Haplotype analysis of molecular markers linked to stem rust resistance genes in Ethiopian improved durum wheat varieties and tetraploid wheat landraces

The recent emergence of wheat stem rust race Ug99 (TTKSK) and related strains threaten Ethiopian as well as world wheat production because they overcome widely used resistance genes that had been effective for many years. The major cause which aggravates the ineffectiveness of Ethiopian wheat varieties against stem rust is the narrow genetic base on which the breeding for resistance has been founded, however, little is known about the resistance genotypes of Ethiopian durum wheat varieties and tetraploid wheat landraces. The objective of the study was to identify stem rust resistance genes that are present in the Ethiopian tetraploid wheat landraces and improved durum wheat varieties using molecular markers and assess which genes are effective for current Ethiopian stem rust races of Puccinia graminis f. sp. tritici including Ug99. The investigated 58 tetraploid wheat accessions consisted of 32 (Triticum durum s.l. incl. Triticum aethiopicum Jakubz., Triticum polonicum) landraces and 22 registered T. durum varieties released in Ethiopia between 1966 and 2009 and four T. durum varieties from ICARDA. A total of 17 molecular markers (SSR, EST and InDel) linked or diagnostic for stem rust resistance genes Sr2, Sr13, Sr22 and Sr35 were used for genotyping. Haplotype analysis indicated that only few of the Ethiopian durum wheat varieties carried Sr13. The resistant variety ‘Sebatel’ showed a haplotype for Sr2 and Sr22 and variety ‘Boohai’ for Sr22, however further evaluation is needed for the diagnostic value of these haplotypes. This study is the first report on the presence of stem rust resistance (Sr) genes in Ethiopian durum wheat varieties and tetraploid wheat landraces based on linked or associated molecular markers. Thus it might help in the identification of varieties carrying resistant alleles that provide valuable genetic material for the development of new improved varieties in further breeding programmes.     

Genetic diversity in Ethiopian hexaploid and tetraploid wheat germplasm assessed by microsatellite markers

The genetic diversity of a subset of the Ethiopian genebank collection maintained at the IPK Gatersleben was investigated applying 22 wheat microsatellites (WMS). The material consisted of 135 accessions belonging to the species T. aestivum L. (69 accessions), T. aethiopicum Jacubz. (54 accessions) and T. durum Desf. (12 accessions), obtained from different collection missions. In total 286 alleles were detected, ranging from 4 to 26 per WMS. For the three species T. aestivum, T. aethiopicum and T. durum on average 9.9, 7.9 and 7.9 alleles per locus, respectively, were observed. The average PIC values per locus were highly comparable for the three species analysed. Considering the genomes it was shown that the largest numbers of alleles per locus occurred in the B genome (18.4 alleles per locus) compared to A (10.1 alleles per locus) and D (8.2 alleles per locus) genomes. Genetic dissimilarity values between accessions were used to produce a dendrogram. All accessions could be distinguished, clustering in two large groups. Whereas T. aestivum formed a separate cluster, no clear discrimination between the two tetraploid species T. durum and T. aethiopicum was observed.     

Characterization of stem rust resistance in old tetraploid wheat landraces from the Watkins collection

Stem rust is one of the important diseases of tetraploid wheats worldwide. One hundred and five landraces from the Watkins collection were assessed for seedling and adult plant stem rust response variation. Seedling resistance genes Sr8a, Sr8b, Sr9e, Sr9g, Sr12, Sr13, Sr17 and Sr23 were postulated in 28 genotypes using Australian and Indian Puccinia graminis f. sp. tritici pathotypes. Four genotypes possessed either uncharacterized seedling stem rust resistance gene(s) or combinations of known stem rust resistance genes with compensating avirulences among pathotypes used. Adult plant stem rust response assessments were made on 73 seedling susceptible genotypes in Australia and Ethiopia. Adult plant stem rust responses varied from 1 (very resistant) to 7 (moderately susceptible) on a 1–9 scale. The Ethiopian nursery was exposed to Ug99 (TTKSK) and JRCQC (virulent on Sr9e and Sr13). Some genotypes that exhibited high responses in Australia were scored low in Ethiopia. These results demonstrated the effectiveness of some resistance gene(s) that were ineffective in Australia. The opposite trend was also noted. Over 50 % genotypes exhibited commercially acceptable rust responses varying between 2 and 5 across sites and years. Genotyping with the Sr2-linked molecular marker csSr2 did not detect Sr2 in any of the genotypes. The marker gwm533 however detected the presence of Sr2 in eight genotypes. Stem rust resistant genotypes that carried varying levels of APR could carry new genes. Genetic analysis and deployment of these uncharacterized sources of seedling and APR in new cultivars will ensure durable stem rust control through increased diversity.     

Genetic erosion of Ethiopian tetraploid wheat landraces in Eastern Shewa,Central Ethiopia

Ethiopia is a centre of diversity and hosts rich genetic resources of tetraploid wheats. Through time, the wheat materials were subject to genetic erosion. Closer investigation was made to assess the status of loss, and identify the possible causes by studying two districts from East Shewa. Information from primary and secondary sources was reviewed and analysed. Farmers identified 26 tetraploid wheat landraces (21 from Akaki and 17 from Ejere), which were once widely grown in the area. Of these, only six were currently available. Compared to the formerly available number of landraces, the loss of diversity in the study area was estimated to be 77%. In the Ejere locality, the loss was 100% before the launching of the on farm landrace conservation programme, and for Akaki it was 95%. Major factors that contributed to the loss include: (1) introduction and expansion of bread wheat varieties; (2) expansion of tef; (3) lack of a mechanism to re-supply seeds of tetraploid wheat landraces; (4) decline in size of landholdings; (5) changes in land use and cropping patterns; (6) lack of policy support; and (7) expansion of improved tetraploid wheat varieties. The lessons from this study underscore the importance of strengthening the local seed supply system as a prerequisite for sustaining on farm conservation of landraces. Moreover, it is necessary to initiate diversity studies focusing on the distribution and status of tetraploid wheat landraces across the country. Complementing these by molecular analyses is essential in order to assess the genetic distinctness of the landraces.     

Assessment of EST-microsatellites markers for discrimination and genetic diversity in bread and durum wheat landraces from Afghanistan

Accurate and reliable means for identification are necessary to assess the discrimination between landraces of tetraploid wheat [T.␣turgidum L. subsp. durum (Desf.) Husn.] and hexaploid wheat (T. aestivum L. em. Thell.). In Afghanistan, farmers usually cultivate mixed landraces, and thus distinction between bread and durum is difficult. A set of 18 microsatellites derived from the DuPont EST-database were used to describe genetic diversity in a sample of 82 Afghan wheat landraces. A total of 101 alleles were detected, with allele number per locus ranging from 2 to 13, and a mean allele number of 6.31. The percentage of polymorphic loci was 89%. The EST-SSRs markers showed different level of gene diversity: the highest Polymorphism Information Content value (0.921) was observed with DuPw 221. Our results demonstrated that with a reasonable number of expressed sequences target microsatellites (EST-SSRs) it is possible to discriminate between T. durum and T. aestivum species of wheat germplasm. Our results showed that EST-databases could be a useful source for species-specific markers and have the potential for new genic microsatellites markers that could enhance screening germplasm in gene banks.     

Genetic diversity assessment of Bulgarian durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> Desf.) landraces and modern cultivars using microsatellite markers

The genetic diversity in a Triticum durum Desf. collection, consisting of 102 Bulgarian landraces, nine Bulgarian and 25 introduced cultivars was studied using 14 highly polymorphic microsatellite markers. A total of 100 alleles were identified, with an average of 7.14 alleles per marker. The gene diversity values (He) of the markers for the total samples ranged from 0.23 (WMS357 and WMS631) to 0.77 (WMS46), with an average of 0.52. Within the landraces that were collected from 18 sites in Southern Bulgaria showed 2–11 alleles per locus with an average of 6.07. The microsatellite analysis suggests that the genetic diversity among landraces is lower compared to the diversity levels for durum wheat in countries close to the main centers of wheat domestication. Breeding activities have caused significant reduction of the allelic polymorphism, elimination of rare alleles, and increase in the number of common alleles and the frequency of dominating alleles.     

Patterns of phenotypic diversity for phenologic and qualitative traits in Ethiopian tetraploid wheat germplasm

A total of 4,840 entries of tetraploid wheat germplasm collections representing 121 accessions from Ethiopia were evaluated for phenologic and qualitative trait diversity. The objectives were to assess the diversity patterns of the germplasm with respect to regions, species and altitudinal class. High values of Shannon–Weaver Diversity Index (H′) were recorded for most traits in each region, altitudinal classes, and species. Monomorphism was also high at accession levels. Both H′ and Nei’s gene diversity value for the entire data set (total gene diversity H _T = 0.572; the within accessions gene diversity H _S = 0.112; and gene diversity among accessions D _ST = 0.460) showed the variation for the trait is mainly among accessions/populations rather than within accessions/population. The least mean H′ value over all the traits used for the study was obtained from released varieties (among the origin groups) and Triticum dicoccon (among species). Triticum durum exhibit the highest H′ for a number of traits. Accessions collected from altitudinal class III (2401–2800 m a.s.l.) and class II (2001–2400 m a.s.l.) showed the highest H′ values for a valuable number of traits. Thus classifications using various phenology and qualitative traits enable to identify adaptation of a genotype and would improve the evaluation of genotype for potential adaptation.     

Focused identification of germplasm strategy (FIGS) detects wheat stem rust resistance linked to environmental variables

Recent studies have shown that novel genetic variation for resistance to pests and diseases can be detected in plant genetic resources originating from locations with an environmental profile similar to the collection sites of a reference set of accessions with known resistance, based on the Focused Identification of Germplasm Strategy (FIGS) approach. FIGS combines both the development of a priori information based on the quantification of the trait-environment relationship and the use of this information to define a best bet subset of accessions with a higher probability of containing new variation for the sought after trait(s). The present study investigates the development strategy of the a priori information using different modeling techniques including learning-based techniques as a follow up to previous work where parametric approaches were used to quantify the stem rust resistance and climate variables relationship. The results show that the predictive power, derived from the accuracy parameters and cross-validation, varies depending on whether the models are based on linear or non-linear approaches. The prediction based on learning techniques are relatively higher indicating that the non-linear approaches, in particular support vector machine and neural networks, outperform both principal component logistic regression and generalized partial least squares. Overall there are indications that the trait distribution of resistance to stem rust is confined to certain environments or areas, whereas the susceptible types appear to be limited to other areas with some degree of overlapping of the two classes. The results also point to a number of issues to consider for improving the predictive performance of the models.     

利用改进Faster-RCNN识别小麦条锈病和黄矮病

条锈病和黄矮病是严重威胁小麦生产的重大病害,病害的早期识别对病害防控具有重要意义。现有病害识别模型对相似表型症状识别困难,对早期病害的识别准确度低。为此,该研究构建了一种改进的快速区域卷积神经网络（Faster Regions with CNN Features,Faster-RCNN）的病害识别方法。该方法采用卷积核拆解和下采样延迟策略优化了深度残差网络（Deep Residual Neural Network,ResNet-50）,用优化后的ResNet-50作为主干特征提取网络以增强所提取特征的表达力,同时简化模型的参数;并采用ROI （Region of Interest）Align改进ROI迟化层以降低特征量化误差,提升识别的精度。在自建的涵盖200余种不同发病时期、不同抗感性的小麦叶部图像数据集上进行试验,结果表明：改进的Faster-RCNN识别方法比其他SSD （Single Shot Multi-Box Detector）、YOLO（You Only Look Once）和Faster-RCNN网络模型的平均精度均值（mean Average Precision,mAP）分别提升了9.26个百分点、7.64个百分点和14.97个百分点。对小麦条锈病、黄矮病、健康小麦和其他黄化症状小麦识别的平均精度均值可达98.74%;对小麦条锈病和黄矮病轻、重症识别的平均精度均值可达91.06%。同时,模型损失函数值降低更快,整体性能表现更优。进一步开发小麦病害智能识别系统部署研究模型,使用微信小程序进行田间小麦病害的识别。在最大并发100的条件下,小程序平均返回时延为5.02 s,识别返回成功率为97.85%,对两种小麦病害及其细分轻重症识别的平均准确率为93.56%,能够有效满足实际应用需求,可用于指导病害的科学防控。     

The potential of microsatellites for high throughput genetic diversity assessment in wheat and barley

Microsatellite (SSR) profiles from 65 wheats and 135 barleys have been generated, involving 14 and 22 loci, respectively. The wheat and barley varieties were chosen to represent the bulk of the area sown to these crops in the UK over the past 70 years. The profiling identified genotypic mixtures in some seed samples. Null alleles were common in wheat, but rare in barley. We describe attempts to increase the efficiency of data acquisition. High resolution agarose gel electrophoresis was unable to satisfactorily resolve 1–2 repeat unit differences in the common size range for SSR loci, and was therefore unsuitable for mass screening of allelic variants. Multiplex PCR was very dependent on the choice of primer combinations and seldom produced amplifications as consistently as when primer pairs were used individually. Background (non-specific) amplification was common to many primer pairs, and this hindered the use of both multiplex PCR and multiple sample loading. Sequential sample loading was the most effective strategy, although this was the least time-efficient of the measures used.     

Diversity and geographic distribution of stem solidness and environmental stress tolerance in a collection of durum wheat landraces from Turkey

Variation and geographic distribution of stem solidness and environmental stress tolerance were assessed under dryland conditions in a collection of 2420 durum wheat landrace morphotypes collected in 28 administrative provinces of Turkey. The former trait is known to confer resistance to wheat stem sawfly – a serious pest insect in West Asia and North Africa. Both characters were scored on a visual five-level scale. Score mean values and frequency distributions in the different provinces were significantly different for both traits according to analysis of variance and chi-square test, respectively. Two groups of provinces emerged with high frequency of solid-stemmed, and hollow-stemmed accessions, respectively. The former could be of interest in durum wheat breeding against the sawfly. The group of solid-stemmed province germplasms was collected at lower altitude than the other, being also different on average for higher temperatures and evapotranspiration, and shorter growing season at sites of origin. Overall frequency of morphotypes tolerant to stress conditions prevailing in the evaluation site was rather low (10%). This low frequency may be due to low adaptation of materials in this harsher environment than those of origin where rainfall is usually higher and temperatures markedly lower. Nonetheless, differences among provinces in frequency distribution of stress tolerant morphotypes were evidenced. The relatively more tolerant gene pools originated either along the western coast or in the southern part of the country, along the Fertile Crescent. The least tolerant province germplasms originated either in the North along the Black Sea or in the highlands of the eastern Anatolian Plateau. Germplasm from provinces Malatya and Izmir combined good levels of both stem solidness and stress tolerance.     

Evaluation of Aegilops tauschii Coss. for resistance to wheat stem rust and inheritance of resistance genes in hexaploid wheat

With the objective to identify new sources of resistance to wheat stem rust, a collection of 169 accessions ofAegilops tauschii, obtained from the IPK genebank at Gatersleben, Germany, were screened for resistance undercontrolled conditions. Fourteen (8%) accessions were resistant to stem rust among which 10 were highly resistant(IT 5 ; and 1) and four exhibited a moderately resistance reaction (IT 5 2). From the synthetic hexaploids whichwere produced by hybridizing resistant Ae. tauschii with susceptible Triticum durum, six synthetics expressed ahigh level of stem rust resistance similar to their corresponding diploid parents, while five displayed either areduced or complete susceptibility compared to their Ae. tauschii parents. This suppression of resistance at thehexaploid level suggests the presence of suppressor genes in the A and/ or B genomes of the T. durum parents.Inheritance of resistance from crosses of five stem rust resistant synthetic hexaploids with two susceptible T.aestivum genotypes revealed that three of the synthetics (syn 101, syn 601 and syn 801) possessed one dominantgene each, syn 111 has two different dominant genes and syn 116 has two complementary interacting genes forstem rust resistance. Intercrosses among the four stem rust resistant synthetic hexaploids indicated that the putativegenes conferring stem rust resistance in each of the synthetics are neither allelic nor closely linked to each other.     

Genetic diversity and structure of Ethiopian,Yemen and Brazilian C<Emphasis Type="Italic">offea arabica</Emphasis> L. accessions using microsatellites markers

Genetic diversity among 115 coffee accessions from the Coffea Germplasm Collection of IAC was assessed using SSR markers. The germplasm represents 73 accessions of Coffea arabica derived from spontaneous and subspontaneous plants in Ethiopia and Eritrea, species center of origin and diversity, 13 commercial cultivars of C. arabica developed by the Breeding Program of IAC, 1 accession of C. arabica cv. ‘Geisha’, 13 accessions of C. arabica from Yemen, 5 accessions of C. eugenioides, 4 accessions of C. racemosa and 6 accessions of C. canephora. Genetic analysis was performed using average number of alleles per locus (A), proportion of polymorphic loci (P), Shannon’s genetic index (H′ and G′_ST) and clustering analysis. All evaluated species were distinguished by a cluster analysis based on Jaccard’s coefficient. Differentiation between the cultivated plants of C. arabica and accessions derived from spontaneous and subspontaneous plants was observed. Spontaneous and subspontaneous accessions from Ethiopia were separated according to the geographical origin: east and west of the Great Rift Valley. Cultivated plants showed a low genetic diversity with a division in two groups: accessions from Yemen (H′=0,028) and Brazilian commercial cultivars (H′=0,030). The results agreed with previously reported narrow genetic basis of cultivated plants of C. arabica and supported the hypotheses about domestication of the species. This study also showed a significant genetic diversity among accessions from Ethiopia and Eritrea present in the Germplasm Collection of IAC. This diversity is specially observed in accessions from Sidamo (H′=0,143), Kaffa (H′=0,142) and Illubabor (H′=0,147) indicating their importance as source of genetic variability for coffee breeding programs.     

Analysis of genetic diversity in Tunisian durum wheat cultivars and related wild species by SSR and AFLP markers

Thirty-four durum wheat cultivars representing the Tunisian durum (Triticum durum Desf.) wheat collection and seven wild species of wheat relatives (Triticum turgidum L., T. dicoccon Schrank., T. dicoccoides (Körn) Schweinf., T. araraticum Jakubz., T. monococcum L., Aegilops geniculata Roth, and Aegilops ventricosa Tausch) were analysed with amplified fragment length polymorphism (AFLP) and microsatellite (SSR) markers. Both marker systems used were able to differentiate durum wheat cultivars from the wild relatives and to specifically fingerprint each of the genotypes studied. However, the two marker systems differed in the amount of detected polymorphisms. The 15 SSR markers were highly polymorphic across all the genotypes. The total number of amplified fragments was 156 and the number of alleles per locus ranged from 3 to 24 with an average of 10.4. Two SSR markers alone, Xwms47 and Xwms268, were sufficient to distinguish all 34 durum wheat genotypes. The five AFLP primer pair combinations analysed yielded a total of 293 bands, of which 31% were polymorphic. The highest polymorphic information content (PIC) value was observed for SSRs (0.68) while the highest marker index (MI) value was for AFLPs (7.16) reflecting the hypervariability of the first and the distinctive nature of the second system. For durum wheat cultivars, the genetic similarity values varied between 31.3 and 81% for AFLPs (with an average of 54.2%), and between 3.6 and 72.7% for SSRs (with an average of 19.9%). The rank correlation between the two marker systems was moderate, with r = 0.57, but highly significant. Based on SSR markers, highest genetic similarity (GS) values were observed within the modern cultivars (37.3%), while the old cultivars showed a low level of GS (19.9%). Moreover, the modern cultivars showed low PIC and MI values. UPGMA Cluster analysis based on the combined AFLP and SSR data separated the wild wheat species from the durum wheat cultivars. The modern cultivars were separated from the old cultivars and form a distinct group.     

Phenotypic diversity analysis of grain yield and yellow pigment content in germplasm collected from Iranian durum wheat (Triticum turgidum L.) landraces

This study was undertaken to compare patterns of agronomic characters and yellow pigment content diversity between Iranian durum landraces and modern varieties. In four field experiments, a collection of 127 accessions of landraces and 23 modern cultivars tetraploid wheat representing Iranian and global genetic diversity were tested under optimum and rain-fed conditions. Across the environments, the yellow pigment concentrations ranged from 1.85 mg kg^?1 to 8.95 mg kg^?1 in the field-grown samples. Multivariate analysis detected five groups, four including landraces and one comprising modern cultivars. As a group, modern cultivars were the most productive and showed high mean values for harvest index, grains per spike and yellow pigment, but they had the lowest plant height. Landraces had the highest mean number of spikes per m² and 1000-kernel weight, but were characterized by low plant height and yellow pigment content. Clustering analysis also showed that, based on yield and yellow pigment content, the accessions in different groups were of different origins, suggesting that there was no clear relationship between accessions and geographical diversity. According to our results, Iranian landraces can be particularly useful as germplasm in breeding programmes to improve spikes per m² and 1000-kernel weights.     

Assessment of genetic diversity in emmer (Triticum dicoccon Schrank) × durum wheat (Triticum durum Desf.) derived lines and their parents using mapped and unmapped molecular markers

In the last few years, the renewed interest for emmer wheat (Triticum dicoccon Schrank) in Italy has stimulated breeding programs for this crop releasing improved genotypes obtained not only by selection from landraces, but even by crosses with durum wheat (Triticum durum Desf.) varieties. The purpose of this work has been to uncover the genetic make-up of some emmer × durum derivatives, specifically by comparing the differences from their parents. Genetic diversity of advanced breeding lines and varieties derived from a durum × emmer cross has been evaluated on the basis of AFLP and SSR markers in comparison with the corresponding emmer and durum wheat parent for addressing the seminal question of how much ‘wild’ variation remains after selection for agronomic type.     

Crossability of tetraploid wheat landraces native to Sichuan, Shaanxi, Gansu and Xinjiang provinces, China with rye

The crossability percentages of 131 landraces of wheat (Triticum turgidum L.) native to Sichuan, Shaanxi, Gansu and Xinjiang provinces of China with rye (Secale cereale L.) were tested. 45 landraces showed crossability percentages higher than 5% with 5 landraces expressing crossability percentages over 60%. The geographical distribution of crossability of these tetraploid wheat landraces was reported. Some utilization of high crossability resources was also discussed.