Fifty new microsatellite loci for the wheat genetic map

 Hexaploid bread wheat (Triticum aestivum) has low levels of RFLP. Simple sequence repeats, however, show high levels of polymorphism and are therefore especially useful in intervarietal breeding applications. We present 53 newly mapped microsatellite loci for the wheat genetic map, 41 primary loci and 12 additional loci from these same primer pairs. Markers have been accredited with a quality score on a scale of 1–5 which describes the complexity of the amplification product profile from each primer pair. Received: 29 June 1997 / Accepted: 4 February 1998     

Characterisation of two gene subunits on the 1R chromosome of rye as orthologs of each of the Glu-1 genes of hexaploid wheat

The visco-elastic properties of bread flour are firmly associated with the presence or absence of certain HMW subunits coded by the Glu-1 genes. Identifying allelic specific molecular markers (AS-PCR) associated with the presence of Glu-1 genes can serve as a valuable tool for the selection of useful genotypes. This paper reports the use of primers designed from nucleotide sequences of the Glu-D1 gene of wheat (AS-PCR for Glu-D1y10) that recognise and amplify homologous sequences of the Glu-R1 gene subunits of rye. The primers amplify the complete coding regions and provided two products of different size in rye, in wheats carrying the substitution 1R(1D) and in rye-wheat aneuploid lines carrying the long arm of chromosome 1R. The location, the molecular characterisation of these sequences and their expression during grain ripening seem to demonstrate that the amplification products correspond to structural genes encoding the high-molecular-weight (HMW) glutenins of rye. The homology of the rye gene to subunits encoding HMW glutenins in wheat was confirmed by Southern blots and sequencing. The amplification-products were cloned, sequenced and characterised, and the sequences compared with the main glutenin subunits of wheat and related species. Further, an RT-PCR experiment was performed using primers designed from the sequence of both amplified products. This assay demonstrated that both sequences are expressed in endosperm during grain ripening. The results of these analyses suggest that both gene subunits correspond to x- and y-type genes of the Glu-R1 locus of rye. Received: 11 December 2000 / Accepted: 17 April 2001     

Transferability of wheat microsatellites to diploid Triticeae species carrying the A, B and D genomes

Hexaploid wheat (Triticum aestivum L em Thell) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. In this study, we evaluated the ability of microsatellite sequences from T. aestivum to be revealed on different ancestral diploid species more or less closely related, i.e. to test for their transferability. Fifty five primer pairs, evenly distributed all over the genome, were investigated. Forty three of them mapped to single loci on the hexaploid wheat genetic map although only 20 (46%) gave single PCR products; the 23 others (54%) gave more than one band with either only one being polymorphic, the others remaining monomorphic, or with several co-segregating polymorphic bands. The other 12 detected two (9) or three (3) different loci. From the 20 primer pairs which gave one amplification pro- duct on hexaploid wheat, nine (45%) also amplified products on only one of the diploid species, and seven (35%) on more than one. Four microsatellites (20%) which mapped to chromosomes from the B genome of wheat, did not give any amplification signal on any of the diploid species. This suggests that some regions of the B genome have evolved more rapidly compared to the A or D genomes since the emergence of polyploidy, or else that the donor(s) of this B genome has(have) not yet been identified. Our results confirm that Triticum monococcum ssp. urartu and Triticum tauschii were the main donors of the A and D genomes respectively, and that Aegilops speltoides is related to the ancestor(s) of the wheat polyploid B genome. Received: 21 June 2000 / Accepted: 15 November 2000     

Isolation and characterisation of microsatellites from hexaploid bread wheat

 The development of large panels of simple-to-analyse genetic markers for tagging agronomically important genes and diversity studies in hexaploid bread wheat is an important goal in applied cereal genetic research. We have isolated and sequenced over 200 clones containing microsatellites from the wheat genome and have tested 153 primer pairs for genetic polymorphism using a panel of ten wheat varieties, including the parents of our main mapping cross. A subset comprising 49 primer pairs detects 76 loci, of which 74 can be unequivocably allocated to one of the wheat chromosomes. A relatively low frequency of the loci detected are from the D genome, and these loci show less polymorphism than those from the A and B genomes. Generally, the microsatellites show high levels of genetic polymorphism and an average of 3.5 alleles per locus with an average polymorphism information content (PIC), value of 0.51. The observed levels of polymorphism are positively correlated with the length of the microsatellite repeats. A high proportion, approximately two-thirds, of primer pairs designed to detect simple sequence repeat (SSR) variation in wheat do not generate the expected amplification products and, more significantly, often generate unresolvable PCR products. In general, our results agree closely with those obtained from other recent studies using microsatellites in plants. Received: 19 March 1996 / Accepted: 28 June 1996     

Low levels of DNA sequence variation among adapted genotypes of hexaploid wheat

 PCR products from regions corresponding to sequences hybridising to wheat RFLP probes were sequenced in order to establish the level of DNA sequence variation among adapted wheat genotypes. Hexaploid bread wheat shows a very low rate of nucleotide polymorphism, approximately 1 polymorphic nucleotide per 1000 basepairs. Differences in PCR product length can be exploited to design genome-specific amplicons, which may have use in gene tagging or in diagnostic applications. Interpretation of results may be complicated by the simultaneous amplification of orthologous and paralogous sequences. These findings have significant implications for the use of STS markers in wheat and other polyploid species. Received: 3 October 1998 / Accepted: 28 November 1998     

The applicability of consensus PCR primers across species and genera: the use of wheat Em sequences to develop markers for orthologues in rye

Two wheat consensus primer sets, directed to ”early-methionine-labelled” (Em) gene sequences, were tested for their ability to amplify beyond their original source. A range of widely diverse templates, including other Triticeae species and sample monocot and dicot species, was assayed. Primer set EMC5/EMC3, amplifying the entire coding region with its intron and part of the 3’ untranslated region, targets Triticeae and sorghum Em sequences. The other set, EMC5/EMCO31, directed to the coding region and its intron, amplifies templates from all the grass species. Both primer sets fail to amplify Em sequences from more distant monocots and the dicots. Using set EMC5/EMC3, we isolated and sequenced ten members of the rye Em gene family from five different rye sources. Significant DNA sequence variation between wheat and rye sequences in the non-coding regions was found, and this was used to develop seven sequence-specific primers. Twelve primer combinations were analysed, 7 of which were Em-R1-specific, amplifying a product in at least one of the tested rye or rye-carrying genotypes but not in wheat. Four sets exhibited clear amplification length polymorphisms which allowed discrimination between and within the rye sources. The primers also discriminated between wheat-rye recombinants with proximal 1RL rye chromatin and those carrying distal 1RL rye chromatin. These results show that wheat consensus primer sets can be used to isolate orthologous sequences, especially from species that are used for alien gene transfer in wheat. Subsequently, species-specific assays can be designed that are useful tools for this application. Received: 7 December 1998 / Accepted: 21 July 1999     

Detection of loci controlling seed dormancy on group 4 chromosomes of wheat and comparative mapping with rice and barley genomes

Three quantitative trait loci (QTLs) controlling seed dormancy were detected on group 4 chromosomes of wheat (Triticum aestivum L.) using 119 doubled haploid lines (DHLs) derived from a cross between AC Domain and Haruyutaka. A major QTL, designated QPhs.ocs-4A.1, was identified within the marker interval between Xcdo795 and Xpsr115 in the proximal region of the long arm of chromosome 4A. Two minor QTLs, QPhs.ocs-4B.2 on 4B and QPhs.ocs-4D.2 on 4D, were flanked by common markers, Xbcd1431.1 and Xbcd1431.2 in the terminal region of the long arms, suggesting a homoeologous relationship. These three QTLs explained more than 80% of the total phenotypic variance in seed dormancy of DHLs grown in the field and under glasshouse conditions. The AC Domain alleles at the three QTLs contributed to increasing seed dormancy. Comparative maps across wheat, barley and rice demonstrated the possibility of a homoeologous relationship between QPhs.ocs-4A.1 and the barley gene SD4, while no significant effects of the chromosome regions of wheat and barley orthologous to rice chromosome 3 region carrying a major seed dormancy QTL were detected. Received: 5 June 2000 / Accepted: 31 August 2000     

Identification of a chromosome-specific probe that maps within the Ph1 deletions in common and durum wheat

 The Ph1 (pairing homoeologous) gene is the major factor that determines the diploid-like chromosome behavior of polyploid wheat. This gene, which is located on the long arm of chromosome 5B (5BL), suppresses homoeologous pairing at meiosis while allowing exclusive homologous pairing. In an effort to tag the specific chromosomal region where this gene is located, we have previously microdissected chromosome arm 5BL from bread wheat and produced a plasmid library by random PCR amplification and cloning. In this work we isolated from this library a 5BL-specific probe, WPG90, and mapped it within the interstitial deleted chromosome fragments carrying Ph1 in common and durum wheat. A PCR assay of Ph1 based on WPG90 was developed that allows an easy identification of homozygous genotypes deficient for this gene. Received: 19 June 1996 / Accepted: 18 October 1996     

A moderately repeated DNA sequence of wheat and rye genomes

A 371 base pair segment (bordered by Hind III and Eco RI cutting sites) of wheat embryo nuclear DNA has been cloned and sequenced. It is AT-rich (68%), shares some sequence features with autonomously replicating sequence (ARS) elements, and occurs in approximately 7600 copies per haploid genome. When used as probe for blot hybridization to Hind III-digested wheat DNA, it gives an irregular series of hybridization bands. Essentially the same hybridization pattern was observed for rye DNA. It is concluded that this segment is distributed irregularly but, apparently, according to the same rule in both wheat and rye genomes.     

Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat

Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10⁻¹²) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity. Received: 19 December 2000 / Accepted: 17 April 2001     

Microsatellites confirm the authenticity of inter-varietal chromosome substitution lines of wheat (Triticum aestivum L.)

Ninety-five wheat microsatellite markers (WMS) were used to verify the authenticity of the set of Saratovskaya 29/Yanetzkis Probat inter-varietal wheat chromosome substitution lines developed using Saratovskaya 29 as the recipient variety. Polymorphic markers were available for all chromosome arms except 4DS, 6DS and 7DS. Each chromosome substitution line was tested by 2–8 microsatellite markers. The results demonstrate that most of the lines are correct. Out of 21 lines tested 17 showed the expected microsatellite pattern of the donor variety. Two entire chromosomes, 1B and 7A, and two chromosome arms, 3AL and 6DS, were not substituted with Yanetzkis Probat in their respective lines. Three microsatellite markers located in the distal regions of chromosome arms 4AL, 3BS and 5BL in the corresponding substitution lines did not reveal the expected microsatellite pattern of the recipient variety. The possible causes of the incorrect substitution line development and the appearance of incorrect distal microsatellite markers are discussed. The data confirm the idea that microsatellite markers provide ideal tools for testing the authenticity of genetic stocks of wheat. Received: 27 August 1999 / Accepted: 8 October 1999     

Comparative molecular mapping of GA insensitive Rht loci on chromosomes 4B and 4D of common wheat (Triticum aestivum L.)

 The two GA-insensitive dwarfing gene loci Rht-B1 and Rht-D1 were mapped using three F₂ populations, segregating for Rht-B1c (Rht3), Rht-D1b (Rht2) or Rht-D1c (Rht10). Rht-B1c was mapped on chromosome 4BS in the centromere region, distal and closely linked to the RFLP markers Xpsr144 (11.9 cM) and Xpsr584 (17.8 cM), but proximal to Xmwg634 (30 cM). Rht-D1c, however, was found to be closely linked to the distally located markers Xpsr921 (0.8 cM) and Xmwg634 (1.5 cM). The homoeologous relationships between the GA-insensitive dwarfing genes within the Triticeae are discussed. Received: 2 May 1997 / Accepted: 9 June 1997     

Characterization of trinucleotide SSR motifs in wheat

Length differences among trinucleotide-based microsatellite alleles can be more easily detected and frequently produce fewer ”stutter bands” as compared to dinucleotide-based microsatellite markers. Our objective was to determine which trinucleotide motif(s) would be the most-polymorphic and abundant source of trinucleotide microsatellite markers in wheat (Triticum aestivum L.). Four genomic libraries of cultivar ’Chinese Spring’ were screened with nine trinucleotide probes. Based on the screening of 28550 clones, the occurrences of (CTT/GAA) _n , (GGA/CCT) _n , (TAA/ATT) _n , (CAA/GTT) _n , (GGT/CCA) _n , (CAT/GTA) _n , (CGA/GCT) _n , (CTA/GAT) _n , and (CGT/GCA) _n repeats were estimated to be 5.4×10⁴, 3.5×10⁴, 3.2×10⁴, 1.2×10⁴, 6.3×10³, 4.9×10³, 4.5×10³, 4.5×10³ and 3.6×10³, i.e., once every 293 kbp, 456 kbp, 500 kbp, 1.3 Mbp, 2.6 Mbp, 3.2 Mbp, 3.6 Mbp, 3.6 Mbp and 4.5 Mbp in the wheat genome, respectively. Of 236 clones selected for sequencing, 38 (93%) (TAA/ATT) _n , 30 (43%) (CTT/GAA) _n , 16 (59%) (CAA/GTT) _n , 3 (27%) (CAT/GTA) _n and 2 (4%) (GGA/CCT) _n clones contained microsatellites with eight or more perfect repeats. From these data, 29, 27 and 16 PCR primer sets were designed and tested to the (TAA/ATT) _n , (CTT/GAA) _n and (CAA/GTT) _n microsatellites, respectively. A total of 12 (41.4%) primers designed to (TAA/ATT) _n , four (14.8%) to (CTT/GAA) _n , and two (12.5%) to (CAA/GTT) _n resulted in polymorphic markers. The results indicated that (TAA/ATT) _n microsatellites would provide the most-abundant and the most-polymorphic source of trinucleotide microsatellite markers in wheat. Received: 17 February 2001 / Accepted: 31 May 2001     

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)_n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity. Received: 15 May 1999 / Accepted: 27 July 1999     

Single-stranded DNA in the genetic transformation of wheat (Triticum aestivum L.): transformation frequency and integration pattern

Two non-linked marker genes (gus and bar) were co-introduced by microprojectile bombardment into wheat cells. Four different DNA structures were compared with respect to ability to integrate into the wheat genome: circular or linear (l) DNA as a single- or double-stranded plasmid (ss and ds, respectively). In eight independent experiments, linearized DNA integrated in the ds or ss form with a high efficiency of up to 14% for l-ssDNA. Molecular analyses by Southern blotting showed that all DNA forms gave a similar complicated integration pattern of the bar gene. Received: 20 July 1998 / Accepted: 30 January 1999     

A molecular genetic map of the long arm of chromosome 6R of rye incorporating the cereal cyst nematode resistance gene, CreR

 A genetic map of the long arm of chromosome 6R of rye was constructed using eight homoeologous group-6 RFLP clones and five PCR markers derived from the rye-specific dispersed repetitive DNA family, R173. The map was developed using a novel test-cross F₁ (TC-F₁) population segregating for resistance to the cereal cyst nematode. Comparisons were made between the map generated with other rye and wheat group-6 chromosome maps by the inclusion of RFLP clones previously mapped in those species. Co-linearity was observed for common loci. This comparison confirmed a dramatic reduction in recombination for chromosome 6R in the TC-F₁ population. The CreR locus was included in the linkage map via progeny testing of informative TC-F₁ individuals. CreR mapped 3.7 cM distal from the RFLP locus, XksuF37. Comparative mapping should allow the identification of additional RFLP markers more closely linked to the CreR locus. Received: 14 April 1998 / Accepted: 29 April 1998     

Conservation of microsatellite loci within the genus Vitis

Eleven microsatellites isolated from grapevine (Vitis vinifera) were used to study the degree of conservation of these sequences across different Vitis species. Nine microsatellites were newly isolated, the remaining two (VVS2 and VVS5) came from the literature. A preliminary assay on the conservation of priming sites was carried out on 14 non-V. vinifera species, including relevant taxa for breeding. Parthenocissus quinquefolia was added as representative of a related genus. Cross-species amplification was obtained in 94% of the 176 genotype×locus tested combinations. Three microsatellite loci were then cloned and sequenced in ten species. The microsatellite repeat was found present in all cases. The repeat region was often longer in V. vinifera than in the other species. Furthermore the non-source species showed interruptions in the repeat. In spite of these constraints, which could reduce the polymorphism of microsatellites in non-source species, the results demonstrate the possibility of extending the use of microsatellite markers to wild germplasm and inter-specific hybrids. Point mutations have been found in microsatellite flanking regions and these variations have been used to investigate the genetic relationship among taxa. The Neighbor-joining tree that was obtained on the basis of ten nucleotide variations, showed that there is not a clear cut difference between American, Asian and European species and that the actual taxonomy which reflects the geographical distribution of species must most likely be revised. Moreover, in general, nucleotide variations which occur in microsatellite flanking regions provide new molecular tools for investigating the evolution of species. Received: 24 October 1999 / Accepted: 11 November 1999     

Construction and characterisation of a large DNA insert library from the D genome of wheat

 A large DNA fragment library consisting of 144 000 clones with an average insert size of 119 kb was constructed from nuclear DNA isolated from root and leaf tissue from Triticum tauschii (syn. Aegilops tauschii), the D-genome progenitor of wheat. The library was made in a binary vector that had previously been shown to stably maintain large inserts of foreign DNA in Escherichia coli. The use of root nuclei reduced considerably the proportion of the library containing clones derived from chloroplast DNA. Several experimental parameters were investigated and optimised, leading to a high cloning efficiency. Only three ligations were needed to construct the library which was estimated to be equivalent to 3.7 haploid genomes. The accuracy of this estimation was demonstrated by screening this library with three well-defined probes. One probe containing a glutenin gene sequence identified 5 clones covering at least 230 kb of the Glu-D1 locus and contained the two tightly linked high-molecular-weight glutenin genes Glu-D1x and -D1y. Each of the other two single-copy probes derived from the Cre3 cereal cyst nematode resistance gene locus hybridised with 4 clones containing gene sequences encoding nucleotide binding sites and a leucine-rich region. This is the first representative large-insert DNA library for wheat, and the results indicated that large molecules of wheat DNA can be efficiently cloned, stably maintained and manipulated in a bacterial system. Received: 28 August 1998 / Accepted: 28 November 1998     

Evaluation of allelic diversity at chloroplast microsatellite loci among common wheat and its ancestral species

Twenty four chloroplast microsatellite loci having more than ten mononucleotide repeats were identified from the entire chloroplast DNA sequence of common wheat, Triticum aestivum cv Chinese Spring. For each microsatellite, a pair of primers were designed to produce specific PCR products in the range of 100– 200 bp. The allelic diversity at the microsatellite loci was evaluated using 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploid evolution. Polymorphic banding patterns were obtained at 21 out of 24 chloroplast microsatellite loci. The three monomorphic microsatellites were found to be located in coding regions. For the polymorphic microsatellites, the number of alleles per microsatellite ranged from 2 to 7 with an average of 4.33, and the diversity values (H) ranged from 0.05 to 0.72 with an average of 0.47. Significant correlations (P<0.01) were observed between the number of repeats and the number of alleles, and between the number of repeats and diversity value, respectively. The genetic diversity explained by chloroplast microsatellites and nuclear RFLP markers were compared using 22 tetraploid accessions. Although the number of alleles for nuclear RFLP markers was found to be higher than that for chloroplast microsatellites, similar diversity values were observed for both types of markers. Among common wheat and its ancestral species, the percentages of common chloroplast microsatellite alleles were calculated to examine their phylogenetic relationships. As a result, Timopheevi wheat species were clearly distinguished from other species, and Emmer and common wheat species were divided into two main groups, each consisting of a series of wild and cultivated species from tetraploid to hexaploid. This indicates that the two types of chloroplast genomes of common wheat might have independently originated from the corresponding types of wild and cultivated Emmer wheat species. Received: 6 October 2000 / Accepted: 13 March 2001     

Discrimination of the closely related A and B genomes in AABB tetraploid species of Avena

Fluorescent in situ (FISH) and Southern hybridization procedures were used to investigate the chromosomal distribution and genomic organization of the satellite DNA sequence As120a (specific to the A-genome chromosomes of hexaploid oats) in two tetraploid species, Avena barbata and Avena vaviloviana. These species have AB genomes. In situ hybridization of pAs120a to tetraploid oat species revealed elements of this repeated family to be distributed over both arms of 14 of the 28 chromosomes of these species. Genomes A and B were subsequently distinguished, indicating an allopolyploid origin for A. barbata. This was confirmed by assigning the satellited chromosomes to individual genomes, using the satellite itself and two ribosomal probes in simultaneous and sequential in situ hybridization analyses. Differences between A. barbata and A. vaviloviana genomes were also revealed by both FISH and Southern techniques using pAs120a probes. Whereas two B-genome chromosome pairs were found to be involved in intergenomic translocations in A. vaviloviana, FISH detected no intergenomic rearrangements in A. barbata. When using pAs120a as a probe, Southern hybridization also revealed differences in the hybridization patterns of the two genomes. A 1300-bp EcoRV fragment was present in A. barbata but absent in A. vaviloviana. This fragment was also detected in Southern analyses of A-genome diploid and hexaploid oat species. Received: 27 November 2000 / Accepted: 28 February 2001