Specific features in using SNP markers developed for allopolyploid wheat

In this work, we analyzed 54 domestic cultivars of hexaploid (common) wheat Triticum aestivum L. (AABBDD genome) and accessions of tetraploid wheats of the Timopheevi group (AAGG) and rye Secale cereale (RR) using 21 SNP markers for common wheat. It was demonstrated that application of the SNP markers developed and verified for particular common wheat cultivars in allele-specific PCR analysis of other cultivars with different geographic origins could lead to an incorrect estimation of the similarity between the genotypes tested. The studied SNP markers of common wheat are inappropriate for analyzing genomes of other cereal species, in particular, T. timopheevii wheats and rye S. cereale.     

Production of wheat-rye substitution lines based on winter rye cultivars with karyotype identification by means of C-banding,GISH, and SSR markers

The study presents a continuation of the research aimed at producing of wheat-rye substitution lines (2n = 42) based on the cross (Triticum aestivum L. × Secale sereale L.) × Triticum aestivum L., and using winter rye cultivars Vyatka and Vietnamskaya Mestnaya. In BC 1 F 5 two lines were identified, having karyotypes in which a pair of homologous wheat chromosomes was substituted by a homeologous pair of rye chromosomes. The chromosome composition of these lines was analyzed using C-banding, GISH, and SSR markers. It was demonstrated that karyotype of each line included a single pair of rye chromosomes and lacked wheat-rye translocations. The rye chromosomes were identified, and the chromosomes of wheat, at which the substitutions occurred, were determined. The lines generated by crosses with rye of Vyatka and Vietnamskaya Mestnaya cultivars were designated 1Rv(1A) and 5Rviet(5A), respectively. Chromosome identification and classification of the lines makes it possible to use them in breeding programs and genetic studies.     

Evolutionary relationships in the genus <Emphasis Type="Italic">Secale</Emphasis> revealed by DArTseq DNA polymorphism

Till this day, there is not much known about the phylogeny of the Secale genus; therefore, in our research, we tried to shed some lights on the issue of rye (Secale genus) taxonomy. The genetic diversity and phylogenetic relationships were evaluated using 13,842 DArTseq? polymorphic markers. The model-based clustering (STRUCTURE software) separated our 84 samples into three main clusters: perennial cluster, annual cluster, and S. sylvestre cluster. The same result was obtained using Neighbour Joining tree and self-organizing maps. Secale sylvestre, S. strictum, and S. cereale are the three main species of the Secale genus. Three samples of rye are in basal positions in phylogenetic trees. These accessions share ancient morphological characters and are probably the ancestors of different lineages within Secale. Annual Secale taxa, with the exception of S. sylvestre, create one mutual taxon. We have found out that the semi-perennial taxa of S. cereale var. multicaule and S. strictum subsp. ciliatoglume are genetically closest to the annual species of S. cereale. Phylogenetic signals for semi-perennial and annual taxa are also present in S. strictum subsp. africanum. SNP-based analysis revealed that evolution of annual S. cereale has already begun in S. strictum subsp. africanum. The results showed that S. vavilovii cannot be considered as a separate species but a subspecies of S. cereale Secale cereale subsp. dighoricum is a hybrid. It is still not clear whether we can consider S. strictum subsp. strictum and S. strictum subsp. kuprijanovii as two separate species.     

Molecular genetic diversity and association mapping of nut and kernel traits in Slovenian hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis>) germplasm

European hazelnut (Corylus avellana L.), cultivated in several areas of the world including Europe, Anatolia, and the USA, is an economically important nut crop due to its high mineral, oleic acid, amino acid, and phenolic compound content and pleasant flavor. This study examined molecular genetic diversity and population structure of 54 wild accessions and 48 cultivars from the Slovenian national hazelnut collection using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Eleven AFLP primer combinations and 49 SSR markers yielded 532 and 504 polymorphic fragments, respectively. As expected for a wind-pollinated, self-incompatible species, levels of genetic diversity were high with cultivars and wild accessions having mean dissimilarity values of 0.50 and 0.60, respectively. In general, cultivars and wild accessions clustered separately in dendrogram, principal coordinate, and population structure analyses with regional clustering of the wild material. The accessions were also characterized for ten nut and seven kernel traits and some wild accessions were shown to have breeding potential. Morphological principal component analysis showed distinct clustering of cultivars and wild accessions. An association mapping panel composed of 64 hazelnut cultivars and wild accessions had considerable variation for the nut and kernel quality traits. Morphological and molecular data were associated to identify markers controlling the traits. In all, 49 SSR markers were significantly associated with nut and kernel traits [P < 0.0001 and LD value (r ²) = 0.15–0.50]. This work is the first use of association mapping in hazelnut and has identified molecular markers associated with important quality parameters in this important nut crop.     

Molecular diversity and genetic relationships in <Emphasis Type="Italic">Secale</Emphasis>

The objective of this study was to quantify the molecular diversity and to determine the genetic relationships among Secale spp. and among cultivars of Secale cereale using RAPDs, ISSRs and sequence analysis of six exons of ScMATE1 gene. Thirteen ryes (cultivated and wild) were genotyped using 21 RAPD and 16 ISSR primers. A total of 435 markers (242 RAPDs and 193 ISSRs) were obtained, with 293 being polymorphic (146 RAPDs and 147 ISSRs). Two RAPD and nine ISSR primers generated more than 80% of polymorphism. The ISSR markers were more polymorphic and informative than RAPDs. Further, 69% of the ISSR primers selected achieved at least 70% of DNA polymorphism. The study of six exons of the ScMATE1 gene also demonstrated a high genetic variability that subsists in Secale genus. One difference observed in exon 1 sequences from S. vavilovii seems to be correlated with Al sensitivity in this species. The genetic relationships obtained using RAPDs, ISSRs and exons of ScMATE1 gene were similar. S. ancestrale, S. kuprijanovii and S. cereale were grouped in the same cluster and S. segetale was in another cluster. S. vavilovii showed evidences of not being clearly an isolate species and having great intraspecific differences.     

Population structure analysis and association mapping of bacterial blight resistance in <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accessions

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a serious disease in rice production worldwide. To understand the genetic diversity of bacterial blight resistance a population consisting of 175 indica accessions from nine countries was collected and detected their association between SSR (Simple Sequence Repeat) markers and resistance to six bacterial races. The resistance phenotypes of various rice accessions were evaluated through artificial inoculation under controlled conditions in 2013 and 2014. Association analysis showed that 17 SSR markers were significantly associated with resistance to four bacterial races and the phenotypic variations explained (PVE) ranged from 7.43 to 15.05%. Among the 17 associated SSR markers, two SSR markers located in previously reported genes regions, and 15 SSR markers were newly identified in this study. These results validated a new approach to map resistance genes of rice to bacterial blight. These markers could be used for marker-assisted selection (MAS) in rice bacterial blight resistance breeding programs.     

Assessment of genetic diversity among okra genotypes using SSR markers

Okra (Abelmoschus esculentus) is an important nutritious vegetable. Despite its high economic and industrial value, very little attention has been paid to assess genetic diversity of okra at molecular level. For effective conservation and proper deployment of germplasm, a study on diversity analysis of okra germplasm was conducted with DNA markers. Microsatellite/Simple sequence repeat (SSR) markers were utilized to evaluate the genetic diversity among 96 accessions of Abelmoschus, of which 92 accessions were of A. esculentus and one accession each of A. tuberculatus, A. moschatus, A. moschatus subspecies tuberosus and A. manihot. A set of 40 SSR primers were tested, of which 30 primers gave reproducible amplification which were used further for diversity analysis. With a mean of 7.1 bands per SSR, DNA amplification with 30 SSRs generated a total 213 bands, of which 60.66 % were recorded polymorphic. Polymorphic information content ranged between 0.11 and 0.80 with an average of 0.52, indicating that the majority of primers were informative. The Jaccard’s coefficient ranged from 0.107 to 0.969. The UPGMA analysis grouped Abelmoschus genotypes into three main clusters at a cut-off of 0.20. Results of present study revealed that sufficient variation exists among the studied accessions and GAO-5 which was found highly diverse can be exploited for okra improvement. The outcome of present research would assist to make use of Ablemoschus germplasm for okra breeding.     

Genetic Diversity in Various Accessions of Pineapple [<Emphasis Type="Italic">Ananas comosus</Emphasis> (L.) Merr.] Using ISSR and SSR Markers

Inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers were used to assess the genetic diversity of 36 pineapple accessions that were introduced from 10 countries/regions. Thirteen ISSR primers amplified 96 bands, of which 91 (93.65%) were polymorphic, whereas 20 SSR primers amplified 73 bands, of which 70 (96.50%) were polymorphic. Nei’s gene diversity (h = 0.28), Shannon’s information index (I = 0.43), and polymorphism information content (PIC = 0.29) generated using the SSR primers were higher than that with ISSR primers (h =  0.23, I = 0.37, PIC = 0.24), thereby suggesting that the SSR system is more efficient than the ISSR system in assessing genetic diversity in various pineapple accessions. Mean genetic similarities were 0.74, 0.61, and 0.69, as determined using ISSR, SSR, and combined ISSR/SSR, respectively. These results suggest that the genetic diversity among pineapple accessions is very high. We clustered the 36 pineapple accessions into three or five groups on the basis of the phylogenetic trees constructed based on the results of ISSR, SSR, and combined ISSR/SSR analyses using the unweighted pair-group with arithmetic averaging (UPGMA) method. The results of principal components analysis (PCA) also supported the UPGMA clustering. These results will be useful not only for the scientific conservation and management of pineapple germplasm but also for the improvement of the current pineapple breeding strategies.     

Assessment of genetic diversity in Ethiopian cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.] germplasm using simple sequence repeat markers

The genetic diversity of cowpea (Vigna unguiculata L. Walp.) in Ethiopia was analyzed using 19 uniform accessions, 62 variable accessions (yielding 185 sub-types), and two mungbean (Vigna radiata) accessions (four subtypes) as outgroup. A set of 23 polymorphic simple sequence repeat (SSR) markers was identified, and polymorphism in the various accessions was scored by determining amplicon variability. Allele frequency, genetic diversity, and polymorphism information content (PIC) were determined for each SSR marker, and a neighbor joining dendrogram was generated to show the genetic relationship among the individual accessions. A total of 75 allelic variants was defined, with the average number of alleles per locus calculated to be three. The average genetic diversity (D) was 0.47, and PIC was 0.4. Three main clusters were identified by phylogenetic analysis, and the clusters and sub-grouping were supported by STRUCTURE and principal component analysis. This grouping had a moderate fixation index value of 0.075 and gene flow (Nm) of 3.176, indicating that the accessions possess wide diversity within individuals and populations. The accessions showed no clustering by geographical origins. Three well-characterized molecular markers (SSR1, C42-2B, and 61RM2) for race specific resistance to Striga gesnerioides in the cowpea cultivar B301 were used to evaluate the accessions for their potential for use in genetic improvement against this pest. Based on this analysis, only two accessions, 222890–2 from Gambela and 286–2 from the Southern Nations, Nationalities, and Peoples (SNNP) region, were found to cluster with B301 and contain the SSR1 resistance allele. These findings will assist in germplasm conservation efforts by the Institute of Biodiversity and Conservation of Ethiopia, and contribute to future studies aimed at the genetic improvement of local germplasm for improved overall agronomic performance as well as Striga resistance in particular.     

Genetic Diversity,Genotype Discrimination,and Population Structure of Mexican <Emphasis Type="Italic">Opuntia</Emphasis> sp., Determined by SSR Markers

The Opuntia (prickly pear) genus, an important horticultural crop in Mexico, is essentially a fruit crop with two variants: sweet (“tunas”) or acid (“xoconostles”) fruits; it is also a source of vegetables “nopalitos” or fodder for livestock, among other uses. Its taxonomical classification has been reported as complex, although few studies on the genetic structure of Mexican Opuntia are available, and genetic differences between the two types of fruits are unknown. Opuntia genotype identification and classification are still mainly based on morphological characters. In this study, the genetic diversity of Mexican Opuntia germplasm with agronomic and economic importance was revealed, using 88 accessions and 13 SSR markers, in an attempt to explore the genetic relationships among them. A total of 159 alleles were detected ranging from 7 to 23 per locus with an average of 12.2. The SSR markers generated unique fingerprints for each Opuntia accession confirming their usefulness for genetic analysis. The accessions’ grouping was defined by several complementary clustering methods, and the moderate incongruences between the different methods did not influence the overall clustering. DAPC and STRUCTURE analyses grouped the accessions into five groups, thus confirming the incorrect delimitation of species in this genus. The following species had no clear boundaries: Opuntia ficus-indica, Opuntia albicarpa, Opuntia megacantha, Opuntia streptacantha, Opuntia lasiacantha, and Opuntia hyptiacantha. However, Opuntia robusta was separated from the rest of the species. Opuntia joconostle and Opuntia matudae, which produce acid fruits, tended to differ from the others. Median-joining simulation classified all genotypes into a complex network, and both linear and reticular ties between Mexican Opuntia genotypes were revealed. The genetic distance revealed in the present study shows the importance of Mexican accessions for conservation and use in breeding programs.     

Molecular Analysis of the Triticale Lines with Different <Emphasis Type="Italic">Vrn</Emphasis> Gene Systems Using Microsatellite Markers and Hybridization In Situ

Hexaploid triticale (×Triticosecale Wittmack) lines were examined using molecular markers and the hybridization in situ technique. Triticale lines were generated based on wheat varieties differing by the Vrn gene systems and the earing times. Molecular analysis was performed using Xgwm and Xrms microsatellite markers with the known chromosomal localization in the common wheat Triticum aestivum, and rye Secale cereale genomes. Comparative molecular analysis of triticale lines and their parental forms showed that all lines contained A and B genomes of common wheat and also rye homoeologous chromosomes. In the three lines the presence of D genome markers, mapped to the chromosomes 2D and 7D, was demonstrated. This was probably the consequence of the translocations of homoeologous chromosomes from wheat genomes, which took part during the process of triticale formation. The data obtained by use of genomic in situ hybridization supported the data of molecular genetic analysis. In none of the lines wheat-rye translocations or recombinations were observed. These findings suggest that the change of the period between the seedling appearance and earing time in triticale lines compared to the initial wheat lines, resulted from the inhibitory effect of rye genome on wheat vernalization genes.     

Molecular and cytological characterization of the global <Emphasis Type="Italic">Musa</Emphasis> germplasm collection provides insights into the treasure of banana diversity

Bananas (Musa spp.) are one of the main fruit crops grown worldwide. With the annual production reaching 144 million tons, their production represents an important contribution to the economies of many countries in Asia, Africa, Latin-America and Pacific Islands. Most importantly, bananas are a staple food for millions of people living in the tropics. Unfortunately, sustainable banana production is endangered by various diseases and pests, and the breeding for resistant cultivars relies on a far too small base of genetic variation. Greater diversity needs to be incorporated in breeding, especially of wild species. Such work requires a large and thoroughly characterized germplasm collection, which also is a safe depository of genetic diversity. The largest ex situ Musa germplasm collection is kept at the International Transit Centre (ITC) in Leuven (Belgium) and currently comprises over 1500 accessions. This report summarizes the results of systematic cytological and molecular characterization of the Musa ITC collection. By December 2015, 630 accessions have been genotyped. The SSR markers confirmed the previous morphological based classification for 84% of ITC accessions analyzed. The remaining 16% of the genotyped entries may need field verification by taxonomist to decide if the unexpected classification by SSR genotyping was correct. The ploidy level estimation complements the molecular data. The genotyping continues for the entire ITC collection, including newly introduced accessions, to assure that the genotype of each accession is known in the largest global Musa gene bank.     

In silico mining of EST-SSRs in <Emphasis Type="Italic">Arachis hypogaea</Emphasis> L. and their utilization for genetic structure and diversity analysis in cultivars/breeding lines in Odisha,India

A total of 26,685 unutilized public domain expressed sequence tags (ESTs) of Arachis hypogaea L. were analyzed to give a total of 4442 EST-SSRs, in which 517 ESTs contained more than one simple sequence repeat (SSR). Of these EST-SSRs, 2542 were mononucleotide repeats (MNRs), 803 were dinucleotide repeats (DNRs), 1043 were trinucleotide repeats (TNRs), 40 were tetranucleotide repeats (TtNRs), six were pentanucleotide repeats (PNRs) and eight were hexanucleotide repeats (HNRs). Out of these 4442 EST-SSRs, only 1160 were found to be successful in non-redundant primer design; 1060 were simple SSRs, while the remaining 100 were compound forms. Among all the motifs, MNRs were abundant, followed by TNRs and DNRs. The AAG/CTT motif was the most abundant (~33 %) TNR, while AG/CT was the most abundant DNR. For redundancy and novelty, a stringent criterion deploying three different strategies was used and a total of 782 novel EST-SSRs were added to the public domain of peanut. These novel EST-SSR markers will be useful for qualitative and quantitative trait mapping, marker-assisted selection and genetic diversity studies in cultivated peanut as well as related Arachis species. A subset of 30 novel EST-SSRs was further randomly selected for validation and genotyping studies with eight well-known cultivars and 32 advanced breeding lines (ADBX lines, ADBY lines and ADBZ lines) from Odisha state, India. The number of polymorphic markers among accessions of A. hypogaea was low; however, a set of informative EST-SSR markers detected considerable levels of genetic variability in peanut cultivars and uncharacterized breeding lines collected from Odisha. The 30 newly developed EST-SSRs from Arachis spp. showed ~97 % amplification in Cicer arientinum and 93 % in pigeon pea. Thus, the EST-SSRs developed in this study will be a very useful asset for genetic analysis, comparative genome mapping, population genetic structure and phylogenetic inferences among wild and allied species of Arachis.     

Analysis of genetic diversity among medicinal therapist <Emphasis Type="Italic">Trigonella foenum</Emphasis>-<Emphasis Type="Italic">graecum</Emphasis> L. genotypes through RAPD and SSR markers

Trigonella is recognized as a medicinal therapist throughout the globe due to its multifaceted rare medicinal properties. It is indigenous from Iran to Northern India but has gained global acceptance towards cultivation and consumption for its yellow-to-amber colored seed which substantially contributes to food, pharmaceutical, nutraceutical and cosmetic industry. Genetic diversity serves as an excellent tool for developing improved crop varieties with breeder preferred traits. Unfortunately, very little information available on variability existing in commercial Trigonella genotypes considerably impedes the crop improvement. In this study, ninety Trigonella genotypes belonging to most productive North Indian states were subjected to multilocus genotyping using RAPD (49) and SSR (13) primers and detected an average of 55.60 and 50.16% polymorphism, respectively. The percentage polymorphism range (RAPD, 16.7–90.90; SSR, 33.30–66.66) average band informativeness (RAPD, 0.182–0.85; SSR, 0.21–0.91) and resolving power (RAPD, 0.95–9.984; SSR, 1.68–7.28) obtained revealed the wide range of diversity prevailing among these genotypes. Hierarchical clustering of genotypes in nine different clusters showed Trigonella’s genetic variability has wide genetic distribution across different agro-climatic zones. No consistency was observed while grouping Trigonella varieties based on eco-geographical region. Eventually, knowledge of these genetic differences significantly contributes in designing intra-specific crosses with potential interest to spice breeding programs. To the best of our knowledge, this is the first report of genetic diversity using SSR molecular markers in Trigonella foenum-graecum L.     

The southwestern origin and eastward dispersal of pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>) in East Asia revealed by comprehensive genetic structure analysis with SSR markers

Pyrus pyrifolia is considered one of the most important cultivated Pyrus species. Hundreds of landraces and bred cultivars have been developed through the natural and artificial hybridizations necessary due to self-incompatibility. In this study, the genetic diversity of 478 Pyrus accessions, including Chinese landraces, bred cultivars, and wild samples, as well as introduced pear cultivars from Japan and Korea, was investigated with a set of 17 simple sequence repeat (SSR) markers distributed across all 17 linkage groups of the pear genome. A total of 121 alleles were detected, including 4 rare alleles with a frequency lower than 5%. Diversity statistics indicated a high level of genetic variation as quantified by the average values of the observed heterozygosity, the expected heterozygosity, and Wright’s fixation index, at 0.76, 0.78, and 0.02, respectively. Population structure and discriminant analysis of principal component analysis implied extensive genetic communication between sand pears in China and revealed four contiguous geographical clusters with overlapping geographical regions. The diversity of the four clusters and approximate Bayesian computation (ABC) indicated that sand pear spread from west to east along the Pearl River and Yangtze River valleys. High diversity and polyphyletic genetic components of cultivars in southwestern China further support southwestern China as the probable center of divergence for Pyrus species. A core collection of 80 out of 470 cultivars was selected, accounting for about 17% of accessions, and capturing 91% of all alleles, including all rare alleles. Our research provides a comprehensive understanding of sand pear germplasm in East Asia and constructs a preliminary core collection, which will be useful for association genetics studies, germplasm conservation, and breeding programs.     

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers for genetic studies in tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most popular non-alcoholic beverage crops worldwide. The availability of complete genome sequences for the Camellia sinensis var. ‘Shuchazao’ has provided the opportunity to identify all types of simple sequence repeat (SSR) markers by genome-wide scan. In this study, a total of 667,980 SSRs were identified in the ~?3.08 Gb genome, with an overall density of 216.88 SSRs/Mb. Dinucleotide repeats were predominant among microsatellites (72.25%), followed by trinucleotide repeats (15.35%), while the remaining SSRs accounted for less than 13%. The motif AG/CT (49.96%) and AT/TA (40.14%) were the most and the second most abundant among all identified SSR motifs, respectively; meanwhile, AAT/ATT (41.29%) and AAAT/ATTT (67.47%) were the most common among trinucleotides and tetranucleotides, respectively. A total of 300 primer pairs were designed to screen six tea cultivars for polymorphisms of SSR markers using the five selected repeat types of microsatellite sequences. The resulting 96 SSR markers that yielded polymorphic and unambiguous bands were further deployed on 47 tea cultivars for genetic diversity assessment, demonstrating high polymorphism of these SSR markers. Remarkably, the dendrogram revealed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or places of origin. The identified genome-wide SSRs and newly developed SSR markers will provide a powerful means for genetic researches in tea plant, including genetic diversity and evolutionary origin analysis, fingerprinting, QTL mapping, and marker-assisted selection for breeding.     

Characterization,genetic diversity,phylogenetic relationships,and expression of the aluminum tolerance <Emphasis Type="Italic">MATE1</Emphasis> gene in <Emphasis Type="Italic">Secale</Emphasis> species

Aluminum (Al) is the main limiting factor for crop production in acidic soils. Efflux of organic acids is one of the mechanisms that determine Al-tolerance, and an Al-activated citrate transporter (multidrug and toxic compound extrusion) MATE1 gene is involved in different species. The contribution of the rye MATE1 gene (ScMATE1) depends on the rye (Secale cereale L.) cultivars and the crosses analyzed; there is no information about different rye species. The cDNA sequences, phylogenetic relationships, Al-tolerance, citrate exudation, and expression of the ScMATE1 gene were analyzed in several cultivars and wild species/subspecies of the Secale genus. Genotypes highly tolerant to Al were found within this genus. For the first time, sequences of the cDNA of the ScMATE1 gene were isolated and characterized in wild ryes. At least two copies of this gene were found likely to be related to Al-tolerance. The sequence comparison of 13 exons of ScMATE1 revealed variability between species, but also inter- and intra-cultivars. Variations were found in the Al-induced expression of ScMATE1 gene, as well as its contribution to Al-tolerance. The pattern of citrate exudation was inducible in most of the species/subspecies studied and constitutive in few. The phylogenetic analysis indicated that ScMATE1 is orthologue of two genes (HvMATE1 and TaMATE1) involved in the Al stress response in barley and wheat, respectively, but not orthologue of SbMATE, implicated in Al-tolerance in sorghum. ScMATE1 is involved in the response to Al stress in ryes, but its contribution to Al-tolerance is complex, and like in other species, there are tolerant and sensitive alleles in the different cultivars and species studied.