Genetic diversity analysis in the section Caulorrhizae (genus Arachis) using microsatellite markers

Diversity in 26 microsatellite loci from section Caulorrhizae germplasm was evaluated by using 33 accessions of A. pintoi Krapov. & W.C. Gregory and ten accessions of Arachis repens Handro. Twenty loci proved to be polymorphic and a total of 196 alleles were detected with an average of 9.8 alleles per locus. The variability found in those loci was greater than the variability found using morphological characters, seed storage proteins and RAPD markers previously used in this germplasm. The high potential of these markers to detect species-specific alleles and discriminate among accessions was demonstrated. The set of microsatellite primer pairs developed by our group for A. pintoi are useful molecular tools for evaluating Section Caulorrhizae germplasm, as well as that of species belonging to other Arachis sections.     

Evaluation of genetic variability in the collared peccary Pecari tajacu and the white-lipped peccary Tayassu pecari by microsatellite markers

In this study, the microsatellite technique was used to evaluate the genetic variability in populations of collared and white-lipped peccaries kept in captivity. Six primers developed for domestic pigs were used and amplified in both species. They revealed the presence of five polymorphic loci and one monomorphic locus. The polymorphic loci included 4 of the 16 alleles in collared peccaries, and 3 of the 10 alleles in the white-lipped peccaries. Polymorphic information content (PIC) in both species and all the loci was highly informative. The probability of paternity exclusion (PEC), if one of the parents is known, was almost as high in white-lipped peccaries (95.53%) as in the collared (99,48%). The Fst values for collared (0.042) and white-lipped (0.1387) peccaries showed that both populations are not structured. The Fis values for all loci, except ACTG2 in white-lipped peccaries (-0.0275) and in both species (0.1985 to 0.9284 in collared peccaries and 0.3621 to 0.4754 in the white-lipped), revealed a high level of homozygosis, probably caused by inbreeding. Data on heterologous amplification and genetic variability in collared and white-lipped peccaries are presented for the first time.     

Search for microsatellite markers associated with water-stress tolerance in wheat through bulked segregant analysis

We used bulked segregant analysis (BSA) to identify microsatellite markers associated with water-stress tolerance in wheat. Two DNA pools (tolerant and sensitive) were established from the selected F₂ individuals of crosses between water-stress-tolerant and -sensitive wheat parental genotypes on the basis of the paraquat (PQ) tolerance, leaf size, and relative water content. All three traits were previously shown to be associated with water-stress tolerance on segregating F₂ progeny of the wheat crosses used in this study. Microsatellite analysis was then performed on the established DNA pools, using 35 primer pairs that included all of the chromosome group 5 (5A, 5B, 5D) markers, to detect microsatellite fragments that were present, absent, or both in the DNA pools and their parental lines. We identified one microsatellite fragment that was present in tolerant parent wheat and the tolerant bulk but absent in the sensitive parent wheat and sensitive bulk. We then followed the segregation of this marker in the tolerant F₂ individuals. Use of this marker may significantly enhance the success of selection for PQ- and water-stress-tolerant genotypes in wheat breeding programs.     

Retention of D genome chromosomes in pentaploid wheat crosses

The transfer of genes between Triticum aestivum (hexaploid bread wheat) and T. turgidum (tetraploid durum wheat) holds considerable potential for genetic improvement of both these closely related species. Five different T. aestivum/T. turgidum ssp. durum crosses were investigated using Diversity Arrays Technology (DArT) markers to determine the inheritance of parental A, B and D genome material in subsequent generations derived from these crosses. The proportions of A, B and D chromosomal segments inherited from the hexaploid parent were found to vary significantly among individual crosses. F(2) populations retained widely varying quantities of D genome material, ranging from 99% to none. The relative inheritance of bread wheat and durum alleles in the A and B genomes of derived lines also varied among the crosses. Within any one cross, progeny without D chromosomes in general had significantly more A and B genome durum alleles than lines retaining D chromosomes. The ability to select for and manipulate this non-random segregation in bread wheat/durum crosses will assist in efficient backcrossing of selected characters into the recurrent durum or hexaploid genotype of choice. This study illustrates the utility of DArT markers in the study of inter-specific crosses to commercial crop species.     

Waxy genes from spelt wheat: new alleles for modern wheat breeding and new phylogenetic inferences about the origin of this species

Background and Aims

Waxy proteins are responsible for amylose synthesis in wheat seeds, being encoded by three waxy genes (Wx-A1, Wx-B1 and Wx-D1) in hexaploid wheat. In addition to their role in starch quality, waxy loci have been used to study the phylogeny of wheat. The origin of European spelt (Triticum aestivum ssp. spelta) is not clear. This study compared waxy gene sequences of a Spanish spelt collection with their homologous genes in emmer (T. turgidum ssp. dicoccum), durum (T. turgidum ssp. durum) and common wheat (T. aestivum ssp. aestivum), together with other Asian and European spelt that could be used to determine the origin of European spelt.

Methods

waxy genes were amplified and sequenced. Geneious Pro software, DNAsp and MEGA5 were used for sequence, nucleotide diversity and phylogenetic analysis, respectively.

Key Results

Three, four and three new alleles were described for the Wx-A1, Wx-B1 and Wx-D1 loci, respectively. Spelt accessions were classified into two groups based on the variation in Wx-B1, which suggests that there were two different origins for the emmer wheat that has been found to be part of the spelt genetic make-up. One of these groups was only detected in Iberian material. No differences were found between the rest of the European spelt and the Asiatic spelt, which suggested that the Iberian material had a different origin from the other spelt sources.

Conclusions

The results suggested that the waxy gene variability present in wheat is undervalued. The evaluation of this variability has permitted the detection of ten new waxy alleles that could affect starch quality and thus could be used in modern wheat breeding. In addition, two different classes of Wx-B1 were detected that could be used for evaluating the phylogenetic relationships and the origins of different types of wheat.     

Genetic diversity of carotenoid-rich bananas evaluated by Diversity Arrays Technology (DArT)

The aim of this work was to evaluate the carotenoid content and genetic variability of banana accessions from the Musa germplasm collection held at Embrapa Cassava and Tropical Fruits, Brazil. Forty-two samples were analyzed, including 21 diploids, 19 triploids and two tetraploids. The carotenoid content was analyzed spectrophotometrically and genetic variability was estimated using 653 DArT markers. The average carotenoid content was 4.73 μg.g (-1) , and ranged from 1.06 μg.g (-1) for the triploid Nanica (Cavendish group) to 19.24 μg.g (-1) for the triploid Saney. The diploids Modok Gier and NBA-14 and the triploid Saney had a carotenoid content that was, respectively, 7-fold, 6-fold and 9-fold greater than that of cultivars from the Cavendish group (2.19 μg.g (-1)). The mean similarity among the 42 accessions was 0.63 (range: 0.24 to 1.00). DArT analysis revealed extensive genetic variability in accessions from the Embrapa Musa germplasm bank.     

Genetic structure and a selected core set of Brazilian soybean cultivars

Soybean is one of the most valuable and profitable oil crop species and a thorough knowledge of the genetic structure of this crop is necessary for developing the best breeding strategies. In this study, a representative collection of soybean cultivars recommended for farming in all Brazilian regions was genotyped using 27 simple sequence repeat (SSR) loci. A total of 130 alleles were detected, with an average allelic number of 4.81 per locus. These alleles determined the core set that best represented this soybean germplasm. The Bayesian analysis revealed the presence of two clusters or subgroups within the whole collection (435 soybean cultivars) and the core set (31 entries). Cultivars of similar origin (ancestral) were clustered into the same groups in both analyses. The genetic diversity parameters, based on the SSR loci, revealed high similarity between the whole collection and core set. Differences between the two clusters detected in the core set were attributed more to the frequency of their ancestors than to their genetic base. In terms of ancestry, divergent groups were presented and a panel is shown which may foster efficient breeding programs and aid soybean breeders in planning reliable crossings in the development of new varieties.     

Genetic variability of Old Portuguese bread wheat cultivars assayed by IRAP and REMAP markers

Retrotransposons (RTNs) constitute informative molecular markers for plant species as a result of their ability of integrating into a multitude of loci throughout the genome and thereby generating insertional polymorphisms between individuals. Inter-retrotransposon amplified polymorphisms (IRAPs) and the retrotransposon-microsatellite amplified polymorphisms (REMAPs) are marker systems based on long terminal repeats (LTRs) RTNs, developed for plants, that have been widely used for evolution, genetic diversity, DNA fingerprinting of cultivars and varieties, genetic mapping linkage and for detection of genetic rearrangements induced by polyploidisation. In the present study, we aimed to analyse the genetic variability among 48 Old Portuguese bread wheat cultivars using both IRAP and REMAP markers. Five IRAP and six REMAP primer combinations were used. IRAP produced 103 polymorphic fragments in a total of 113 bands. On average, 22.6 bands were amplified per IRAP primer combination. The bands ranged in size from 250 to 5000 bp. The REMAP primer combinations allowed the amplification of 53 bands, 51 of them polymorphic. An average of 8.8 REMAP bands was scored per primer combination. The REMAP bands ranged from 250 to 3000 bp. Both marker systems presented high percentages of polymorphism. However, IRAP markers were suitable for detecting genetic variability at the individual level and did not differentiate higher taxa. The REMAP maker system allowed the clustering by botanical variety and identified most of the homonym bread wheat cultivars.     

Engineering greater aluminium resistance in wheat by over-expressing TaALMT1

MethodsParticle bombardment was used to transform wheat with TaALMT1, the Al³⁺ resistance gene from wheat, using the maize ubiquitin promoter to drive expression. TaALMT1 expression, malate efflux and Al³⁺ resistance were measured in the T₁ and T₂ lines and compared with the parental line and an Al³⁺-resistant reference genotype, ET8.ConclusionsThe Al³⁺ resistance of wheat was increased by enhancing TaALMT1 expression with biotechnology. This is the first report of a major food crop being stably transformed for greater Al³⁺ resistance. Transgenic strategies provide options for increasing food supply on acid soils.     

Genetic diversity in Capsicum germplasm based on microsatellite and random amplified microsatellite polymorphism markers

A sound knowledge of the genetic diversity among germplasm is vital for strategic germplasm collection, maintenance, conservation and utilisation. Genomic simple sequence repeats (SSRs) and random amplified microsatellite polymorphism (RAMPO) markers were used to analyse diversity and relationships among 48 pepper (Capsicum spp.) genotypes originating from nine countries. These genotypes covered 4 species including 13 germplasm accessions, 30 improved lines of 4 domesticated species and 5 landraces derived from natural interspecific crosses. Out of 106 SSR markers, 25 polymorphic SSR markers (24 %) detected a total of 76 alleles (average, 3.04; range, 2–5). The average polymorphic information content (PIC) was 0.69 (range, 0.29–0.92). Seventeen RAMPO markers produced 87 polymorphic fragments with average PIC of 0.63 (range, 0.44–0.81). Dendrograms based on SSRs and RAMPOs generated two clusters. All 38 Capsicum annuum genotypes and an interspecific landrace clustered together, whereas nine non-annuum (three Capsicum frutescens, one Capsicum chinense, one Capsicum baccatum and four interspecific landraces) genotypes clustered separately. Genetic variation within non-annuum genotypes was greater than the C. annuum genotypes. Distinctness of interspecific derivative landraces grown in northeast India was validated; natural crossing between sympatric Capsicum species has been proposed as the mechanism of their origin.     

Genetic diversity in Chinese modern wheat varieties revealed by microsatellite markers

Genetic diversity of 1680 modern varieties in Chinese candidate core collections was analyzed at 78 SSR loci by fluorescence detection system. A total of 1336 alleles were detected, of which 1253 alleles could be annotated into 71 loci. For these 71 loci, the alleles ranged from 4 to 44 with an average of 17.6, and the PIC values changed from 0.19 to 0.89 with an average of 0.69. (1) In the three genomes of wheat, the average genetic richness was B>A>D, and the genetic diversity indexes were B>D>A. (2) Among the seven homoeologous groups, the average genetic richness was 2=7>3>4>6>5>1, and the genetic diversity indexes were 7>3>2>4>6>5>1. As a whole, group 7 possessed the highest genetic diversity, while groups 1 and 5 were the lowest. (3) In the 21 wheat chromosomes, 7A, 3B and 2D possessed much higher genetic diversity, while 2A, 1B, 4D, 5D and 1D were the lowest. (4) The highest average genetic diversity index existed in varieties bred in the 1950s, and then it declined continually. However, the change tendency of genetic diversity among decades was not greatly sharp. This was further illustrated by changes of the average genetic distance between varieties. In the 1950s it was the largest (0.731). Since the 1960s, it has decreased gradually (0.711, 0.706, 0.696, 0.695). The genetic base of modern varieties is becoming narrower and narrower. This should be given enough attention by breeders and policy makers.     

Genetic diversity in Chinese modern wheat varieties revealed by microsatellite markers

Genetic diversity of 1680 modern varieties in Chinese candidate core collections was analyzed at 78 SSR loci by fluorescence detection system. A total of 1336 alleles were detected, of which 1253 alleles could be annotated into 71 loci. For these 71 loci, the alleles ranged from 4 to 44 with an average of 17.6, and the PIC values changed from 0.19 to 0.89 with an average of 0.69. (1) In the three genomes of wheat, the average genetic richness was B>A>D, and the genetic diversity indexes were B>D>A. (2) Among the seven homoeologous groups, the average genetic richness was 2=7>3>4>6>5>1, and the genetic diversity indexes were 7>3>2>4>6>5>1. As a whole, group 7 possessed the highest genetic diversity, while groups 1 and 5 were the lowest. (3) In the 21 wheat chromosomes, 7A, 3B and 2D possessed much higher genetic diversity, while 2A, 1B, 4D, 5D and 1D were the lowest. (4) The highest average genetic diversity index existed in varieties bred in the 1950s, and then it declined continually. However, the change tendency of genetic diversity among decades was not greatly sharp. This was further illustrated by changes of the average genetic distance between varieties. In the 1950s it was the largest (0.731). Since the 1960s, it has decreased gradually (0.711, 0.706, 0.696, 0.695). The genetic base of modern varieties is becoming narrower and narrower. This should be given enough attention by breeders and policy makers.     

Genetic diversity assessment in greek Medicago truncatula genotypes using microsatellite markers

In this study we examined the genetic diversity and geographic scale of genotype distribution within the model legume species Medicago truncatula widely distributed in pasture and marginal agricultural lands in Greece and other Mediterranean countries. Thirty one Medicago truncatula and Medicago littorialis accessions were chosen on the basis of their geographical distributions and studied using 9 polymorphic simple sequence repeats (SSR) markers. The number of alleles per locus varied between 3 and 7. A total of 42 alleles were detected with a mean value of 4.66 alleles per locus. Geographic origin was not related with genotypic similarity among accessions. However, there were instances of close genetic relatedness between accessions from neighboring locations in a geographic compartment. In conclusion, the presented data revealed extensive M. truncatula genotype dispersal in Greece pointing to the significance of preserving local genetic resources in their natural environment.     

Abundance, variability and chromosomal location of microsatellites in wheat

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)_n blocks was estimated to be 3.6 x 10⁴ and the number of (GT)_n blocks to be 2.3 x 10⁴ per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.     

Genetic relatedness of Portuguese almond cultivars assessed by RAPD and ISSR markers

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyse the genetic diversity of Portuguese Prunus dulcis cultivars and their relationship to important foreign cultivars. Of the primers tested, 6 (out of 60) RAPD and 5 (out of 18) ISSR primers were selected for their reproducibility and high polymorphism. Out of 124 polymerase chain reaction fragments that were scored, 120 (96.8%) were polymorphic. All the plants could be discriminated and constitute a very heterogeneous group. Five unidentified almond plants found in the region of Foz Côa (north Portugal) and wild almond (P. webbii) from Italy and Spain were also included. Four main groups of plants could be distinguished: P. dulcis cultivars; one Foz Côa plant; P. webbii; and P. persica (outgroup). The segregating Foz Côa plant may represent a feral individual or a hybrid between P. dulcis and P. webbii.Abbreviations dNTP Deoxynucleotide triphosphate - CTAB Cetyltrimethylammonium bromide - ISSR Inter-simple sequence repeats - PCR Polymerase chain reaction - RAPD Randomly amplified polymorphic DNA - RASTM Regional Agricultural Services of Trás-os-Montes - TE Tris-EDTA buffer - UPGMA Unweighted pair group method with arithmetical averagesCommunicated by P. Puigdoménech     

Assessment of genetic diversity in Brazilian barley using SSR markers

Barley is a major cereal grown widely and used in several food products, beverage production and animal fodder. Genetic diversity is a key component in breeding programs. We have analyzed the genetic diversity of barley accessions using microsatellite markers. The accessions were composed of wild and domesticated barley representing genotypes from six countries and three breeding programs in Brazil. A total of 280 alleles were detected, 36 unique to Brazilian barley. The marker Bmag120 showed the greatest polymorphism information content (PIC), with the highest mean value found on chromosome three, and the lowest on chromosomes four and six. The wild accessions presented the highest diversity followed by the foreign genotypes. Genetic analysis was performed using Principal Coordinates Analysis, UPGMA clustering, and Bayesian clustering analysis implemented in Structure. All results obtained by the different methods were similar. Loss of genetic diversity has occurred in Brazilian genotypes. The number of alleles detected in genotypes released in 1980s was higher, whereas most of the cultivars released thereafter showed lower PIC and clustered in separate subgroups from the older cultivars. The use of a more diverse panel of genotypes should be considered in order to exploit novel alleles in Brazilian barley breeding programs.     

Genetic diversity for grain nutrients in wild emmer wheat: potential for wheat improvement

Background and Aims

Micronutrient malnutrition, particularly zinc and iron deficiency, afflicts over three billion people worldwide due to low dietary intake. In the current study, wild emmer wheat (Triticum turgidum ssp. dicoccoides), the progenitor of domesticated wheat, was tested for (1) genetic diversity in grain nutrient concentrations, (2) associations among grain nutrients and their relationships with plant productivity, and (3) the association of grain nutrients with the eco-geographical origin of wild emmer accessions.

Methods

A total of 154 genotypes, including wild emmer accessions from across the Near Eastern Fertile Crescent and diverse wheat cultivars, were characterized in this 2-year field study for grain protein, micronutrient (zinc, iron, copper and manganese) and macronutrient (calcium, magnesium, potassium, phosphorus and sulphur) concentrations.

Key Results

Wide genetic diversity was found among the wild emmer accessions for all grain nutrients. The concentrations of grain zinc, iron and protein in wild accessions were about two-fold greater than in the domesticated genotypes. Concentrations of these compounds were positively correlated with one another, with no clear association with plant productivity, suggesting that all three nutrients can be improved concurrently with no yield penalty. A subset of 12 populations revealed significant genetic variation between and within populations for all minerals. Association between soil characteristics at the site of collection and grain nutrient concentrations showed negative associations between soil clay content and grain protein and between soil-extractable zinc and grain zinc, the latter suggesting that the greatest potential for grain nutrient minerals lies in populations from micronutrient-deficient soils.

Conclusions

Wild emmer wheat germplasm offers unique opportunities to exploit favourable alleles for grain nutrient properties that were excluded from the domesticated wheat gene pool.