DNA Fingerprinting and Genetic Characterization of Anatolian Triticum spp. using AFLP Markers

In this study, genetic analysis of Triticum spp. was carried out using AFLP markers. Six AFLP selective combinations were scored as presence and absence of bands for all the individual samples obtained from a single seed of each accession (70 accessions); T. baeoticum (21), T. monococcum (5), T. urartu (16), T. araraticum (7), T. dicoccoides (16) and T. dicoccon (5), resulting in 506 polymorphic AFLP bands. The phylogenetic tree showed two major clusters; one was composed of T. monococcum (AA) and T. baeoticum (AA), and the other cluster included T. araraticum (AAGG), T. dicoccon (AABB), T. dicoccoides (AABB), and T. urartu (AA). T. urartu, although having a diploid AA genome, did not cluster with other A genome diploids such as T. monococcum and T. baeoticum; instead it clustered together with the tetraploid species, confirming that T. urartu is the A genome progenitor. The extent of variations within and among species is discussed.     

Microsatellite markers – a new tool for distinguishing diploid wheat species

Triticum baeoticum and T. urartu are very similar morphologically. By using microsatellite markers it was possible to distinguish between these two species. Microsatellite markers are, therefore, a powerful new tool to support the determination of critical races in diploid wild wheat species. They also allow the discussion of evolutionary pathways within Triticum.     

Reaction to powdery mildew and stripe rust in related species and landraces of wheat

Field and controlled environmental tests indicated that the 49 accessions of closely related species and 12 landraces of wheat (Triticum aestivum L. em. Thell.) from the National Gene Bank of China showed different reactions to powdery mildew (Blumeria graminis (DC.) E. O. Speer. f. sp. tritici) and stripe rust (Puccinia striiformis Westend f. sp. tritici) at adult and seedling stages. Unknown Pm genes or alleles were postulated with Triticum baeoticum Boiss. accessions BO 3 and Triticum monococcum L. MO 4 and MO 5 when inoculated with 21 powdery mildew isolates at seedling stage. Fourteen accessions of T. baeoticum, T. monococcum, Triticum durum, and wheat landraces were inoculated with 30 stripe rust isolates at seedling stage. Unknown Yr genes or alleles were postulated with T. baeoticum Boiss. accession BO 5, as well as wheat landraces Xiaobaimai, Laomangmai, and Shaanxibai. Heterogeniety in reaction to powdery mildew isolates and stripe rust races were observed in related species and landraces of wheat.     

Genetic Variation Among Portuguese Landraces of ‘Arrancada’ Wheat and <Emphasis Type="Italic">Triticum petropavlovskyi</Emphasis> by AFLP-based Assessment

Portuguese wheat landraces, ‘Arrancada’ were collected from the Aveiro region, Portugal before the 1950s. We found in eight accessions of `Arrancada' hexaploid wheat with the long glume phenotype. We assessed the comparative genetic diversity among Portuguese `Arrancada' wheat and Triticum petropavlovskyi Udacz. et Migusch. using AFLP assays and discuss the origin of long glumed `Arrancada' wheat. With the four primer pairs a total of 4885 visible bands were scored corresponding to 99 AFLP markers as putative loci, of which 55 markers (54%) were polymorphic. UPGMA clustering and PCO grouping showed that long glumed ‘Arrancada’ wheat and T. petropavlovskyi were genetically diverse. Long glumed ‘Arrancada’ hexaploid wheat separated into two clusters (groups) in both the UPGMA dendrogram and in PCO analysis. Four long glumed accessions fell in the cluster of tetraploid wheat. A similar argument could be made for another four accessions which belong to the cluster of hexaploid wheat. The substantial level of genetic variation indicated that long glumed ‘Arrancada’ wheat and T. petropavlovskyi originated independently. It is most likely that the P-gene of long glumed ‘Arrancada’ hexaploid wheat was introduced from T. turgidum ssp. polonicum (L.) Thell. to T. aestivum via natural introgression or breeding. We suggest that the long glumed ‘Arrancada’ hexaploid wheat did not originate from T. aestivum through spontaneous mutation at the P locus     

Cloning and phylogenetic analysis of polyphenol oxidase genes in common wheat and related species

Cloning and phylogenetic analysis of polyphenol oxidase (PPO) genes in common wheat and its relatives would greatly advance the understanding of molecular mechanisms of grain PPO activity. In the present study, six wheat relative species, including T. urartu, T. boeoticum, T. monococcum, T. dicoccoides, T. durum and Ae. tauschii, were sampled to isolate new alleles at Ppo-A1 and Ppo-D1 loci corresponding to common wheat PPO genes, and seven new alleles were identified from these species, which were designated as Ppo-A1c (from T. urartu), Ppo-A1d (T. boeoticum), Ppo-A1e (T. monococcum and T. durum), Ppo-A1f (T. dicoccoides), Ppo-A1g (T. durum), Ppo-D1c (Ae. tauschii) and Ppo-D1d (Ae. tauschii), respectively. Five out of the seven alleles detected in the wheat relatives contained an open reading frame (ORF) of 1,731 bp, encoding a polypeptide of 577 residues, which is the same as those of Ppo-A1 and Ppo-D1 genes in common wheat, whereas, the full-length ORF of the allele Ppo-A1g from T. durum was not obtained, and a 73-bp deletion occurred in the third exon of Ppo-D1d, an allele from Ae. tauschii, resulting in a shorter polypeptide of 466 amino acids. The 191-bp insertion in the first intron reported previously in common wheat was also found in T. dicoccoides lines, implying that more than one tetraploid wheat lines may be involved in the origination of common wheat. Phylogenetic trees were constructed using the genomic DNA sequences of the seven alleles, together with four from common wheat and four partial PPO gene sequences deposited in GenBank. The genome tribe A was divided into two clusters, one of which contained Ppo-A1d and Ppo-A1e, and the other included the remaining five alleles at Ppo-A1 locus. The alleles from different clusters showed high sequence divergences, indicated by dozens of SNPs and five to six InDels. The genome tribe D comprised the alleles Ppo-D1a, Ppo-D1c, Ppo-D1d and Ppo-D1b, and the former three were clustered together, showing significant sequence divergence from Ppo-D1b. In addition, the relationships between these allelic variants and grain PPO activities were also discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Optimum Sample Size for Estimating Gene Diversity in Wild Wheat using AFLP Markers

Genetic diversity in five wild types of wheat was estimated using Simpson's index (based on heterozygosity) applied to data from AFLP markers. For such studies, the cost of obtaining the required information increases both with the number of samples required to estimate diversity and with the number of markers used. When the population studied is in Hardy–Weinberg equilibrium (HWE), allelic frequencies follow the binomial expansion and parametric methods can be used to calculate the variance of the diversity index in terms of the number of individuals sampled. Inbred species are never in HWE. With regard to such populations, this study addresses the question of the sample size required to estimate gene diversity using a distribution-free re-sampling method. We studied populations of five wild species (Aegilops speltoides, Triticum urartu, Triticum boeoticum, Triticum dicoccoides, and Triticum araraticum) as sources of diversity. We used bootstrap re-sampling with varying sample sizes to develop a relationship between the precision of the diversity estimate and the sample size. Such a relationship was used to determine the samples required for capturing a given amount of diversity and its precision. We found that 5–6 samples are sufficient to obtain a standard error equal to 10% of the diversity in the populations of the species Ae. speltoides, T. dicoccoides and T. araraticum. However, more than 12 samples would be needed for populations of T. urartu and T. boeoticum. The procedure presented here can be used to obtain the optimum sample size for other crop species as well.     

Polymorphism and Genetic Diversity for the Seed Storage Proteins in Spanish Cultivated Einkorn Wheat (Triticum monococcum L. ssp. monococcum)

The seed storage protein composition of one collection of cultivated einkorn wheat (Triticum monococcum L. ssp. monococcum) of Spanish origin has been analysed by SDS-PAGE and A-PAGE. Three allelic variants were detected for the Glu-A1^m, whereas up to six alleles were detected for the Glu-A3^m. For the gliadins, 7 and 14 alleles for the Gli-A1^m and Gli-A2^m were found between the evaluated accessions. Internal variability was detected in some of these materials, which could be related to the landrace nature of them. Up to 48 different genotypes based on the origin and seed storage protein composition have been identified. Further researches on these materials must be carried out for determining the variability degree in morphological traits that could complement the evaluation for their safeguard.     

Combined use of gliadins and SSRs to analyse the genetic variability of the Spanish collection of cultivated diploid wheat (Triticum monococcum L. ssp. monococcum)

This work studied the combined use of gliadins and SSRs to analyse inter- and intra-accession variability of the Spanish collection of cultivated einkorn (Triticum monococcum L. ssp. monococcum) maintained at the CRF-INIA. In general, gliadin loci presented higher discrimination power than SSRs, reflecting the high variability of the gliadins. The loci on chromosome 6A were the most polymorphic with similar PIC values for both marker systems, showing that these markers are very useful for genetic variability studies in wheat. The gliadin results indicated that the Spanish einkorn collection possessed high genetic diversity, being the differentiation large between varieties and small within them. Some associations between gliadin alleles and geographical and agro-morphological data were found. Agro-morphological relations were also observed in the clusters of the SSRs dendrogram. A high concordance was found between gliadins and SSRs for genotype identification. In addition, both systems provide complementary information to resolve the different cases of intra-accession variability not detected at the agro-morphological level, and to identify separately all the genotypes analysed. The combined use of both genetic markers is an excellent tool for genetic resource evaluation in addition to agro-morphological evaluation.     

Genetic relationship of diploid wheat (<Emphasis Type="Italic">Triticum</Emphasis> spp.) species assessed by SSR markers

Genetic diversity of 139 accessions of diploid Triticum species including Triticum urartu, Triticum boeoticum and Triticum monococcum was studied using 11 SSR (simple sequence repeats) markers. A total of 111 alleles with an average of 10 alleles per locus were detected. The polymorphism information content (PIC) of each SSR marker ranged from 0.30 to 0.90 with an average value of 0.62. Among the three Triticum species T. urartu had the highest number of total alleles (Na?=?81), private alleles (Npa?=?15) and showed higher genetic diversity (Hex?=?0.58; PIC?=?0.54). The genotypes from Turkey exhibited the highest genetic diversity (PIC?=?0.6), while the least diversity was observed among 4 Georgian accessions (PIC?=?0.11). Cluster analysis was able to distinguish 139 wheat accessions at the species level. The highest genetic similarity (GS) was noted between T. boeticum and T. monococcum (GS?=?0.84), and the lowest between T. urartu and T. monococcum (GS?=?0.46). The grouping pattern of the PCoA analysis corresponded with cluster analysis. No significant differences were found in clustering of T. urartu and T. monococcum accessions with respect to their geographic regions, while within T. boeoticum species, accessions from Iran were somewhat associated with their geographical origin and clustered as a close and separate group. The results from our study demonstrated that SSR markers were good enough for further genetic diversity analysis in einkorn wheat species.     

Divergent evolution of wild and cultivated subspecies of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> as revealed by the study of <Emphasis Type="Italic">PolA1</Emphasis> gene

Triticum timopheevii (genome symbol AAGG) comprises two subspecies, cultivated ssp. timopheevii, and wild ssp. armeniacum. These two subspecies are considered as allotetraploids of AA genome from Triticum diploid species and SS genome from Aegilops species. The difference in genome symbol (G vs. S) is due to wide genetic variations among four SS genome species, Ae. bicornis, Ae. longissima, Ae. searsii, and Ae. speltoides. In order to study the origin of T. timopheevii, we compared 19th intron (PI19) sequence of the PolA1 gene, encoding the largest subunit of RNA polymerase I. Two different sized DNA fragments containing PI19 sequences (PI19A and PI19G) were amplified both in ssp. timopheevii and ssp. armeniacum. Shorter PI19A (112 bp) sequences of both subspecies were identical to PI19 sequences of two AA species, T. monococcum and T. urartu. Interestingly, the longer PI19G (241–243 bp) sequences of ssp. armeniacum showed more similarity to PI19 sequences of Ae. speltoides whereas ssp. timopheevii showed more similarity to PI19 sequences of other three SS genome species. The results indicated that two subspecies of T. timopheevii, ssp. armeniacum or ssp. timopheevii, might have arisen independently by allotetraploidization of AA genome with Ae. speltoides or one of the remaining three Aegilops species, respectively.     

Maintenance of Squash (<Emphasis Type="Italic">Cucurbita</Emphasis> spp.) Landrace Diversity by Farmers' Activities in Mexico

Squash (Cucurbita spp.) is a common component in traditional cropping systems in Mexico, mainly in the agroecosystem known as the “milpa”, in which squash is cultivated in association with maize (Zea mays), the main crop. Using a questionnaire, 80 farmers were interviewed about crop production and selection practices in order to understand how these factors affect genetic diversity of local squash populations. We found that the most of the farmers who cultivate squash were elderly 59.8 ± 14.5 (mean ± SD; n = 78) years old. Squash varieties in the area were exclusively locally adapted landraces, and had not been replaced by modern squash cultivars. Two cultivated squash species, C. argyrosperma ssp. argyrosperma and C. moschata, had been grown intercropped with maize by 97.5% of the interviewed farmers, but only 50.0% were still producing squash at the time of the study. Farmers recognize typical characteristics of particular varieties within each of the local cultivated squash species, and selection is directed to maintain their identity. Nearly two thirds of the farmers (62.0%) had exchanged seeds of squash for planting, a practice that serves to increase genetic variability in the populations. All of the interviewed farmers were conscious of the possible hybridization between the wild gourd (C. argyrosperma ssp. sororia) and their cultivated squash. Despite various natural and human managed factors identified as contributing to enhancement of genetic diversity in these populations, results of the study show that genetic erosion of Cucurbita is likely in the region in the near future.     

Biodiversity of Diploid D-Genome Aegilops Tauschii Coss. in Iran Measured Using Microsatellites

Microsatellite markers were used to analyse the biodiversity of 57 accessions of different subspecies and varieties of wild Aegilops tauschii (2n = 2x = 14; D genome) collected across the major areas where it grows in Iran. Levels of diversity were high, with numbers of alleles averaging 7.3 (ranging up to 12) and polymorphism information contents averaging 0.6591. One accession was notably more similar to two of the D genome in hexaploid wheats (Triticum aestivum) used as outgroups. Within the Ae. tauschii accessions, no markers were characteristic for taxa or geographical origin, suggesting high gene flow between the subspecies and varieties, although some groupings, which could be related to geographical origin, were evident. This survey demonstrates the high diversity present in wild goatgrass in Iran, and indicates that there is value in sampling for useful genes for wheat breeding.     

A wide range of genetic diversity in pear (<Emphasis Type="Italic">Pyrus ussuriensis</Emphasis> var. <Emphasis Type="Italic">aromatica</Emphasis>) genetic resources from Iwate,Japan revealed by SSR and chloroplast DNA markers

Iwateyamanashi (Pyrus ussuriensis var. aromatica) is one of the Pyrus species which grows wild in Japan. The number of Iwateyamanashi trees has been decreasing, so conservation and evaluation is urgently needed. Over 500 accessions of Pyrus species collected from Iwate in northern Tohoku region are maintained at Kobe University as an Iwateyamanashi germplasm collection. In order to investigate the genetic diversity, five SSR (simple sequence repeat) markers, developed from Japanese and European pear were examined for 86 Pyrus individuals including 58 accessions from Iwate. These SSR loci could discriminate between all the Iwate accessions except for 10 that bear seedless fruit, as well as determine the genetic diversity in Iwateyamanashi germplasms. High levels of variation were detected in 41 alleles and the mean observed heterozygosity across 5 loci was 0.50 for the Iwate accessions. Seedless accessions sharing identical SSR genotype with the local pear variety “Iwatetanenashi” were supposed to have been propagated vegetatively via grafting. In an UPGMA phenogram, Japanese pear varieties (P. pyrifolia) were clustered into two groups with some Iwate accessions including seedless ones. Another 38 Iwate accessions were not clustered clearly, and there was no clear relationship between these accessions and geographical distribution or morphological characters. Allele frequency revealed that the Iwate accessions were genetically more divergent than the Japanese pear varieties. Most Japanese pears possessed a 219 bp deletion at a spacer region between the accD and psaI genes in the chloroplast DNA (cpDNA), but other Pyrus species and two Iwateyamanashi trees did not. In the Iwate accessions, 79.3% had a deletion type cpDNA and others had a standard type cpDNA without deletion. These results are indicative of the wide range of genetic diversity in the Iwate accessions which include Japanese pear varieties. A combination of SSR and cpDNA analyses revealed high heterogeneity in Iwateyamanashi and coexistence of Iwateyamanashi and hybrid progeny with P. pyrifolia. These could be reasons for the wide range of continuous morphological variation described previously.     

The origin of the A genome donor of wheats (Triticum: Poaceae) – a perspective based on the sequence variation of the 5S DNA gene units

In a prior study on the haplomes of wheat using the 5S rRNA gene we assigned the long A1 and short A1 unit classes to the A haplome in the diploid T. monococcum. The short A1 unit class is absent in the tetraploids T. turgidum and T. timopheevii and in the hexaploid T. aestivum, although present in the hexaploid T. zhukovskyi. Both T. turgidum and T. aestivum contained a different 5S DNA unit class labeled the short A2.The purpose of this paper was to study the short A2 units in the two diploid species to shed light on the theory that the A haplome donor of T. turgidum and T. aestivum was T. urartu. Fifty eight clones were obtained from 12 accessions, sequenced and analyzed. As expected T. baeoticum, which is often classified as a subspecies of T. monococcum, contained the long A1 and the short A1 5S DNA units. Unexpectedly, T. urartu had the long A1 and the short G1 unit classes instead and other units not found so far in Triticum. These findings support the hypothesis that the donor of the A genome in T. zhukovskyi was T. monococcum, as identified by the short A1 units. However, the short A1 units are absent in T. timopheevii, also a carrier of the A genome. The short G1 units found in T. urartu also identify it as a possible donor of the G genome to T. timopheevii. The short G1 units were also found in T. aestivum in our prior study. The long G1 unit class was not found in T. urartu but reported from T. timopheevii and T. zhukovskyi. The implications of these and related findings on the evolution of wheats are discussed.     

Emmer (Triticum dicoccon Schrank) in Oman†

Emmer (Triticum dicoccon ) was collected recently in northern Oman. The material was analyzed morphologically and phenologically. It belongs to the Asiatic emmers (subsp. asiaticum) and not to the Ethiopian ones (subsp. abyssinicum), distributed in Ethiopia and Yemen, as originally expected. The determination of the material resulted in var. haussknechtianum and var. aeruginosum.     

Powdery Mildew Resistance in a Collection of Chinese Barley Varieties

One hundred and forty-seven Chinese barley varieties maintained at the Gene Bank of the National Barley Improvement Centre, Zhejiang, and 84 progenies from these varieties were tested at the seedling stage for their reaction to 32 selected pathotypes of Blumeria graminis f. sp. hordei. Eighteen resistance spectra were identified comprising single or combined resistances from eight known (Ml(Bw), Ml(Ch), Mla7, Mla8, Mla9, Mla13, MlaRu4 and Mlg) and six unknown resistance genes. The most frequent gene was Ml(Bw), which was found in 69 varieties and previously detected in only a few European winter barley varieties. The genes Mla8 and Ml(Ch) were also often present, but other resistance genes were rare. After inoculation, considerably fewer pathogen colonies were observed in ‘Aiganqi’ and one line of ‘Tong5’. Twenty varieties were composed of lines with different resistance genes. China is likely to be a region of origin of the genes Ml(Bw), Mla7, as well as three unknown genes found in original landraces and perhaps another three unknown genes detected in cultivars bred in China. The resistances of varieties from the Zhejiang province and those originating from 11 other Chinese provinces were quite different. Unfortunately, none of the varieties are promising sources of resistance to powdery mildew and China does not seem to be a region suitable for identifying such sources.     

Genetic characterisation of olive trees from Madeira Archipelago using flow cytometry and microsatellite markers

In this study, native olive plants from Olea maderensis (≡ O. europaea ssp. cerasiformis) and O. cerasiformis (≡ O. europaea ssp. guanchica), wild olives (O. europaea ssp. europaea var. sylvestris) and cultivated olives (O. europaea ssp. europaea var. europaea) were analysed with respect to genome size and microsatellite markers. The mean nuclear DNA content of O. maderensis was estimated as 5.97 ± 0.191 pg/2C, while the remaining studied taxa presented mean genome sizes ranging from 2.99 to 3.18 pg/2C. These data and the obtained simple sequence repeats (SSR) profiles, i.e., with 2–4 alleles in O. maderensis and a maximum of two alleles in the other taxa, enabled the identification of a new ploidy level, tetraploidy, for a species belonging to the Olea genus. Cluster analysis of the microsatellite data revealed a clear separation of each species in different clusters and a high genetic dissimilarity could be observed among genotypes belonging to different species. This work contributed to a better characterization of olive species and the obtained data can be helpful to support taxonomic studies, and to develop germplasm preservation strategies in endangered populations of O. maderensis from Madeira Archipelago.     

Aegilops tauschii: Genetic Variation in Iran

All the 79 Aegilops tauschii Coss. accessions of Iranian origin from Prof. Kihara’s collection were analyzed electrophoretically. Of 23 enzyme-encoding loci studied, 11 were polymorphic. In Iran Ae. tauschii is presented by ssp. tauschii and ssp. strangulata which distinctly differ genetically, morphologically and ecologically. Variation patterns of low polymorphic locus Aco2 and highly polymorphic Ep are similar in both subspecies. In contrast, variation of Acph1, Ak, Est2, Est5, Got1, Got2, Got3 and Lap is a set of diverse patterns which markedly differ between subspecies and natural regions also, implying that natural selection is involved.     

Distribution and diversity of Aegilops tauschii in Iran

The wide morphological variation of Aegilops tauschii has led to the distinction of different subspecies; a typical ssp. tauschii and a second ssp. strangulata. However some researchers pointed out the existance of the intermediate form among morphologically distinguished subspecies. Distribution, diversity and the relationship between different subspecies and the intermediate form were evaluated in the Iranian Ae. tauschii collection. This collection was classified to 15 different populations according to morphological similarities and the collecting origin of accessions. The highest variation was found in tauschii population of Golestan followed by tauschii populations of Gilan and Ardebill, whereas the lowest variation was observed in tauschii populations of central Iran. Two discriminant functions suggested that the length of rachis node and spikelet glume, particularly, the length/width ratios of these traits had the highest impact on identification of different forms. Mahalanobis distances (D ² ) between the two subspecies along with intermediate form on the multidimensional scaling plot showed that the intermediate form is more similar to ssp. tauschii than ssp. strangulata. Although, the diversity within the ssp. strangulata was not very high, it widely affected the diversity of Iranian accessions of Ae. tauschii through continues crossing with the more diversed subspecies, tauschii, during thousands of years. This fact had lead to expansion of its distribution from its origin to Northern Khorasan, Northern Semnan and Eastern Ardebill by producing the intermediate form.     

Genetic diversity in populations of wild diploid wheat Triticum urartu Tum. ex. Gandil. revealed by isozyme markers

Genetic variation and its distribution within and among 23 populations of Triticum urartu collected from Syria, Lebanon, Turkey, Armenia, and Iran was estimated using isozyme markers at eight polymorphic loci. The number of alleles per locus (A= 1.21), percentage polymorphic loci (P= 20.1%), and mean gene diversity (H_e= 0.024) were relatively low. In a population from Lebanon, a high number of alleles per locus (A= 2.13) and percentage polymorphic loci (P= 87.5%) was found. On average, genetic variation among populations (G_ST= 0.407) was smaller than within-population variation (0.593). However, different patterns of genetic structure were found among various geographic regions. Interpopulation variation was highest for the Iranian populations (0.89) followed by the Turkish populations (0.66). A reverse pattern was observed for the Syrian (0.11) and for the Lebanese (0.13) populations. The Armenian populations exhibited similar interpopulation and within-population variation. Principal component and cluster analyses resulted in distinct grouping of the geographically proximal populations, with the exception of the two Iranian populations. The Turkish populations were different from the neighboring Armenian populations compared to other countries. The populations from southern Syria and those from Lebanon also exhibited a high degree of genetic diversity. The two most heterozygous loci, Mdh-2 and Pgi-2, separated the populations along the first and second principal components, respectively. Most of the rare alleles were scattered sporadically throughout the geographic regions. Rare alleles with high frequencies were found in the Turkish and Armenian populations. These results indicated that different geographic regions require specific sampling procedures in order to capture the range of genetic variation observed in T. urartu populations.