Evaluation of Polymorphism at Microsatellite Loci of Spring Durum Wheat (Triticum durum Desf.) Varieties and the Use of SSR-Based Analysis in Phylogenetic Studies

Polymorphism at 28 SSR loci was analyzed and described in 45 cultivars of spring durum wheat created in the former USSR and Russia during the last 80 years. Each cultivar was shown to have a unique allele combination. This allows SSR markers to be used to identify durum wheat varieties. Meanwhile, these markers can hardly be used to detect phylogenetic relationships among varieties and to specify their pedigrees, because genetic distances calculated on the basis of these markers do not correlate with the distance calculated by coefficient of parentage.     

Evaluation of polymorphism at microsatellite loci of spring durum wheat (Triticum durum Desf.) varieties and the use of SSR-based analysis in phylogenetic studies

Polymorphism at 28 SSR loci was analyzed and described in 45 cultivars of spring durum wheat created in the former USSR and Russia during the last 80 years. Each cultivar was shown to have a unique allele combination. This allows SSR markers to be used to identify durum wheat varieties. Meanwhile, these markers can hardly be used to detect phylogenetic relationships among varieties and to specify their pedigrees, because genetic distances calculated on the basis of these markers do not correlate with the distance calculated by coefficient of parentage.     

An integrated DArT-SSR linkage map of durum wheat

Genetic mapping in durum wheat (Triticum durum Desf.) is constrained by its large genome and allopolyploid nature. We developed a Diversity Arrays Technology (DArT) platform for durum wheat to enable efficient and cost-effective mapping and molecular breeding applications. Genomic representations from 56 durum accessions were used to assemble a DArT genotyping microarray. Microsatellite (SSR) and DArT markers were mapped on a durum wheat recombinant inbred population (176 lines). The integrated DArT-SSR map included 554 loci (162 SSRs and 392 DArT markers) and spanned 2022 cM (5 cM/marker on average). The DArT markers from durum wheat were positioned in respect to anchor SSRs and hexaploid wheat DArT markers. DArT markers compared favourably to SSRs to evaluate genetic relationships among the durum panel, with 1315 DArT polymorphisms found across the accessions. Combining DArT and SSR platforms provides an efficient and rapid method of generating linkage maps in durum wheat.     

AFLP and pedigree-based genetic diversity estimates in modern cultivars of durum wheat [ Triticum turgidum L. subsp. durum (Desf.) Husn.

A substantial amount of between and within cultivar genetic variation was detected in all the 13 registered modern Canadian durum wheat (Triticum turgidum L. ssp. durum (Desf.) Husn.) cultivars based upon amplified restriction fragment polymorphism (AFLP). Of the approximately 950 detected AFLP markers, only 89 were polymorphic, with 41 between cultivars whereas the remaining 48 showed polymorphism within at least one cultivar. The ancestry of Canadian durum wheat cultivars was traced back to 125 cultivars, selections, and breeding lines including 17 landraces. Mean pair-wise genetic distance based on the kinship coefficient was 0.76. On the other hand, AFLP-based mean pair-wise genetic distance was 0.40. Even though there was a large difference between the means of the two diversity measures, a moderate positive correlation (r=0.457, p<0.002) was detected between the two distance matrices. Cluster analysis with the entire AFLP data divided all cultivars into three major groups reflecting their breeding origins. One group contained ’Pelissier’ alone, which was a selection from a landrace introduced into the US from Algeria. On the other hand such groupings among cultivars were not evident when KIN was used for genetic diversity measures instead. The level of genetic variation among individuals within a cultivar at the breeders’ seed level was estimated based on an inter-haplotypic distance matrix derived from the AFLP data. We found that the level of genetic variation within the most-developed cultivars is fairly substantial despite rigorous selection pressure aimed at cultivar purity in breeding programs. Comparison of AFLP and pedigree-based genetic diversity estimates in crop species such as durum wheat can provide important information for plant improvement. Received: 26 January 2001 / Accepted: 31 May 2001     

Nucleotide-binding site (NBS) profiling of genetic diversity in durum wheat.

Molecular markers are effective tools to investigate genetic diversity for resistance to pathogens. NBS (nucleotide-binding site) profiling is a PCR (polymerase chain reaction)-based approach to studying genetic variability that specifically targets chromosome regions containing R-genes and R-gene analogues. We used NBS profiling to measure genetic diversity among 58 accessions of durum wheat. Mean polymorphism rates detected using MseI and AluI as restriction enzymes were 34% and 22%, respectively. Mean number of polymorphisms per enzyme-primer combination was equal to 23.8 +/- 5.9, ranging from 13 to 31 polymorphic bands. In total, 96 markers over 190 indicated a good capacity to discriminate between accessions (the polymorphic index content ranging from 0.30 to 0.50). The results obtained with NBS profiling were compared with simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) data of the same set of accessions. The genetic distances computed with 190 NBS profiling markers were in close agreement with those obtained with AFLP and SSR markers (r = 0.73 and 0.76, respectively). Our results indicate that NBS profiling provides an effective means to investigate genetic diversity in durum wheat.     

Development of a SCAR marker associated with salt tolerance in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>) from a semi-arid region

Durum wheat (Triticum turgidum ssp. durum) is one of the main species of cultivated wheat. In arid and semi-arid areas, salinity stress reduces durum wheat productivity. This study used 26 durum wheat accessions from semi-arid regions in Tunisia to analyze plant tolerance to salt stress. Salt stress was experimentally applied by regularly submerging pots in NaCl solution. The salt tolerance trait index (STTI) and salt tolerance index (STI) of various growth parameters were used as criteria to select for salt tolerance. Analysis of genetic relationships was carried out to determine the genetic distance between durum wheat accessions. Based on simple sequence repeats analysis, a molecular marker for salt stress resistance in durum wheat was developed. Salt-treated plants had reduced morphological traits compared to control plants. Most STTIs in all genotypes were below 100 %. Based on STI, 8 accessions were found to be salt-resistant, 16 were salt-moderate, two were salt-susceptible. Analysis of the genetic relationships among 28 Tunisian durum wheat accessions revealed that landraces of the same nominal type are closely related. Of the 94 SSR primers investigated, three were selected and used to design sequence characterized amplified region (SCAR) primers. One SCAR primer pair, KUCMB_Xgwm403_2, produced a 207 bp band that was present in salt-resistant durum wheat lines but absent in salt-susceptible lines. The results suggest that KUCMB_Xgwm403_2 could be a potential genetic tag for salt-tolerant durum wheats.     

Relationships among durum wheat accessions. I. Comparative analysis of SSR, AFLP, and phenotypic data.

The determination of genetic relatedness among elite materials of crop species allows for more efficient management of breeding programs and for the protection of breeders' rights. Seventy simple sequence repeats (SSRs) and 234 amplified fragment length polymorphisms (AFLPs) were used to profile a collection of 58 durum wheat (Triticum durum Desf.) accessions, representing the most important extant breeding programs. In addition, 42 phenotypic traits, including the morphological characteristics recommended for the official distinctness, uniformity, and stability tests, were recorded. The correlation between the genetic similarities obtained with the 2 marker classes was high (r = 0.81), whereas lower values were observed between molecular and phenotypic data (r = 0.46 and 0.56 for AFLPs and SSRs, respectively). Morphological data, even if sampled in high numbers, largely failed to describe the pattern of genetic similarity, according to known pedigree data and the indications provided by molecular markers.     

Molecular Characterization and Comparative Analysis of Six Durum Wheat Accessions Including Graziella Ra

Genetic identity and relatedness of the durum wheat Graziella Ra, four Italian commercial durum cultivars (Cappelli, Grazia, Flaminio and Svevo) and Kamut were evaluated using amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs) and α-gliadin gene sequence analysis. Our primary objective was to study molecular genetic diversity in such a set of wheats including three modern (Grazia, Flaminio and Svevo) and three older (Cappelli, Kamut and Graziella Ra) durum accessions. Specifically, we aimed at determining the relationship between the historic accession Graziella Ra and Kamut, which is considered an ancient relative of the durum subspecies. Obtained results revealed that (1) both AFLP and SSR molecular markers detected highly congruent patterns of genetic diversity among the accessions showing nearly similar efficiency; (2) for AFLPs, percentage of polymorphic loci within accession ranged from 6.57% to 19.71% (mean, 12.77%) and for SSRs, from 0% to 57.14% (mean, 28.57%); (3) principal component analysis of genetic distance among accessions showed the first two axes accounting for 58.03% (for AFLPs) and 61.60% (for SSRs) of the total variability; (4) for AFLPs, molecular variance was partitioned into 80% (variance among accessions) and 20% (within accession) and for SSRs, into 73% (variance among accessions) and 27% (within accession); (5) cluster analysis of AFLPs and SSRs datasets displayed Graziella Ra and Kamut constantly grouped into the same cluster; and (6) molecular comparison of α-gliadin gene sequences showed Graziella Ra and Kamut in separate clusters. All these findings support the hypothesis that Graziella Ra and Kamut, although very similar, at least in the little part of the genome investigated by molecular markers employed in this study, might be considered as distinct accessions.     

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     

Genetic diversity of durum wheat as determined by AFLP in fluorescence

The DNA of fifteen Italian cultivars of durum wheat (Triticum turgidum L. ssp. durum) were analyzed by in fluorescence amplified fragment length polymorphism (fAFLP) in order to obtain the characteristic fingerprintings of genotypes and assess their genetic relatedness. Among 64 combinations of fluorescence labelled primers, three different combinations were chosen as producing a total of 6630 AFLP fragments, 2277 (34.3 %) of them being polymorphic. By using this fAFLP methodology a DNA fingerprinting of each durum wheat cultivar was generated for genotype identification. Analysis of the genetic relationships show the low variability among durum wheat cultivars.     

Microsatellite analysis reveals a progressive widening of the genetic basis in the elite durum wheat germplasm

It has been argued that the level of genetic diversity in the modern durum wheat (Triticum turgidum L. var. durum) elite germplasm may have declined due to the high selection pressure applied in breeding programs. In this study, 58 accessions covering a wide spectrum of genetic diversity of the cultivated durum wheat gene pool were characterized with 70 microsatellite loci (or simple sequence repeats, SSRs). On average, SSRs detected 5.6 different allelic variants per locus, with a mean diversity index (DI) equal to 0.56, thus revealing a diversity content comparable to those previously observed with SSRs in other small-grain cereal gene pools. The mean genetic similarity value was equal to 0.44. A highly diagnostic SSR set has been identified. A high variation in allele size was detected among SSR loci, suggesting a different suitability of these loci for estimating genetic diversity. The B genome was characterized by an overall polymorphism significantly higher than that of the A genome. Genetic diversity is organised in well-distinct sub-groups identified by the corresponding foundation-genotypes. A large portion (92.7%) of the molecular variation detected within the group of 45 modern cvs was accounted for by SSR alleles tracing back to ten foundation-genotypes; among those, the most recent CIMMYT-derived founders were genetically distant from the old Mediterranean ones. On the other hand, rare alleles were abundant, suggesting that a large number of genetic introgressions contributed to the foundation of the well-diversified germplasm herein considered. The profiles of recently released varieties indicate that the level of genetic diversity present in the modern durum wheat germplasm has actually increased over time.Communicated by F. Salamini     

Genetic Diversity and Population Structure of Tetraploid Wheats (Triticum turgidum L.) Estimated by SSR,DArT and Pedigree Data

Levels of genetic diversity and population genetic structure of a collection of 230 accessions of seven tetraploid Triticum turgidum L. subspecies were investigated using six morphological, nine seed storage protein loci, 26 SSRs and 970 DArT markers. The genetic diversity of the morphological traits and seed storage proteins was always lower in the durum wheat compared to the wild and domesticated emmer. Using Bayesian clustering (K = 2), both of the sets of molecular markers distinguished the durum wheat cultivars from the other tetraploid subspecies, and two distinct subgroups were detected within the durum wheat subspecies, which is in agreement with their origin and year of release. The genetic diversity of morphological traits and seed storage proteins was always lower in the improved durum cultivars registered after 1990, than in the intermediate and older ones. This marked effect on diversity was not observed for molecular markers, where there was only a weak reduction. At K >2, the SSR markers showed a greater degree of resolution than for DArT, with their identification of a greater number of groups within each subspecies. Analysis of DArT marker differentiation between the wheat subspecies indicated outlier loci that are potentially linked to genes controlling some important agronomic traits. Among the 211 loci identified under selection, 109 markers were recently mapped, and some of these markers were clustered into specific regions on chromosome arms 2BL, 3BS and 4AL, where several genes/quantitative trait loci (QTLs) are involved in the domestication of tetraploid wheats, such as the tenacious glumes (Tg) and brittle rachis (Br) characteristics. On the basis of these results, it can be assumed that the population structure of the tetraploid wheat collection partially reflects the evolutionary history of Triticum turgidum L. subspecies and the genetic potential of landraces and wild accessions for the detection of unexplored alleles.     

Domesticated emmer wheat [T. turgidum L. subsp. dicoccon (Schrank) Thell.] as a source for improvement of zinc efficiency in durum wheat

Modern durum wheat (AABB) is more sensitive to zinc (Zn) deficiency than bread wheat (AABBDD). One strategy to increase productivity and expansion of durum wheat industry in Zn-deficient soils is to improve its ability to grow and yield in such soils. This ability is termed Zn efficiency. In a growth room experiment using soil culture, we assessed the potential of Triticum turgidum L. subsp. dicoccon (Shrank) Thell. (domesticated emmer wheat, AABB) as a genetic resource for further improvement of Zn efficiency in modern durum wheat. Twenty four accessions of domesticated emmer wheat, four durum landraces/cultivars, and two bread wheat cultivars/ advanced breeders lines of known Zn efficiency were tested under Zn deficiency and Zn sufficiency. Significant variation was observed among genotypes in Zn deficiency symptoms, dry matter production, shoot Zn concentration, shoot Zn content and Zn utilisation efficiency (physiological efficiency). We identified domesticated emmer wheat accessions with greater Zn efficiency than modern durum wheat and even bread wheat genotypes. These accessions could be used in breeding programs to improve Zn efficiency of durum wheat. The results suggest that Zn efficiency of durum or bread wheat is likely to be determined collectively by its progenitors.     

Global diversity of durum wheat Triticum durum Desf. for alleles of gliadin-coding loci

Genetic diversity for the alleles of gliadin-coding loci was studied with 465 durum wheat accessions from 42 countries. A total of 108 alleles were identified for four loci; 60 alleles were described for the first time. Broad diversity of rare gliadin-coding alleles was observed. The highest genetic diversity was characteristic of durum wheat accessions from the Middle East, Trans-Caucasia, the Pyrenean Peninsula, and the Balkans. Two genetically isolated ancient branches of durum wheat were isolated. A “southern” branch included mostly accessions from the Mediterranean region, the Middle East, and Trans-Caucasia. A “northern” branch included Russian and Ukrainian durum wheat accessions and varieties obtained on their basis. An additional group included durum wheat accessions that had been obtained in several past decades on the basis of the material of international breeding centers (CIMMYT and ICARDA) and had low genetic diversity.     

Assessment of genetic diversity and relationship among a collection of US sweet sorghum germplasm by SSR markers

Sweet sorghum (Sorghum bicolor L.) is a type of cultivated sorghums and has been recognized widely as potential alternative source of bio-fuel because of its high fermentable sugar content in the stalk. A substantial variation of sugar content and related traits is known to exist in US sweet sorghum. The objectives of the study were to assess the genetic diversity and relationship among the US sweet sorghum cultivars and lines using SSR markers and to examine the genetic variability within sweet sorghum accessions for sugar content. Sixty-eight sweet sorghum and four grain sorghum cultivars and lines were genotyped with 41 SSR markers that generated 132 alleles with an average of 3.22 alleles per locus. Polymorphism information content (PIC) value, a measure of gene diversity, was 0.40 with a range of 0.03–0.87. The genetic similarity co-efficient was estimated based on the segregation of the 132 SSR alleles. Clustering analysis based on the genetic similarity (GS) grouped the 72 sorghum accessions into 10 distinct clusters. Grouping based on clustering analysis was in good agreement with available pedigree and genetic background information. The study has revealed the genetic relationship of cultivars with unknown parentage to those with known parentage. A number of diverse pairs of sweet sorghum accessions were identified which were polymorphic at many SSR loci and significantly different for sugar content as well. Information generated from this study can be used to select parents for hybrid development to maximize the sugar content and total biomass, and development of segregating populations to map genes controlling sugar content in sweet sorghum.     

全国7省本地早橘基因组间相关性的AFLP分析

采用AFLP分子标记对全国７省的12份本地早橘资源进行遗传多样性分析。使用了28对选择性引物组合,共扩增出882个遗传位点,其中192条（21.8％）为多态性位点;以黄岩本地早橘为对照,各地的本地早橘的多态性位点数不多（3.5%～10.54%）,且来源于浙江省外的本地早橘样品的AFLP多态性位点数要多于浙江省内的,说明区域生态差异对遗传多样性有着一定的影响。对12个样品进行聚类分析结果表明,样品间的遗传距离最大为0.229,说明差异不大,引种后的变异率不高。这些结果有助于揭示其遗传变异与生态条件的关系,对柑橘品种的遗传改良提供参考意见。      

Association mapping of leaf rust response in durum wheat

Resistance to leaf rust (Puccinia triticina Eriks.) is a main objective for durum wheat (Triticum durum Desf.) breeding. Association mapping on germplasm collections is now being used as an additional approach for the discovery and validation of major genes/QTLs. In this study, a collection of 164 elite durum wheat accessions suitable for association mapping has been tested for leaf rust response at the seedling stage and under field conditions (adult plant stage). Seedling tests were carried out with 25 selected isolates from durum wheat, bread wheat and triticale, while field experiments were carried out in artificially inoculated plots in Italy and in Mexico. The collection has been profiled with 225 simple sequence repeat (SSR) loci of known map position and a PCR assay targeting Ppd-A1. Associations showing highly consistent experiment-wise significances across leaf rust isolates and field trials were mainly detected for the 7BL distal chromosome (chr.) region (harbouring Lr14 from cultivar Llareta INIA and QLr.ubo-7B.2 from cultivar Creso) and for two chr. regions located in chrs. 2A and 2B. Additionally, isolate-specific associations and/or associations with smaller effects in the field trials were identified in most of the chromosomes. The chr. 7BL distal region was investigated in detail through haplotyping with 15 SSR markers, revealing that the Creso and Llareta INIA alleles are identical by descent at 6 adjacent SSR loci in the most distal 7BL region spanning 8 cM. Association mapping allowed us to further refine the map location of the Lr14/QLr.ubo-7B.2 resistance gene to the most distal region of the linkage group, tagged by Xcfa2257.2, Xgwm344.2 and Xwmc10. The resistant haplotype is present in a number of accessions (ca. 15% of the accessions included in the collection) from the Italian, CIMMYT and ICARDA breeding programmes. Therefore, this chr. 7BL region can be considered as the most important source of resistance to leaf rust currently exploited by durum breeders in the Mediterranean areas. Furthermore, the field trials at the adult plant stage allowed us to identify marker associations (e.g. chrs. 2BL and 3BS, proximal regions; chr. 7BS, distal region) which suggest the presence of minor QTLs for slow-rusting resistance.     

Characterization of ��/��- and ��-Gliadins in Commercial Varieties and Breeding Lines of Durum Wheat Using MALDI-TOF and A-PAGE Gels

In this work, gliadin composition has been analyzed in 33 accessions of durum wheat using MALDI-TOF MS and compared with A-PAGE results. The MALDI-TOF MS spectra were 29,900-42,500 Da, which corresponds to the α/β- and γ-gliadin regions in A-PAGE. The average of gliadin peaks per line was 23 for MALDI-TOF MS and only 14.8 bands for A-PAGE. MALDI-TOF MS identified 33 gliadin peaks in the durum wheat collection, 20 of which were unique peaks present in 7 lines. A-PAGE analysis identified 30 bands, of which only 4 were unique. Thus, the MALDI-TOF MS method was more sensitive than A-PAGE for identifying α/β- and γ-gliadins in the 33 durum wheat lines studied. Phylogenetic analyses performed using MALDI-TOF MS data assigned the durum wheat lines to two groups. The utility of MALDI-TOF MS to determine relationships among genotypes and for identification of durum wheat accessions is discussed.     

普通小麦Genomic-SSR和EST-SSR分子标记遗传差异及其与系谱遗传距离的比较研究

采用Genomic-SSR和EST—SSR标记技术,对来自我国北方冬麦区的18份普通小麦品种(系)的遗传多样性进行了探讨,并与系谱遗传距离进行了比较分析。研究发现,平均每个Genomic—SSR检测到的等位基因数为3．34个,明显高于EST-SSR的2．31个。遗传距离(GD)计算结果显示,18个小麦基因型之间的EST—SSR平均遗传距离较小,仅为0．3996,低于Genomic—SSR的GD平均值0．5458。尽管EST-SSR揭示出的多态性明显低于Genomic-SSR,但系谱分析和聚类结果均表明,与Genomic—SSR相比,EST—SSR标记能更准确地反映出不同小麦基因型之间的遗传和亲缘关系。据此可以认为,EST—SSR是评价小麦遗传多样性的一种理想标记形式。研究还证实,一个骨干亲本与由其衍生出来的品种(系)之间的遗传差异一般较小,并对拓宽普通小麦遗传基础的策略和方法进行了讨论。     

SSR-based assessment of genetic diversity in South American Vitis vinifera varieties

Six SSR loci, previously developed for grapevine, were analyzed to evaluate the genetic variability and cultivar relatedness in a collection of 25 autochthonous Vitis vinifera varieties from Perú and Argentina.

The number of alleles per locus ranged from 6 to 13, while the number of microsatellites genotypes varied between 9 and 16. The expected heterozygosity varied between 0.71 and 0.89 and the polymorphism information content ranged from 0.70 to 0.88 indicating that the SSRs were highly informative. It was possible to identify 76 different genotypes, with all accessions showing-at least one-specific combination of alleles. Triallelic loci were observed with some SSR. Sequence analysis revealed that variation in the number of repeats and insertion/deletions (InDels) accounted for the polymorphisms observed. Clustering analysis resulted in four separate groups of varieties sharing at least 75% of the markers. A few cases of synonymies were found within the Peruvian accessions. Varieties were clustered following a general pattern of shared morphological and enological traits, rather than geographical origin.