Xingfei Zheng

Development of microsatellite markers by transcriptome sequencing in two species of Amorphophallus (Araceae)

BackgroundAmorphophallus is a genus of perennial plants widely distributed in the tropics or subtropics of West Africa and South Asia. Its corms contain a high level of water-soluble glucomannan; therefore, it has long been used as a medicinal herb and food source. Genetic studies of Amorphophallus have been hindered by a lack of genetic markers. A large number of molecular markers are required for genetic diversity study and improving disease resistance in Amorphophallus. Here, we report large scale of transcriptome sequencing of two species: Amorphophallus konjac and Amorphophallus bulbifer using deep sequencing technology, and microsatellite (SSR) markers were identified based on these transcriptome sequences.ResultscDNAs of A. konjac and A. bulbifer were sequenced using Illumina HiSeq™ 2000 sequencing technology. A total of 135,822 non-redundant unigenes were assembled from about 9.66 gigabases, and 19,596 SSRs were identified in 16,027 non-redundant unigenes. Di-nucleotide SSRs were the most abundant motif (61.6%), followed by tri- (30.3%), tetra- (5.6%), penta- (1.5%), and hexa-nucleotides (1%) repeats. The top di- and tri-nucleotide repeat motifs included AG/CT (45.2%) and AGG/CCT (7.1%), respectively. A total of 10,754 primer pairs were designed for marker development. Of these, 320 primers were synthesized and used for validation of amplification and assessment of polymorphisms in 25 individual plants. The total of 275 primer pairs yielded PCR amplification products, of which 205 were polymorphic. The number of alleles ranged from 2 to 14 and the polymorphism information content valued ranged from 0.10 to 0.90. Genetic diversity analysis was done using 177 highly polymorphic SSR markers. A phenogram based on Jaccard’s similarity coefficients was constructed, which showed a distinct cluster of 25 Amorphophallus individuals.ConclusionA total of 10,754 SSR markers have been identified in Amorphophallus using transcriptome sequencing. One hundred and seventy-seven polymorphic markers were successfully validated in 25 individuals. The large number of genetic markers developed in the present study should contribute greatly to research into genetic diversity and germplasm characterization in Amorphophallus.

QTL Mapping of Combining Ability and Heterosis of Agronomic Traits in Rice Backcross Recombinant Inbred Lines and Hybrid Crosses

Background Combining ability effects are very effective genetic parameters in deciding the next phase of breeding programs. Although some breeding strategies on the basis of evaluating combining ability have been utilized extensively in hybrid breeding, little is known about the genetic basis of combining ability. Combining ability is a complex trait that is controlled by polygenes. With the advent and development of molecular markers, it is feasible to evaluate the genetic bases of combining ability and heterosis of elite rice hybrids through QTL analysis. Methodology/Principal Findings In the present study, we first developed a QTL-mapping method for dissecting combining ability and heterosis of agronomic traits. With three testcross populations and a BCRIL population in rice, biometric and QTL analyses were conducted for ten agronomic traits. The significance of general combining ability and special combining ability for most of the traits indicated the importance of both additive and non-additive effects on expression levels. A large number of additive effect QTLs associated with performance per se of BCRIL and general combining ability, and dominant effect QTLs associated with special combining ability and heterosis were identified for the ten traits. Conclusions/Significance The combining ability of agronomic traits could be analyzed by the QTL mapping method. The characteristics revealed by the QTLs for combining ability of agronomic traits were similar with those by multitudinous QTLs for agronomic traits with performance per se of BCRIL. Several QTLs (1–6 in this study) were identified for each trait for combining ability. It demonstrated that some of the QTLs were pleiotropic or linked tightly with each other. The identification of QTLs responsible for combining ability and heterosis in the present study provides valuable information for dissecting genetic basis of combining ability.

De novo transcriptome and small RNA analyses of two amorphophallus species.

Konjac is one of the most important glucomannan crops worldwide. The breeding and genomic researches are largely limited by the genetic basis of Amorphophallus. In this study, the transcriptomes of A. konjac and A. bulbifer were constructed using a high-throughput Illumina sequencing platform. All 108,651 unigenes with average lengths of 430 nt in A. konjac and 119,678 unigenes with average lengths of 439 nt were generated from 54,986,020 reads and 52,334,098 reads after filtering and assembly, respectively. A total of 54,453 transcripts in A. konjac and 55,525 in A. bulbifier were annotated by comparison with Nr, Swiss-Prot, KEGG, and COG databases after removing exogenous contaminated sequences. A total of 80,332 transcripts differentially expressed between A. konjac and A. bulbifer. The majority of the genes that are associated with konjac glucomannan biosynthetic pathway were identified. Besides, the small RNAs in A. konjac leaves were also obtained by deep sequencing technology. All of 5,499,903 sequences of small RNAs were obtained with the length range between 18 and 30 nt. The potential targets for the miRNAs were also predicted according to the konjac transcripts. Our study provides a systematic overview of the konjac glucomannan biosynthesis genes that are involved in konjac leaves and should facilitate further understanding of the crucial roles of carbohydrate synthesis and other important metabolism pathways in Amorphophallus.

Development and characterisation of EST-SSR markers by transcriptome sequencing in taro ( Colocasia esculenta (L.) Schoot)

Taro (Colocasia esculenta) is an important crop with a long history of cultivation. In this study 5278 SSRs were identified in taro transcriptome data. A total of 2858 primer pairs were designed for marker development. 100 primers were randomly selected and synthesized. Among them, 72 primer pairs were successfully amplified and 62 were polymorphic in taro accessions. The number of alleles ranged from 2 to 14 for each different polymorphic locus and the polymorphism information content valued ranged from 0.01 to 0.82. The phylogenetic tree was also constructed to analyse the genetic diversity in 68 taro accessions. The large number of taro SSR markers developed in the present study will be useful in the researches of genetic diversity, germplasm characterization and molecular breeding etc.

QTL mapping for combining ability in different population-based NCII designs: a simulation study

The NCII design (North Carolina mating design II) has been widely applied in studies of combining ability and heterosis. The objective of our research was to estimate how different base populations, sample sizes, testcross numbers and heritability influence QTL analyses of combining ability and heterosis. A series of Monte Carlo simulation experiments with QTL mapping were then conducted for the base population performance, testcross population phenotypic values and the general combining ability (GCA), specific combining ability (SCA) and Hmp (midparental heterosis) datasets. The results indicated that: (i) increasing the number of testers did not necessarily enhance the QTL detection power for GCA, but it was significantly related to the QTL effect. (ii) The QTLs identified in the base population may be different from those from GCA dataset. Similar phenomena can be seen from QTL detected in SCA and Hmp datasets. (iii) The QTL detection power for GCA ranked in the order of DH(RIL) based > F2 based > BC based NCII design, when the heritability was low. The recombinant inbred lines (RILs) (or DHs) allows more recombination and offers higher mapping resolution than other populations. Further, their testcross progeny can be repeatedly generated and phenotyped. Thus, RIL based (or DH based) NCII design was highly recommend for combining ability QTL analysis. Our results expect to facilitate selecting elite parental lines with high combining ability and for geneticists to research the genetic basis of combining ability.

Pedigree-based genome re-sequencing reveals genetic variation patterns of elite backbone varieties during modern rice improvement

Rice breeding has achieved great productivity improvements by semi-dwarf varieties and hybrid vigour. Due to poor understanding of genetic basis of elite backbone varieties, the continuous increasing in rice yield still faces great challenges. Here, 52 elite rice varieties from three historical representative pedigrees were re-sequenced with 10.1× depth on average, and ~6.5 million single nucleotide polymorphisms (SNPs) were obtained. We identified thousands of low-diversity genomic regions and 0-diversity genes during breeding. Using pedigree information, we also traced SNP transmission patterns and observed breeding signatures in pedigree. These regions included the larger number of key well-known functional genes. Besides, 35 regions spanning 0.16% of the rice gnome had been differentially selected between conventional and restorer pedigrees. These genes identified here will be useful to the further pedigree breeding. Our study provides insights into the genetic basis of backbone varieties and will have immediate implications for performing genome-wide breeding by design.

Integration of transcriptome and proteome analyses reveal molecular mechanisms for formation of replant disease in Nelumbo nucifera

The normal growth of Nelumbo nucifera, a widely planted aquatic crop in Asia, was severely ruined by replant disease. The mechanism of replant disease was still unknown in aquatic crops. Complementary transcriptomic and proteomic analyses were performed by comparing seedings of first-year planting (FP) and consecutive planting (CP). 9810 differentially expressed genes (DEGs) were identified between FP and CP. Additionally, 975 differentially expressed proteins (DEPs) were obtained. The correlation of proteome and transcriptome illustrated phenylpropanoid biosynthesis, flavonoid biosynthesis, metabolic pathways, and MAPK signaling pathways were significantly activated. Peroxidase, determined as one of the key proteins in replant disease of N. nucifera, was phylogenetically analyzed. A new depiction of the molecular mechanism causing replant disease in N. nucifera was illustrated. A consecutive monoculture stimulated the generation of reactive oxygen species (ROS) and ethylene, altered the metabolic balance of lignin and flavonoid, and attenuated the activity of antioxidant enzymes through DNA methylation. Therefore, the accumulation of autotoxic allelochemicals and the deficiency of antioxidant enzymes unavoidably suppressed the normal growth and development of replanted N. nucifera.

Transcriptomics and proteomics reveal genetic and biological basis of superior biomass crop Miscanthus

Miscanthus is a rhizomatous C4 grass which is considered as potential high-yielding energy crop with the low-nutrient requirements, high water-use efficiency, and capability of C mitigation. To better understand the genetic basis, an integrative analysis of the transcriptome and proteome was performed to identify important genes and pathways involved in Miscanthus leaves. At the transcript level, 64,663 transcripts in M. lutarioriparius, 97,043 in M. sacchariflorus, 97,043 in M. sinensis, 67,323 in M. floridulus and 70,021 in M. × giganteus were detected by an RNA sequencing approach. At the protein level, 1964 peptide-represented proteins were identified and 1933 proteins differed by 1.5-fold or more in their relative abundance, as indicated by iTRAQ (isobaric tags for relative and absolute quantitation) analysis. Phylogenies were constructed from the nearly taxa of Miscanthus. A large number of genes closely related to biomass production were found. And SSR markers and their corresponding primers were derived from Miscanthus transcripts and 90% of them were successfully detected by PCR amplification among Miacanthus species. These similarities and variations on the transcriptional and proteomic level between Miscanthus species will serve as a resource for research in Miscanthus and other lignocellulose crops.