo L.) genomic libraries. Forty of the markers (30 melon and 10 cucumber SSRs) were evaluated for length polymorphism in a sample of 13 melon genotypes and 11 cucumber (Cucumis sativus L.) genotypes. PCR-amplification revealed up to six size alleles among the melon genotypes and up to five alleles among the cucumber genotypes, with mean gene-diversity values of 0.52 and 0.28 for melon and cucumber, respectively. These differences are in accordance with the known narrower genetic background of the cucumber. SSR data were applied to phylogenetic analysis among the melon and cucumber genotypes. A clear distinction between the ’exotic’ groups and the sweet cultivated groups was demonstrated in melon. In cucumber, separation between the two sub-species, C.sativus var. sativus and C.sativus var. hardwickii,was obtained. Conservation of SSR loci between melon and cucumber was proven by sequence comparisons. Received: 17 April 2000 / Accepted: 16 May 2000     

A genetic linkage map for watermelon derived from a testcross population: ( Citrullus lanatus var. citroides x C. lanatus var. lanatus) x Citrullus colocynthis

A genetic linkage map was constructed for watermelon using a testcross population [Plant Accession Griffin 14113 (Citrullus lanatus var. citroides) 2 New Hampshire Midget (NHM; C. lanatus var. lanatus)] 2 U.S. Plant Introduction (PI) 386015 (Citrullus colocynthis). The map contains 141 randomly amplified polymorphic DNA (RAPD) markers produced by 78 primers, 27 inter-simple sequence repeat (ISSR) markers produced by 17 primers, and a sequence-characterized amplified region (SCAR) marker that was previously reported as linked (1.6 cM) to race-1 Fusarium wilt [incited by Fusarium oxysporum Schlechtend.:Fr. f. sp. niveum (E.F.Sm.) W.C. Synder &; H.N. Hans] resistance in watermelon. The map consists of 25 linkage groups. Among them are a large linkage group that contains 22 markers covering a mapping distance of 225.6 cM and six large groups each with 10-20 markers covering a mapping distance of 68.8 to 110.8 cM. There are five additional linkage groups consisting of 3-7 markers per group, each covering a mapping distance of 36.5 to 57.2 cM. The 13 remaining linkage groups are small, each consisting of 2-11 markers covering a mapping distance of 3.5-29.9 cM. The entire map covers a total distance of 1,166.2 cM with an average distance of 8.1 cM between two markers. This map is useful for the further development of markers linked to disease resistance and watermelon fruit qualities.     

A high resolution genetic map anchoring scaffolds of the sequenced watermelon genome

As part of our ongoing efforts to sequence and map the watermelon (Citrullus spp.) genome, we have constructed a high density genetic linkage map. The map positioned 234 watermelon genome sequence scaffolds (an average size of 1.41 Mb) that cover about 330 Mb and account for 93.5% of the 353 Mb of the assembled genomic sequences of the elite Chinese watermelon line 97103 (Citrullus lanatus var. lanatus). The genetic map was constructed using an F(8) population of 103 recombinant inbred lines (RILs). The RILs are derived from a cross between the line 97103 and the United States Plant Introduction (PI) 296341-FR (C. lanatus var. citroides) that contains resistance to fusarium wilt (races 0, 1, and 2). The genetic map consists of eleven linkage groups that include 698 simple sequence repeat (SSR), 219 insertion-deletion (InDel) and 36 structure variation (SV) markers and spans ～800 cM with a mean marker interval of 0.8 cM. Using fluorescent in situ hybridization (FISH) with 11 BACs that produced chromosome-specifc signals, we have depicted watermelon chromosomes that correspond to the eleven linkage groups constructed in this study. The high resolution genetic map developed here should be a useful platform for the assembly of the watermelon genome, for the development of sequence-based markers used in breeding programs, and for the identification of genes associated with important agricultural traits.     

Development of genome-wide SSR markers in melon with their cross-species transferability analysis and utilization in genetic diversity study

Simple sequence repeats (SSRs) are one of the most informative and widely used molecular markers in plant research. The melon draft genome has provided a powerful tool for SSR marker development in this species in which there are still not enough SSR markers. We therefore developed genome-wide SSR markers from melon, which were used for genetic diversity analysis in melon accessions and comparative mapping with cucumber and watermelon. A total of 44,265 microsatellites from the melon genome were characterized, of which 28,570 SSR markers were developed. In silico PCR analysis with these SSR markers identified 4002 and 1085 with one amplicon in cucumber and watermelon genome, respectively. With these cross-species transferable melon SSR markers, the chromosome synteny between melon and cucumber as well as watermelon was established, which revealed complicated mosaic patterns of syntenic blocks among them. We experimentally validated 384 SSR markers, from which 42 highly informative SSR ones were selected for genetic diversity and population structure analysis among 118 melon accessions. The large number of melon SSR markers developed in this study provides a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection (MAS) in melon. Furthermore, the cross-species transferable SSR markers could also be useful in various molecular marker-related studies in other closely related species in Cucurbitaceae family in which draft genomes are not yet available.     

Detection and Location of Seed-Borne Inoculum of Didymella bryoniae and its Transmission in Seedlings of Cucumber and Pumpkin

Of the 91 tested cucurbit seed samples from thirteen countries nine from four countries were found to be infected with Didymella bryoniae. The pathogen is located on and in the seed coat including the perisperm and in the tissue of the cotyledons. Primary seedling infection occurred on the radicle, hypoctyl and cotyledons. Infection of the radicle generally caused pre-emergence tot while infection on the hypocotyl and cotyledons developed furthere inoculum for infection of the first true leaves and the stem. Experimentally, all the isolates of D. bryoniae could infect cucumber (Cucumis sativus), oriental melon (Cucumis melo var. makuwa), pumpkin (Cucurbita pepo) and watermelon (Citrullus vulgaris) at different growth stages; the susceptibility of cucumber and pumpkin was markedly influenced by previailing humid conditions.The blotter method was found more suitable for detection of seed-borne infection than the agar plate method.     

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2

Background

Integration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.).

Results

In this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed.

Conclusions

Recombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.     

Construction of High-Density Linkage Maps of Populus deltoides × P. simonii Using Restriction-Site Associated DNA Sequencing

Although numerous linkage maps have been constructed in the genus Populus, they are typically sparse and thus have limited applications due to low throughput of traditional molecular markers. Restriction-site associated DNA sequencing (RADSeq) technology allows us to identify a large number of single nucleotide polymorphisms (SNP) across genomes of many individuals in a fast and cost-effective way, and makes it possible to construct high-density genetic linkage maps. We performed RADSeq for 299 progeny and their two parents in an F₁ hybrid population generated by crossing the female Populus deltoides ‘I-69’ and male Populus simonii ‘L3’. A total of 2,545 high quality SNP markers were obtained and two parent-specific linkage maps were constructed. The female genetic map contained 1601 SNPs and 20 linkage groups, spanning 4,249.12 cM of the genome with an average distance of 2.69 cM between adjacent markers, while the male map consisted of 940 SNPs and also 20 linkage groups with a total length of 3,816.24 cM and an average marker interval distance of 4.15 cM. Finally, our analysis revealed that synteny and collinearity are highly conserved between the parental linkage maps and the reference genome of P. trichocarpa. We demonstrated that RAD sequencing is a powerful technique capable of rapidly generating a large number of SNPs for constructing genetic maps in outbred forest trees. The high-quality linkage maps constructed here provided reliable genetic resources to facilitate locating quantitative trait loci (QTLs) that control growth and wood quality traits in the hybrid population.     

The First High-Density Genetic Map Construction in Tree Peony (Paeonia Sect. Moutan) using Genotyping by Specific-Locus Amplified Fragment Sequencing

Genetic linkage maps, permitting the elucidation of genome structure, are one of most powerful genomic tools to accelerate marker-assisted breeding. However, due to a lack of sufficient user-friendly molecular markers, no genetic linkage map has been developed for tree peonies (Paeonia Sect. Moutan), a group of important horticultural plants worldwide. Specific-locus amplified fragment sequencing (SLAF-seq) is a recent molecular marker development technology that enable the large-scale discovery and genotyping of sequence-based marker in genome-wide. In this study, we performed SLAF sequencing of an F1 population, derived from the cross P. ostti ‘FenDanBai’ × P. × suffruticosa ‘HongQiao’, to identify sufficient high-quality markers for the construction of high-density genetic linkage map in tree peonies. After SLAF sequencing, a total of 78 Gb sequencing data and 285,403,225 pair-end reads were generated. We detected 309,198 high-quality SLAFs from these data, of which 85,124 (27.5%) were polymorphic. Subsequently, 3518 of the polymorphic markers, which were successfully encoded in to Mendelian segregation types, and were in conformity with the criteria of high-quality markers, were defined as effective markers and used for genetic linkage mapping. Finally, we constructed an integrated genetic map, which comprised 1189 markers on the five linkage groups, and spanned 920.699 centiMorgans (cM) with an average inter-marker distance of 0.774 cM. There were 1115 ‘SNP-only’ markers, 18 ‘InDel-only’ markers, and 56 ‘SNP&InDel’ markers on the map. Among these markers, 450 (37.85%) showed significant segregation distortion (P < 0.05). In conclusion, this investigation reported the first large-scale marker development and high-density linkage map construction for tree peony. The results of this study will serve as a solid foundation not only for marker-assisted breeding, but also for genome sequence assembly for tree peony.     

Optimization of linkage mapping strategy and construction of a high-density American lotus linkage map

Background

Lotus is a diploid plant with agricultural, medicinal, and ecological significance. Genetic linkage maps are fundamental resources for genome and genetic study, and also provide molecular markers for breeding in agriculturally important species. Genotyping by sequencing revolutionized genetic mapping, the restriction-site associated DNA sequencing (RADseq) allowed rapid discovery of thousands of SNPs markers, and a crucial aspect of the sequence based mapping strategy is the reference sequences used for marker identification.

Results

We assessed the effectiveness of linkage mapping using three types of references for scoring markers: the unmasked genome, repeat masked genome, and gene models. Overall, the repeat masked genome produced the optimal genetic maps. A high-density genetic map of American lotus was constructed using an F₁ population derived from a cross between Nelumbo nucifera ‘China Antique’ and N. lutea ‘AL1’. A total of 4,098 RADseq markers were used to construct the American lotus ‘AL1’ genetic map, and 147 markers were used to construct the Chinese lotus ‘China Antique’ genetic map. The American lotus map has 9 linkage groups, and spans 494.3 cM, with an average distance of 0.7 cM between adjacent markers. The American lotus map was used to anchor scaffold sequences in the N. nucifera ‘China Antique’ draft genome. 3,603 RADseq markers anchored 234 individual scaffold sequences into 9 megascaffolds spanning 67% of the 804 Mb draft genome.

Conclusions

Among the unmasked genome, repeat masked genome and gene models, the optimal reference sequences to call RADseq markers for map construction is repeat masked genome. This high density genetic map is a valuable resource for genomic research and crop improvement in lotus.     

Genetic linkage map of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) and localization of a major QTL for powdery mildew resistance

Powdery mildew caused by Podosphaera xanthii has become a major problem in melon since it occurs all year round irrespective of the growing system. The TGR-1551 melon genotype was found to be resistant to several melon diseases, among them powdery mildew. However, the corresponding resistance genes have been never mapped. We constructed an integrated genetic linkage map using an F2 population derived from a cross between the multi-resistant genotype TGR-1551 and the susceptible Spanish cultivar ‘Bola de Oro’. The map spans 1,284.9 cM, with an average distance of 3.6 cM among markers, and consists of 354 loci (188 AFLP, 39 RAPD, 111 SSR, 14 SCAR/CAPS/dCAPS, and two phenotypic traits) distributed in 14 linkage groups. QTL analysis identified one major QTL (Pm-R) on LG V for resistance to races 1, 2, and 5 of powdery mildew. The PM4-CAPS marker is closely linked to the Pm-R QTL at a genetic distance of 1.9 cM, and the PM3-CAPS marker is located within the support interval of this QTL. These codominant markers, together with the map information reported here, could be used for melon breeding, and particularly for genotyping selection of resistance to powdery mildew in this vegetable crop species.     

A re-assigned American mink (Neovison vison) map optimal for genome-wide studies

Our previously published second generation genetic map for the American mink (Neovison vison) has been used and redesigned in its best for genome-wide studies with maximum of efficiency. A number of 114 selected markers, including 33 newly developed microsatellite markers from the CHORI-231 mink Bacterial Artificial Chromosome (BAC) library, have been genotyped in a two generation population composed of 1200 individuals. The outcome reassigns the position of some markers on the chromosomes and it produces a more reliable map with a convenient distance between markers. A total of 104 markers mapped to 14 linkage groups corresponding to the mink autosomes. Six markers are unlinked and four markers are allocated to the X chromosome by homology but no linkage was detected. The sex-average linkage map spans 1192 centiMorgans (cM) with an average intermarker distance of 11.4 cM and 1648 cM when the ends of the linkage groups and the autosomal unlinked markers are added. Sex-specific genetic linkage maps were also generated. The male sex-specific map had a total length of 1014.6 cM between the linked markers and an average inter-marker interval of 9.7 cM. The female map has a corresponding length of 1378.6 cM and an average inter-marker interval of 13.3 cM. The study is complemented with additional anchorage for most of the chromosomes of the map by BAC in situ hybridization with clones containing microsatellites strategically selected from the various parts of the genome. This map provides an improved tool for genetic mapping and comparative genomics in mink, also useful for the future assembly of the mink genome sequence when this will be taken forward.     

Construction of a high-density genetic map using specific length amplified fragment markers and identification of a quantitative trait locus for anthracnose resistance in walnut (Juglans regia L.)

Background

Walnut (Juglans regia, 2n = 32, approximately 606 Mb per 1C genome) is an economically important tree crop. Resistance to anthracnose, caused by Colletotrichum gloeosporioides, is a major objective of walnut genetic improvement in China. The recently developed specific length amplified fragment sequencing (SLAF-seq) is an efficient strategy that can obtain large numbers of markers with sufficient sequence information to construct high-density genetic maps and permits detection of quantitative trait loci (QTLs) for molecular breeding.

Results

SLAF-seq generated 161.64 M paired-end reads. 153,820 SLAF markers were obtained, of which 49,174 were polymorphic. 13,635 polymorphic markers were sorted into five segregation types and 2,577 markers of them were used to construct genetic linkage maps: 2,395 of these fell into 16 linkage groups (LGs) for the female map, 448 markers for the male map, and 2,577 markers for the integrated map. Taking into account the size of all LGs, the marker coverage was 2,664.36 cM for the female map, 1,305.58 cM for the male map, and 2,457.82 cM for the integrated map. The average intervals between two adjacent mapped markers were 1.11 cM, 2.91 cM and 0.95 cM for three maps, respectively. ‘SNP_only’ markers accounted for 89.25 % of the markers on the integrated map. Mapping markers contained 5,043 single nucleotide polymorphisms (SNPs) loci, which corresponded to two SNP loci per SLAF marker. According to the integrated map, we used interval mapping (Logarithm of odds, LOD > 3.0) to detect our quantitative trait. One QTL was detected for anthracnose resistance. The interval of this QTL ranged from 165.51 cM to 176.33 cM on LG14, and ten markers in this interval that were above the threshold value were considered to be linked markers to the anthracnose resistance trait. The phenotypic variance explained by each marker ranged from 16.2 to 19.9 %, and their LOD scores varied from 3.22 to 4.04.

Conclusions

High-density genetic maps for walnut containing 16 LGs were constructed using the SLAF-seq method with an F1 population. One QTL for walnut anthracnose resistance was identified based on the map. The results will aid molecular marker-assisted breeding and walnut resistance genes identification.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1822-8) contains supplementary material, which is available to authorized users.     

An expanded genetic linkage map of an intervarietal Agaricus bisporus var. bisporus × A. bisporus var. burnettii hybrid based on AFLP,SSR and CAPS markers sheds light on the recombination behaviour of the species

A genetic linkage map for the edible basidiomycete Agaricus bisporus was constructed from 118 haploid homokaryons derived from an intervarietal A. bisporus var. bisporus × A. bisporus var. burnettii hybrid. Two hundred and thirty-one AFLP, 21 SSR, 68 CAPS markers together with the MAT, BSN, PPC1 loci and one allozyme locus (ADH) were evenly spread over 13 linkage groups corresponding to the chromosomes of A. bisporus. The map covers 1156 cM, with an average marker spacing of 3.9 cM and encompasses nearly the whole genome. The average number of crossovers per chromosome per individual is 0.86. Normal recombination over the entire genome occurs in the heterothallic variety, burnettii, contrary to the homothallic variety, bisporus, which showed adaptive genome-wide suppressed recombination. This first comprehensive genetic linkage map for A. bisporus provides foundations for quantitative trait analyses and breeding programme monitoring, as well as genome organisation studies.     

A physical map covering the nsv locus that confers resistance to Melon necrotic spot virus in melon (Cucumis melo L.)

Melon necrotic spot virus (MNSV) is a member of the genus Carmovirus, which produces severe yield losses in melon and cucumber crops. The nsv gene is the only known natural source of resistance against MNSV in melon, and confers protection against all widespread strains of this virus. nsv has been previously mapped in melon linkage group 11, in a region spanning 5.9 cM, saturated with RAPD and AFLP markers. To identify the nsv gene by positional cloning, we started construction of a high-resolution map for this locus. On the basis of the two mapping populations, F₂ and BC1, which share the same resistant parent PI 161375 (nsv/nsv), and using more than 3,000 offspring, a high-resolution genetic map has been constructed in the region around the nsv locus, spanning 3.2 cM between CAPS markers M29 and M132. The availability of two melon BAC libraries allowed for screening and the identification of new markers closer to the resistance gene, by means of BAC-end sequencing and mapping. We constructed a BAC contig in this region and identified the marker 52K20sp6, which co-segregates with nsv in 408 F₂ and 2.727 BC1 individuals in both mapping populations. We also identified a single 100 kb BAC that physically contains the resistance gene and covers a genetic distance of 0.73 cM between both BAC ends. These are the basis for the isolation of the nsv recessive-resistance gene.     

A genetic map of melon (Cucumis melo L.) based on amplified fragment length polymorphism (AFLP) markers

 Genetic maps facilitate the study of genome structure and evolution, and the identification of monogenic traits or Mendelian components of quantitative traits. We evaluated 228 RAPD, microsatellite and AFLP markers for linkage analysis in melon (Cucumis melo L.) varieties MR-1 (resistant to Fusarium wilt, powdery and downy mildews) and Ananas Yokneum (AY; susceptible to these diseases) and constructed a detailed genetic map. The mapping population consisted of 66 backcross progenies derived from AY×(MR-1×AY). Despite a relatively low level of polymorphism in the species, AFLP markers were found to be more efficient in mapping the melon genome than RAPD or microsatellite markers. The map contains 197 AFLPs, six RAPDs and one microsatellite marker assigned to 14 major and six minor linkage groups, and covers 1942 cM with the average distance between adjacent markers of approximately 10 cM. The maximum distance allowed between markers is 27.5 cM. About 11% of the intervals (20 out of 173) are over 20 cM (but less than 27.5 cM). The map has immediate utility for identifying markers linked to disease resistance genes that are suitable for marker-assisted breeding. The use of microsatellite markers for integration with other maps is also discussed. Received: 12 March 1997 / Accepted: 20 May 1997     

Construction of a genetic linkage map in Fenneropenaeus chinensis using SNP markers

Genetic linkage maps of Fenneropenaeus chinensis were constructed using a “double pseudo-testcross” strategy with 200 single nucleotide polymorphisms (SNPs) markers. This study represents the first SNP genetic linkage map for F. chinensis. The parents and F ₁ progeny of 100 individuals were used as mapping populations. 21 genetic linkage groups in the male and female maps were identified. The male linkage map was composed of 115 loci and spanned 879.7 cM, with an average intermarker spacing of 9.4 cM, while the female map was composed of 119 loci and spanned 876.2 cM, with an average intermarker spacing of 8.9 cM. The estimated coverage of the linkage maps was 51.94% for the male and 53.77% for the female, based on two estimates of genome length. The integrated map contains 180 markers distributed in 16 linkage groups, and spans 899.3 cM with an average marker interval of 5.2 cM. This SNP genetic map lays the foundation for future shrimp genomics and genetic breeding studies, especially the discovery of gene or regions for economically important traits in Chinese shrimp.