Two EST-derived marker systems for cultivar identification in tree peony

Tree peony (Paeonia suffruticosa Andrews), a woody deciduous shrub, belongs to the section Moutan DC. in the genus of Paeonia of the Paeoniaceae family. To increase the efficiency of breeding, two EST-derived marker systems were developed based on a tree peony expressed sequence tag (EST) database. Using target region amplification polymorphism (TRAP), 19 of 39 primer pairs showed good amplification for 56 accessions with amplicons ranging from 120 to 3,000 bp long, among which 99.3% were polymorphic. In contrast, 7 of 21 primer pairs demonstrated adequate amplification with clear bands for simple sequence repeats (SSRs) developed from ESTs, and a total of 33 alleles were found in 56 accessions. The similarity matrices generated by TRAP and EST-SSR markers were compared, and the Mantel test (r = 0.57778, P = 0.0020) showed a moderate correlation between the two types of molecular markers. TRAP markers were suitable for DNA fingerprinting and EST-SSR markers were more appropriate for discriminating synonyms (the same cultivars with different names due to limited information exchanged among different geographic areas). The two sets of EST-derived markers will be used further for genetic linkage map construction and quantitative trait locus detection in tree peony.     

Genetic diversity of switchgrass and its relative species in Panicum genus using molecular markers

Switchgrass (Panicum virgatum), a warm season C4 grass, is a promising crop for bioenergy-dedicated biomass production. Understanding of genetic diversity within Panicum genus will facilitate switchgrass breeding. Genetic relationships of 22 Panicum species from six continents including ninety-one USDA germplasm accessions were investigated by Sequence-Related Amplified Polymorphism (SRAP) and Expressed Sequence Tags-Simple Sequence Repeat (EST-SSR) markers. Eight hundred and twenty-six markers from 28 pairs of SRAP and 25 pairs of EST-SSR Primers were used to differentiate between accessions of a bulk of 25 genotypes. The results showed that there was high genetic diversity found in Panicum species. Most genetic variation was present among the different species and cluster analysis indicated that all the Panicum accessions could be distinguished by SRAP or EST-SSR. Dendrogram results reflected the phylogenetic relationships between Panicum species and Panicum amarum was found to be the closest species to switchgrass. Comparison between molecular markers revealed that SRAP methods were considered more efficient than EST-SSR for screening Panicum accessions.     

Genetic diversity and population structure of common bean (Phaseolus vulgaris) landraces from China revealed by a new set of EST-SSR markers

A new set of EST-SSR markers were developed and employed to analyze the genetic diversity and population structure of Phaseolus vulgaris in China. A total of 2452 microsatellites were identified in 2144 unigenes assembled from P. vulgaris ESTs, indicating that merely 6.9% of the 30,952 unigene sequences contained SSRs. Seventeen of 153 randomly designed EST-SSR primer pairs successfully amplified polymorphic products in 31 landraces from six major production provinces of China, with the mean number of alleles per locus of 2.700 and polymorphism information content of 0.378. The observed and expected heterozygosity ranged from 0.100 to 0.954 and 0.081 to 0.558, respectively. Using these markers, both an unrooted neighbor-joining tree and principal coordinates analysis showed that almost all of the landraces were separated according with their regional distribution. Moreover, population structure analysis revealed that all genotypes formed into three distinct clusters (k = 3), suggesting that geographic and climatic factors could provide diverse degrees of selection pressure. Accordingly, germplasm collection and cross breeding among different regions are suggested to accelerate the process of diverse germplasm creation and broaden germplasm resources of Chinese common bean.     

香合欢EST-SSR标记开发及种间通用性研究

香合欢是我国南方特有的珍贵用材树种。为了对其种质资源开展群体遗传学研究,该研究根据香合欢转录组测序结果设计开发EST-SSR引物,并在黄豆树、南洋楹、黑木相思、格木等近缘树种中进行通用性分析。结果表明：(1)所开发的243对引物有171对能够成功扩增出目的条带,在香合欢、黄豆树、南洋楹、黑木相思、格木中的有效扩增率分别为63.79%、33.75%、45.68%、41.56%、14.81%;多态性比率分别为23.87%、12.20%、9.01%、3.96%、2.78%;5个物种间均通用的引物有18对。(2)通过验证共获得香合欢SSR多态性标记37个,黄豆树和南洋楹多态性标记均为10个,黑木相思多态性标记4个,格木多态性标记1个。(3)所开发的香合欢EST-SSR标记,可以满足开展香合欢群体遗传学相关研究的需要,并在黄豆树、南洋楹等近缘树种中具有较好的通用性和研究实用性。综上认为,EST-SSR标记可在香合欢、黄豆树、南洋楹、黑木相思、格木等树种的种质资源遗传多样性评价、育种材料指纹图谱构建、群体交配系统分析等方面提供可靠的研究工具,对香合欢种质资源的保护和利用具有重要意义。     

3种不同油桐种仁蛋白质提取方法比较研究

一种有效的蛋白质提取方法是蛋白质双向电泳成功分离的关键。油桐种仁可以用来制取工业用桐油,但其中富含大量的能干扰蛋白质提取的物质,并能影响双向电泳图谱的分辨率。本文中对油桐种仁蛋白质采用丙酮提取（方法A）、苯酚抽提（方法B）以及丙酮—苯酚结合提取（方法C）,并通过蛋白质双向电泳技术对这3种方法的提取效果进行比较研究。结果显示：采用方法C所提取的蛋白质样品,其蛋白浓度高达到8.1 μg·μL^-1;并且该方法对油桐种仁中的高分子量、低分子量蛋白均有较强的提取能力;此外,其蛋白样品经过双向电泳所得到的蛋白质点和图谱分辨率也较其余两种方法好。     

Discrimination and genetic diversity of cultivated and wild safflowers (Carthamus spp.) using EST-microsatellites markers

In spite of being one of the major oilseed crops, little is known about genetic diversity and relationships between species of safflower. In this study EST-SSR markers were used to evaluate and characterize 42 genotypes from six species including Carthamus tinctorius, Carthamus palaestinus, Carthamus oxyacanthus, Carthamus lanatus, Carthamus dentatus, and Carthamus boissieri. Thirty three primer pairs produced 123 polymorphic bands with 2–8 alleles per locus. The EST-SSR markers showed different level of gene diversity. The highest Polymorphism Information Content (PIC) values were observed for primers EL510507 and EL390720 (0.49 and 0.45, respectively). The highest genetic diversity and heterozygosity were observed for C. oxyacanthus. Both cluster and principal coordinate analysis (PCoA) clearly separated species into distinct groups. Within each species the accessions were clustered in different subgroups that mainly supported the known origins. The result showed that C. palaestinus had the most genetic similarity with cultivated safflower and C. oxyacanthus was next in this respect. In general, EST-SSR markers effectively revealed the genetic relationships and diversity of Carthamus species. This information is valuable for safflower improvement since C. palaestinus and C. oxyacanthus are both crossable with the cultivated species C. tinctorius.     

An effective protocol to solve the problem in genomic DNA isolation of tung tree

Tung tree (Vernicia fordii) is a native and oil-producing woody plant in China. The oil is industrially important and promising biodiesel raw material. However, until recently the lack of effective protocols for the extraction of genomic DNA had made DNA-based molecular studies of tung tree difficult. Here, four conventional protocols and one novel protocol were compared for their capacity in isolating DNA from tung tree leaves of different age. Our results showed that all the four conventional protocols could isolate DNA from old leaves, two from matured leaves, but none from young leaves. However, the detectable DNA samples contained many contaminations, leading to overestimation of DNA concentration measured by ultraviolet spectrophotometer, also interfering with the downstream PCR reaction. The novel protocol could produce high-yield and good-quality DNA from tung tree leaves regardless of leaf age. Its key steps were that a single leaf tissue sample could be recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations (termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively) were conducted. All the four DNA samples of a single tissue were good template for PCR reaction. The novel protocol is an effective method for genomic DNA isolation of tung tree.     

Developmental Regulation of Diacylglycerol Acyltransferase Family Gene Expression in Tung Tree Tissues

Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms.     

Development of EST-SSR markers through data mining and their use for genetic diversity study in Indian accessions of Jatropha curcas L.: a potential energy crop

Jatropha curcas L. is gaining importance as a potential energy crop. However, lack of sufficient numbers of molecular markers hinder current research on crop improvement in Jatropha. The expressed sequences tags (EST) sequences deposited in public databases, offers an excellent opportunity to identify simple sequence repeats (SSRs) through data mining, for further research on molecular breeding. In the present study 42,477 ESTs of J. curcas were screened, out of which 5,673 SSRs were identified with 48.8 % simple (excluding mononucleotide repeats) and 52.2 % compound repeat motifs. Amongst these repeat motifs, dinucleotide repeats were abundant (26.5 %), followed by trinucleotide (23.1 %) and tetranucleotide repeats (0.8 %). From these microsatellites, 32 EST-SSR (genic microsatellite) primer pairs were designed. These primers were used to analyze the genetic diversity among 42 accessions collected from different parts of India. Out of the 32 EST-SSR primers, 24 primer pairs exhibited polymorphism among the genotypes, with amplicons varying from one to eight, giving an average of 2.33 alleles per polymorphic marker. Polymorphic information content value ranged from 0.02 to 0.5 with an average of 0.402 indicating moderate level of informativeness within these EST-SSRs markers. The EST-SSR markers developed here will serve as a valuable resource for genetic studies, like linkage mapping, diversity analysis, quantitative trait locus/association mapping, and molecular breeding. The current study also revealed low diversity in the screened Indian Jatropha germplasm. Therefore, the future efforts must be made to broaden the gene pool of Jatropha for the creation of genetic diversity that can be further used for crop improvement through breeding.     

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.     

Genetic Diversity and Characterization of a Core Collection of Malus Germplasm Using Simple Sequence Repeats (SSRs)

Simple sequence repeats (SSRs) were used to assess genetic diversity and study genetic relatedness in a large collection of Malus germplasm. A total of 164 accessions from the Malus core collection, maintained at the University of Illinois, were genotyped using apple SSR markers. Each of the accessions was genotyped using a single robust SSR marker from each of the 17 different linkage groups in Malus. Data were subjected to principal component analysis, and a dendrogram was constructed to establish genetic relatedness. As expected, this diverse core collection showed high allelic diversity; moreover, this allelic diversity was higher than that previously reported. Cluster analysis revealed the presence of four distinct clusters of accessions in this collection.     

Grouping of accessions of Mexican races of maize revisited with SSR markers

Mexican races of maize (Zea mays L.) represent a valuable genetic resource for breeding and genetic surveys. We applied simple sequence repeat (SSR) markers to characterize 25 accessions of races of maize from Mexico. Our objectives were to (1) study the molecular genetic diversity within and among these accessions and (2) examine their relationships as assumed previously on the basis of morphological data. A total of 497 individuals were fingerprinted with 25 SSR markers. We observed a high total number of alleles (7.84 alleles per locus) and total gene diversity (0.61), confirming the broad genetic base of the maize races from Mexico. In addition, the accessions were grouped into distinct racial complexes on the basis of a model-based clustering approach. The principal coordinate analyses of the four Modern Incipient hybrids corroborated the proposed parental races of Chalqueño, Cónico Norteño, Celaya, and Bolita on the basis of the morphological data. Consequently, for some of the accessions, hybridizations provide a clue that can further be used to explain the associations among the Mexican races of maize.     

Heterologous gene silencing induced by tobacco rattle virus (TRV) is efficient for pursuing functional genomics studies in woody plants

Virus-induced gene silencing (VIGS) is an effective tool for studying the functions of plant genes, but only a few VIGS vectors available for woody plants were reported so far. Here we present an effective heterologous VIGS system in woody plants based on tobacco rattle virus (TRV) vectors. We first tested whether the TRV-vector can be directly applied to infect woody plant species, such as Vernicia fordii, Populus tomentosa Carr. and Camellia oleifera. The results revealed that TRV-mediated VIGS could be effectively elicited in V. fordii, weakly in P. tomentosa Carr., but not in C. oleifera. TRV-based VIGS vectors with heterologous phytoene desaturase (PDS) sequences from various woody plant species silenced successfully the endogenous PDS gene in Nicotina benthamiana and V. fordii. The photobleached leaf phenotype of silenced plants significantly correlated with the down-regulation of endogenous PDS as compared with controls. To further confirm the reliability of VIGS in V. fordii, we also isolated the cloroplastos alterados 1 gene from P. tomentosa Carr., and the silencing pheotypes of albino leaves were observed in V. fordii 2 weeks after inoculation using a heterologous TRV-based VIGS system. Taken together, we have successfully developed an Agrobacterium-mediated VIGS assay in V. fordii and demonstrated that V. fordii as a heterologous VIGS system provides a valuable tool for functional genomic analysis in woody plant species.     

Development,cross-species/genera transferability of novel EST-SSR markers and their utility in revealing population structure and genetic diversity in sugarcane

Sugarcane (Saccharum spp. hybrid) with complex polyploid genome requires a large number of informative DNA markers for various applications in genetics and breeding. Despite the great advances in genomic technology, it is observed in several crop species, especially in sugarcane, the availability of molecular tools such as microsatellite markers are limited. Now-a-days EST-SSR markers are preferred to genomic SSR (gSSR) as they represent only the functional part of the genome, which can be easily associated with desired trait. The present study was taken up with a new set of 351 EST-SSRs developed from the 4085 non redundant EST sequences of two Indian sugarcane cultivars. Among these EST-SSRs, TNR containing motifs were predominant with a frequency of 51.6%. Thirty percent EST-SSRs showed homology with annotated protein. A high frequency of SSRs was found in the 5′UTR and in the ORF (about 27%) and a low frequency was observed in the 3′UTR (about 8%). Two hundred twenty-seven EST-SSRs were evaluated, in sugarcane, allied genera of sugarcane and cereals, and 134 of these have revealed polymorphism with a range of PIC value 0.12 to 0.99. The cross transferability rate ranged from 87.0% to 93.4% in Saccharum complex, 80.0% to 87.0% in allied genera, and 76.0% to 80.0% in cereals. Cloning and sequencing of EST-SSR size variant amplicons revealed that the variation in the number of repeat-units was the main source of EST-SSR fragment polymorphism. When 124 sugarcane accessions were analyzed for population structure using model-based approach, seven genetically distinct groups or admixtures thereof were observed in sugarcane. Results of principal coordinate analysis or UPGMA to evaluate genetic relationships delineated also the 124 accessions into seven groups. Thus, a high level of polymorphism adequate genetic diversity and population structure assayed with the EST-SSR markers not only suggested their utility in various applications in genetics and genomics in sugarcane but also enriched the microsatellite marker resources in sugarcane.     

Molecular characterization and genetic structure of Quercus acutissima germplasm in China using microsatellites

Quercus acutissima is native to eastern Asia. It has a wide distribution in China and China is an important component in understanding the ecology and genetic structure of this species. Q. acutissima attained high economic value for hardwood product and can be managed as an energy tree species. To investigate the genetic variation of Q. acutissima provenances, 12 microsatellite primer pairs were used to analyze 672 trees sampled from 28 provenances of Q. acutissima in China. All of the tested microsatellite loci proved to be effective for the studied Q. acutissima provenances. The results revealed that allele numbers varied from 5 to 13 per locus, with an average of 8 alleles per locus. The mean observed heterozygosity and expected heterozygosity were 0.4927 and 0.7023, respectively. The relatedness of the provenances was studied using the arithmetic mean algorithm based on Nei’s genetic distance and principal coordinates analysis. Interestingly, both approaches revealed two main groups: one consisted of the eastern Chinese provenances, and the other comprised of the western Chinese provenances. An analysis of molecular variance indicated that most genetic variation was contained within populations (84 %). The two microsatellite markers developed in this study may be employed for genetic characterization of other oak species. Considering the management or breeding programs of Q. acutissima provenances in China, we should treat each main group as a single gene resource.     

Assessment of genetic diversity in the sorghum reference set using EST-SSR markers

Selection and use of genetically diverse genotypes are key factors in any crop breeding program to develop cultivars with a broad genetic base. Molecular markers play a major role in selecting diverse genotypes. In the present study, a reference set representing a wide range of sorghum genetic diversity was screened with 40 EST-SSR markers to validate both the use of these markers for genetic structure analyses and the population structure of this set. Grouping of accessions is identical in distance-based and model-based clustering methods. Genotypes were grouped primarily based on race within the geographic origins. Accessions derived from the African continent contributed 88.6 % of alleles confirming the African origin of sorghum. In total, 360 alleles were detected in the reference set with an average of 9 alleles per marker. The average PIC value was 0.5230 with a range of 0.1379–0.9483. Sub-race, guinea margaritiferum (Gma) from West Africa formed a separate cluster in close proximity to wild accessions suggesting that the Gma group represents an independent domestication event. Guineas from India and Western Africa formed two distinct clusters. Accessions belongs to the kafir race formed the most homogeneous group as observed in earlier studies. This analysis suggests that the EST-SSR markers used in the present study have greater discriminating power than the genomic SSRs. Genetic variance within the subpopulations was very high (71.7 %) suggesting that the germplasm lines included in the set are more diverse. Thus, this reference set representing the global germplasm is an ideal material for the breeding community, serving as a community resource for trait-specific allele mining as well as genome-wide association mapping.