A high-density genetic map of Sorghum bicolor (L.) Moench based on 2926 AFLP®, RFLP and SSR markers

Using AFLP technology and a recombinant inbred line population derived from the sorghum cross of BTx623 × IS3620C, a high-density genetic map of the sorghum genome was constructed. The 1713 cM map encompassed 2926 loci distributed on ten linkage groups; 2454 of those loci are AFLP products generated from either the EcoRI/MseI or PstI/MseI enzyme combinations. Among the non-AFLP markers, 136 are SSRs previously mapped in sorghum, and 203 are cDNA and genomic clones from rice, barley, oat, and maize. This latter group of markers has been mapped in various grass species and, as such, can serve as reference markers in comparative mapping. Of the nearly 3000 markers mapped, 692 comprised a LOD 3.0 framework map on which the remaining markers were placed with lower resolution (LOD <3.0). By comparing the map positions of the common grass markers in all sorghum maps reported to date, it was determined that these reference markers were essentially collinear in all published maps. Some clustering of the EcoRI/MseI AFLP markers was observed, possibly in centromeric regions. In general, however, the AFLP markers filled most of the gaps left by the RFLP/SSR markers demonstrating that AFLP technology is effective in providing excellent genome coverage. A web site, http://SorghumGenome.tamu.edu, has been created to provide all the necessary information to facilitate the use of this map and the 2590 PCR-based markers. Finally, we discuss how the information contained in this map is being integrated into a sorghum physical map for map-based gene isolation, comparative genome analysis, and as a source of sequence-ready clones for genome sequencing projects.     

Linkage group alignment of sorghum RFLP maps using a RIL mapping population.

Several molecular maps have been constructed in sorghum (Sorghum bicolor L. Moench) using a variety of probes from different grass species such as sorghum, maize, sugarcane, rice, oat, and barley. In order to enhance the utility of the existing mapping information by the sorghum research community, alignment and integration of all major molecular maps is necessary. To achieve this objective, a genetic map of 214 loci with a total map distance of 1200 cM was constructed using 98 F7 sorghum recombinant inbred lines (RILs) from a cross between two inbred lines, B35 and Tx7000. Few cDNA clones of sorghum and maize related to photosynthesis and drought stress were mapped on this map for the first time. Five major restriction fragment length polymorphism (RFLP) maps independently developed in this species were used for alignment purpose. The distributions of previously mapped markers were compared with their respective sorghum maps to align each of the linkage groups. In general, consistent linear order among markers was maintained in all the linkage maps. The successful alignment of these RFLP maps will now allow selection of a large number of markers for any region of the sorghum genome with many potential applications ranging from fine mapping and marker-assisted selection to map-based cloning for the improvement of sorghum and related species.     

RFLP inheritance and linkage in walnut

Thirty-two low-copy-number genomic DNA clones from a walnut (Juglans sp.) Pst I genomic library were used to establish a molecular-marker linkage map for walnut. The clones were hybridized to restriction-endonuclease-digested DNA from parent walnut trees involved in an interspecific backcross of (J. hindsii x J. regia) x J. regia in order to identify parental polymorphism. Sixty-three backcross progeny were analyzed to determine the inheritance and linkage of 48 RFLP loci. Sixty-six percent of the walnut cloned sequences detected duplicated, but unlinked, loci. Twelve linkage groups were identified by 42 of the RFLP loci. A Poisson probability method for estimating genome size was utilized to calculate the approximate walnut genome length as 1660 cM and to estimate that 138 markers would be needed to cover 95% of the walnut genome within 20 cM of each marker.     

An SSR genetic map of Sorghum bicolor (L.) Moench and its comparison to a published genetic map.

Sorghum bicolor (L.) Moench is an important grain and forage crop grown worldwide. We developed a simple sequence repeat (SSR) linkage map for sorghum using 352 publicly available SSR primer pairs and a population of 277 F2 individuals derived from a cross between the Westland A line and PI 550610. A total of 132 SSR loci appeared polymorphic in the mapping population, and 118 SSRs were mapped to 16 linkage groups. These mapped SSR loci were distributed throughout 10 chromosomes of sorghum, and spanned a distance of 997.5 cM. More important, 38 new SSR loci were added to the sorghum genetic map in this study. The mapping result also showed that chromosomes SBI-01, SBI-02, SBI-05, and SBI-06 each had 1 linkage group; the other 6 chromosomes were composed of 2 linkage groups each. Except for 5 closely linked marker flips and 1 locus (Sb6_34), the marker order of this map was collinear to a published sorghum map, and the genetic distances of common marker intervals were similar, with a difference ratio 相似文献   

An integrated SSR and RFLP linkage map of Sorghum bicolor (L.) Moench.

We report the development, testing, and use (for genetic mapping) of a large number of polymerase chain reaction (PCR) primer sets that amplify DNA simple sequence repeat (SSR) loci of Sorghum bicolor (L.) Moench. Most of the primer sets were developed from clones isolated from two sorghum bacterial artificial chromosome (BAC) libraries and three enriched sorghum genomic-DNA (gDNA) libraries. A few were developed from sorghum DNA sequences present in public databases. The libraries were probed with radiolabeled di- and trinucleotide oligomers, the BAC libraries with four and six oligomers, respectively, and the enriched gDNA libraries with four and three oligomers, respectively. Both types of libraries were markedly enriched for SSRs relative to a size-fractionated gDNA library studied earlier. However, only 2% of the sequenced clones obtained from the size-fractionated gDNA library lacked a SSR, whereas 13% and 17% of the sequenced clones obtained from the BAC and enriched gDNA libraries, respectively, lacked a SSR. Primer sets were produced for 313 SSR loci. Two-hundred sixty-six (85%) of the loci were amplified and 165 (53%) of the loci were found to be polymorphic in a population composed of 18 diverse sorghum lines. (AG/TC)n and (AC/TG)n repeats comprised 91% of the dinucleotide SSRs and 52% of all of the SSRs at the polymorphic loci, whereas four types of repeats comprised 66% of the trinucleotide SSRs at the loci. Primer sequences are reported for the 165 polymorphic loci and for eight monomorphic loci that have a high degree of homology to genes. Also reported are the genetic map locations of 113 novel SSR loci (including four SSR-containing gene loci) and a linkage map composed of 147 SSR loci and 323 RFLP (restriction fragment length polymorphism) loci. The number of SSR loci per linkage group ranges from 8 to 30. The SSR loci are distributed relatively evenly throughout approximately 75% of the 1406-cM linkage map, but segments of five linkage groups comprising about 25% of the map either lack or contain few SSR loci. Mapping of SSR loci isolated from BAC clones located to these segments is likely to be the most efficient method for placing SSR loci in the segments.     

Linkages between restriction fragment length,isozyme, and morphological markers in lentil

Summary A genetic linkage map of lentil comprising 333 centimorgans (cM) was constructed from 20 restriction fragment length, 8 isozyme, and 6 morphological markers segregating in a single interspecific cross (Lens culinaris × L. orientalis). Because the genotypes at marker loci were determined for about 66 F₂ plants, linkages are only reported for estimates of recombination less than 30 cM. Probes for identification of restriction fragment length polymorphisms (RFLPs) were isolated from a cDNA and EcoRI and PstI partial genomic libraries of lentil. The cDNA library gave the highest frequency of relatively low-copy-number probes. The cDNAs were about twice as efficient, relative to random genomic fragments, in RFLP detection per probe. Nine markers showed significant deviations from the expected F₂ ratios and tended to show a predominance of alleles from the cultigen. Assuming a genome size of 10 Morgans, 50% of the lentil genome could be linked within 10 cM of the 34 markers and the map is of sufficient size to attempt mapping of quantitative trait loci.     

QTL analysis of late blight resistance in a diploid potato family of Solanum phureja × S. stenotomum

Field resistance to Phytophthora infestans (Mont.) de Bary, the causal agent of late blight in potatoes, has been characterized in a potato segregating family of 230 full-sib progenies derived from a cross between two hybrid Solanum phureja × S. stenotomum clones. The distribution of area under the disease progress curve values, measured in different years and locations, was consistent with the inheritance of multigenic resistance. Relatively high levels of resistance and transgressive segregations were also observed within this family. A genetic linkage map of this population was constructed with the intent of mapping quantitative trait loci (QTLs) associated with this late blight field resistance. A total of 132 clones from this family were genotyped based on 162 restriction fragment length polymorphism (RFLP) markers. The genome coverage by the map (855.2 cM) is estimated to be at least 70% and includes 112 segregating RFLP markers and two phenotypic markers, with an average distance of 7.7 cM between two markers. Two methods were employed to determine trait–marker association, the non-parametric Kruskal–Wallis test and interval mapping analysis. Three major QTLs were detected on linkage group III, V, and XI, explaining 23, 17, and 10%, respectively, of the total phenotypic variation. The present study revealed the presence of potentially new genetic loci in this diploid potato family contributing to general resistance against late blight. The identification of these QTLs represents the first step toward their introgression into cultivated tetraploid potato cultivars through marker-assisted selection.     

A RFLP-based linkage map of mustard [Brassica juncea (L.) Czern. and Coss.]

 A genetic linkage map of Brassica juncea was constructed based on restriction fragment length polymorphism (RFLP) detected by anonymous cDNA markers from B. napus, using a segregating F₁-derived doubled haploid (DH) progeny from a cross between a canola-quality mustard line (J90-4317) and a high-oil-content mustard line (J90-2733). The RFLP probes consisted of 229 cDNA probes from B. napus and a B. napus tandem repeat sequence, RDA2. The map consisted of 343 marker loci arranged in 18 major linkage groups plus five small segments with two to five marker loci, covering a total map distance of 2073 cM. Twenty-four percent of the markers were dominant in nature. Sixty-two percent of the marker loci were duplicated, and the majority were involved in inter-linkage group duplications, illustrating that complex duplications and subsequent rearrangements occurred after allopolyploidy. Deviation from the Mendelian segregation ratio for a DH population was observed for 27% of the markers. Two-thirds of these markers with a skewed segregation were clustered in 6 linkage groups and two unassigned segments. The overall average marker interval of the B. juncea map reported here was 6.6 cM, which would provide a marker density satisfactory for efficient use of the map in breeding applications, such as tagging of important agronomic traits and marker-assisted selection. Received: 14 May 1996 / Accepted: 11 October 1996     

Comparative genome analysis of mungbean (Vigna radiata L. Wilczek) and cowpea (V. unguiculata L. Walpers) using RFLP mapping data

Genome relationships between mungbean (Vigna tradiata) and cowpea (V. Unguiculata) based on the linkage arrangement of random genomic restriction fragment length polymorphism (RFLP) markers have been investigated. A common set of probes derived from cowpea, common bean (Phaseolus vulgaris), mungbean, and soybean (Glycine max) PstI genomic libraries were used to construct the genetic linkage maps. In both species, a single F₂ population from a cross between an improved cultivar and a putative wild progenitor species was used to follow the segregation of the RFLP markers. Approximately 90% of the probes hybridized to both mungbean and cowpea DNA, indicating a high degree of similarity in the nucleotide sequences among these species. A higher level of polymorphism was detected in the mungbean population (75.7%) than in the cowpea population (41.2%). Loci exhibiting duplications, null phenotypes, and distorted segregation ratios were detected in both populations. Random genomic DNA RFLP loci account for about 89% of the currently mapped markers with a few cDNA and RAPD markers added. The current mungbean map is comprised of 171 loci/loci clusters distributed in 14 linkage groups spanning a total of 1570cM. On the other hand, 97 markers covered 684 cM and defined 10 linkage groups in the current cowpea map. The mungbean and cowpea genomes were compared on the basis of the copy number and linkage arrangement of 53 markers mapped in common between the two species. Results indicate that nucleotide sequences are conserved, but variation in copy number were detected and several rearrangements in linkage orders appeared to have occurred since the divergence of the two species. Entire linkage groups were not conserved, but several large linkage blocks were maintained in both genomes.     

Molecular genetic maps of the group 6 chromosomes of hexaploid wheat (Triticum aestivum L. em. Thell.).

Restriction fragment length polymorphism (RFLP) maps of chromosomes 6A, 6B, and 6D of hexaploid wheat (Triticum aestivum L. em. Thell.) have been produced. They were constructed using a population of F7-8 recombinant inbred lines derived from a synthetic wheat x bread wheat cross. The maps consist of 74 markers assigned to map positions at a LOD >= 3 (29 markers assigned to 6A, 24 to 6B, and 21 to 6D) and 2 markers assigned to 6D ordered at a LOD of 2.7. Another 78 markers were assigned to intervals on the maps. The maps of 6A, 6B, and 6D span 178, 132, and 206 cM, respectively. Twenty-one clones detected orthologous loci in two homoeologues and 3 detected an orthologous locus in each chromosome. Orthologous loci are located at intervals of from 1.5 to 26 cM throughout 70% of the length of the linkage maps. Within this portion of the maps, colinearity (homosequentiality) among the three homoeologues is strongly indicated. The remainder of the linkage maps consists of three segments ranging in length from 47 to 60 cM. Colinearity among these chromosomes and other Triticeae homoeologous group 6 chromosomes is indicated and a consensus RFLP map derived from maps of the homoeologous group 6 chromosomes of hexaploid wheat, tetraploid wheat, Triticum tauschii, and barley is presented. Key words : RFLP, wheat, linkage maps, molecular markers.     

Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map

Inter-simple sequence repeat (ISSR) analysis was evaluated for its usefulness in generating markers to extend the genetic linkage map of Citrus using a backcross population previously mapped with restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and isozyme markers. ISSR markers were obtained through the simple technique of PCR followed by analysis on agarose gels, using simple sequence repeat (SSR) primers. Optimization of reaction conditions was achieved for 50% of the SSR primers screened, and the primers amplified reproducible polymorphic bands in the parents and progeny of the backcross population. Mendelian segregation of the polymorphic bands was demonstrated, with an insignificant number of skewed loci. Most of the SSR primers produced dominant loci; however co-dominance was observed with loci derived from three primers. A new genetic map was produced by combining the segregation data for the ISSR markers and data for the RFLP, RAPD and isozyme markers from the previous map and creating genetic linkages among all the markers using JoinMap 2.0 mapping software. The new map has an improved distribution of markers along the linkage groups with fewer gaps, and marker order showed partial or complete conservation in the linkage groups. The incorporation of ISSR markers into the genetic linkage map demonstrates that ISSR markers are suitable for genetic mapping in Citrus. Received: 3 February 2000 / Accepted: 12 May 2000     

A comparative map of wild rice (Zizania palustris L. 2n=2x=30)

We present the first genetic map of wild rice (Zizania palustris L., 2n=2x=30), a native aquatic grain of northern North America. This map is composed principally of previously mapped RFLP (restriction fragment length polymorphism) genetic markers from rice (Oryza sativa 2n=2x=24). The map is important as a foundation for genetic and crop improvement studies as well as a reference for genome organization comparisons among species of Gramineae. A comparative mapping approach with rice is especially useful because wild rice is grouped in the same subfamily, Oryzoideae, and no other mapping comparison has yet been made within the subfamily. As rice is the reference point for mapping and gene cloning in cereals, establishing a consensus map within the subfamily identifies conserved and unique regions. The genomes of wild rice and rice differ in total DNA content (wild rice has twice that of rice) and the number of chromosome pairs (wild rice=15 versus rice=12). The wild rice linkage map reported herein consists of 121 RFLP markers on 16 linkage groups spanning 1805 cM. Two linkage groups consist of only two markers. Colinear markers were found representing all rice linkage groups except #12. The majority of rice loci mapped to colinearly arranged arrays in wild rice (92 of 118). Features of the map include duplication of portions of three rice linkage groups and three possible translocations. The map gives basic information on the composition of the wild rice genome and provides tools to assist in the domestication of this important food source. Received: 25 August 1998 / Accepted: 20 February 1999     

Restriction fragment variation in the nuclear and chloroplast genomes of cultivated and wild Sorghum bicolor

Summary Fifty-six accessions of cultivated and wild sorghum were surveyed for genetic diversity using 50 low-copy-number nuclear DNA sequence probes to detect restriction fragment length polymorphisms (RFLPs). These probes revealed greater genetic diversity in wild sorghum than in cultivated sorghum, including a larger number of alleles per locus and a greater portion of polymorphic loci in wild sorghum. In comparison to previously published isozyme analyses of the same accessions, RFLP analysis reveals a greater number of alleles per locus. Furthermore, many RFLP alleles have frequencies between 0.25–0.75, while the vast majority of isozyme alleles are either rare (< 0.25) or near fixation (> 0.75). Correlations between genetic and geographic distances among the accessions were stronger when calculated with RFLP than with isozyme data. Systematic relationships revealed by nuclear and chloroplast restriction site analysis indicate that cultivated sorghum is derived from the wild ssp. arundinaceum. The portion of the wild gene pool most genetically similar to the cultivars is from central-northeastern Africa. Previous published data also suggested that this is most likely the principal area of domestication of sorghum. Introgression between wild and cultivated sorghum was inferred from disconcordant relationships shown by nuclear and chloroplast DNA markers. Introgression apparently occurs infrequently enough that the crop and its wild relatives maintain distinct genetic constitutions.     

An integrative genetic linkage map of winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

We constructed a genetic linkage map based on a cross between two Swiss winter wheat (Triticum aestivum L.) varieties, Arina and Forno. Two-hundred and forty F₅ single-seed descent (SSD)-derived lines were analysed with 112 restriction fragment length polymorphism (RFLP) anonymous probes, 18 wheat cDNA clones coding for putative stress or defence-related proteins and 179 simple-sequence repeat (SSR) primer-pairs. The 309 markers revealed 396 segregating loci. Linkage analysis defined 27 linkage groups that could all be assigned to chromosomes or chromosome arms. The resulting genetic map comprises 380 loci and spans 3,086 cM with 1,131 cM for the A genome, 920 cM for the B genome and 1,036 cM for the D genome. Seventeen percent of the loci showed a significant (P < 0.05) deviation from a 1:1 ratio, most of them in favour of the Arina alleles. This map enabled the mapping of QTLs for resistance against several fungal diseases such as Stagonospora glume blotch, leaf rust and Fusarium head blight. It will also be very useful for wheat genetic mapping, as it combines RFLP and SSR markers that were previously located on separate maps. S. Paillard and T. Schnurbusch contributed equally to the work     

Construction of an RFLP map in sorghum and comparative mapping in maize.

An F2 population derived from a cross between Sorghum bicolor ssp. bicolor ('CK60') and Sorghum bicolor ssp. drummondii ('PI229828') was used to develop an RFLP genetic linkage map of sorghum. The map consists of 201 loci distributed among 10 linkage groups covering a map distance of 1530 cM, with an average 8 cM between adjacent loci. Maize genomic probes (52), maize cDNA probes (124), and sorghum genomic probes (10) were used to define the loci (55, 136, and 10, respectively). Ninety-five percent of the loci fit expected segregation ratios. The loci with distorted segregation ratios were confined almost exclusively to a region of one linkage group. Comparison of sorghum and maize maps indicated high correspondence between the two genomes in terms of loci order and genetic distance. Many loci linked in maize (45 of 55) were also linked in sorghum. Instances of both conserved and rearranged locus orders were detected.     

Allotetraploidy of Zoysia species with 2n=40 based on a RFLP genetic map

 A RFLP linkage map of Zoysia spp. (2n=40), a warm-season turfgrass, was constructed by using the self-pollinated progenies obtained from an interspecific hybrid. Out of 115 DNA clones tested, 100 (87.0%), including 55 genomic clones, 38 cDNA clones, and seven gene clones encoding photosynthetic enzymes showed allelic-RFLP banding patterns among the parental accessions. We found that 26 probes detected two or more loci segregating in the self-pollinated progenies independently. The RFLP linkage map of Zoysia spp. consists of 115 loci in 22 linkage groups ranging in size from 12.5 cM to 141.3 cM with a total map distance of 1506 cM. Six RFLP loci (5.4%) showed significant segregation distortion (P<0.01). Two loci out of six were mapped to linkage group II, and another two loci were mapped to group VII. In the RFLP linkage map of zoysiagrass, five pairs of linkage groups sharing a series of duplicated loci with approximately the same order were identified. Therefore, we conclude that Zoysia spp. with 2n=40 should be considered as allotetraploids, which might have evolved from progenitors with a basic chromosome number of ten (x=10). Received: 20 March 1998 / Accepted: 17 September 1998     

A linkage map of sugar beet (Beta vulgaris L.)

Summary We have established a first linkage map for beets based on RFLP, isozyme and morphological markers. The population studied consisted of 96 F₂ individuals derived from an intraspecific cross. As was expected for outbreeding species, a relatively high degree of polymorphism was found within sugar beet; 47% of the DNA markers were polymorphic for the chosen population. The map consists of 115 independent chromosomal loci designated by 108 genomic DNA probes, 6 isozyme and one morphological marker. The loci cover 789 cM with an average spacing of 6.9 cM. They are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Eighteen markers (15.4%) showed distorted segregation which, in most instances, can be explained by gametic selection of linked lethal loci. The application of the linkage map in sugar beet breeding is discussed.     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

A molecular linkage map of cultivated oat.

A molecular linkage map of cultivated oat composed of 561 loci has been developed using 71 recombinant inbred lines from a cross between Avena byzantina cv. Kanota and A. sativa cv. Ogle. The loci are mainly restriction fragment length polymorphisms detected by oat cDNA clones from leaf, endosperm, and root tissue, as well as by barley leaf cDNA clones. The loci form 38 linkage groups ranging in size from 0.0 to 122.1 cM (mean, 39 cM) and consist of 2-51 loci each (mean, 14). Twenty-nine loci remain unlinked. The current map size is 1482 cM and the total size, on the basis of the number of unlinked loci, is estimated to be 2932.0 cM. This indicates that this map covers at least 50% of the cultivated oat genome. Comparisons with an A-genome diploid oat map and between linkage groups exhibiting homoeology to each other indicate that several major chromosomal rearrangements exist in cultivated oat. This map provides a tool for marker-assisted selection, quantitative trait loci analyses, and studies of genome organization in oat.     

A genetic linkage map of cowpea (Vigna unguiculata) developed from a cross between two inbred, domesticated lines

 We have constructed a genetic linkage map within the cultivated gene pool of cowpea (2n=2x=22) from an F₈ recombinant inbred population (94 individuals) derived from a cross between the inbreds IT84S-2049 and 524B. These breeding lines, developed in Nigeria and California, show contrasting reactions against several pests and diseases and differ in several morphological traits. Parental lines were screened with 332 random RAPD decamers, 74 RFLP probes (bean, cowpea and mung bean genomic DNA clones), and 17 AFLP primer combinations. RAPD primers were twice as efficient as AFLP primers and RFLP probes in detecting polymorphisms in this cross. The map consists of 181 loci, comprising 133 RAPDs, 19 RFLPs, 25 AFLPs, three morphological/classical markers, and a biochemical marker (dehydrin). These markers identified 12 linkage groups spanning 972 cM with an average distance of 6.4 cM between markers. Linkage groups ranged from 3 to 257 cM in length and included from 2 to 41 markers, respectively. A gene for earliness was mapped on linkage group 2. Seed weight showed a significant association with a RAPD marker on linkage group 5. This map should facilitate the identification of markers that “tag” genes for pest and disease resistance and other traits in the cultivated gene pool of cowpea. Received: 16 September 1996 / Accepted: 25 April 1997