Xiuquan Li

The introgression of chromosome 6P specifying for increased numbers of florets and kernels from Agropyron cristatum into wheat.

A wheat (Triticum aestivum L.) line 4844 with superior numbers of florets and grains per spike was derived from the cross between Fukohokomugi wheat and Agropyron cristatum (L.) Gaertn. In order to determine the genetic control of floret and kernel number per spike in this line, chromosome addition and substitution lines that were derived from line 4844 were characterized by means of in situ hybridization, microsatellite (SSR), and gliadin analyses. Genomic in situ hybridization analysis with biotinylated P genomic DNA of A. cristatum as a probe demonstrated that the increased number of florets and grains in a spike was associated with the introgression of an A. cristatum chromosome. Fluorescence in situ hybridization, using a repetitive sequence, pAs1, derived from Aegilops squarrosa L., indicated the replacement of chromosome 6D of wheat in the wheat-A. cristatum chromosome substitution lines. This was confirmed by microsatellite analyses with wheat SSR markers specific for chromosome 6D, suggesting that the A. cristatum chromosome was homoeologous to group 6 and was therefore designated as 6P. This conclvsion was further confirmed by amplification using EST-SSR markers and gliadin analysis. The increased number of florets and kernels within a spike of the wheat-A. cristatum hybrids thus was controlled by gene(s) located on A. cristatum chromosome 6P.

QTL mapping of yield-related traits in the wheat germplasm 3228

The new wheat germplasm 3228, a putative derivative of tetraploid Agropyron cristatum Z559 and the common wheat Fukuhokomugi, has superior features in yield-related traits, particularly in spike morphological traits, such as large spike and superior grain number. To identify favorable alleles of these traits in 3228, 237 F2:3 families were developed from the cross 3228/Jing 4839. A genetic map was constructed using 179 polymorphic SSR and EST-SSR markers. A total of 76 QTL controlling spike number per plant (SNP), spike length (SL), spikelet number per spike (SNS), floret number per spikelet (FNS), grain number per spike (GNS) and thousand-grain weight (TGW) were detected on 16 chromosomes. Each QTL explained 1.24–27.01% of the phenotypic variation, and 9 QTL (28.95%) were detected in two or all environments. Additive effects of 45 QTL were positive with 3228 alleles increasing the QTL effects, 31 QTL had negative effects indicating positive contributions from Jing 4839. Three important clusters involving all traits were located on chromosomes 5A, 6A and 4B, and several co-located QTL were also found. Most of the QTL detected on the three chromosome regions could contribute to the use of 3228 in breeding for grain yield improvement.

De novo transcriptome sequencing of Agropyron cristatum to identify available gene resources for the enhancement of wheat

Agropyron cristatum is a wild grass of the tribe Triticeae that is widely grown in harsh environments. As a wild relative of wheat, A. cristatum carries many resistance genes that could be used to broaden the genetic diversity of wheat. Here, we report the transcriptome sequencing of the flag leaf and young spike tissues of a representative tetraploid A. cristatum. More than 90 million reads from the two tissues were assembled into 73,664 unigenes. All unigenes were functionally annotated against the KEGG, COG, and Gene Ontology databases and predicted long non-coding RNAs. Pfam prediction demonstrates that A. cristatum carries an abundance of stress resistance genes. The extent of specific genes and rare alleles make A. cristatum a vital genetic reservoir for the improvement of wheat. Altogether, the available gene resources in A. cristatum facilitate efforts to harness the genetic diversity of wild relatives to enhance wheat.

Genetic rearrangements of six wheat-agropyron cristatum 6P addition lines revealed by molecular markers.

Agropyron cristatum (L.) Gaertn. (2n = 4x = 28, PPPP) not only is cultivated as pasture fodder but also could provide many desirable genes for wheat improvement. It is critical to obtain common wheat–A. cristatum alien disomic addition lines to locate the desired genes on the P genome chromosomes. Comparative analysis of the homoeologous relationships between the P genome chromosome and wheat genome chromosomes is a key step in transferring different desirable genes into common wheat and producing the desired alien translocation line while compensating for the loss of wheat chromatin. In this study, six common wheat–A. cristatum disomic addition lines were produced and analyzed by phenotypic examination, genomic in situ hybridization (GISH), SSR markers from the ABD genomes and STS markers from the P genome. Comparative maps, six in total, were generated and demonstrated that all six addition lines belonged to homoeologous group 6. However, chromosome 6P had undergone obvious rearrangements in different addition lines compared with the wheat chromosome, indicating that to obtain a genetic compensating alien translocation line, one should recombine alien chromosomal regions with homoeologous wheat chromosomes. Indeed, these addition lines were classified into four types based on the comparative mapping: 6PI, 6PII, 6PIII, and 6PIV. The different types of chromosome 6P possessed different desirable genes. For example, the 6PI type, containing three addition lines, carried genes conferring high numbers of kernels per spike and resistance to powdery mildew, important traits for wheat improvement. These results may prove valuable for promoting the development of conventional chromosome engineering techniques toward molecular chromosome engineering.

Isolation and characterization of two putative cytokinin oxidase genes related to grain number per spike phenotype in wheat

Cytokinin oxidases are involved in the regulation of plant cytokinin levels, which are important in regulating plant growth and development, and may affect the yield of cereals. Here, we report the isolation and characterization of two putative cytokinin oxidase genes, TaCKX2.1 and TaCKX2.2, from wheat. Both TaCKX2.1 and TaCKX2.2 are mapped to the 0.24–0.55 region of the short arm of wheat chromosome 3D and their coding proteins are most closely related to OsCKX2. Phylogenetic tree analysis reveals that TaCKX2.1 and TaCKX2.2 belong to the clustered clade I of monocot plants. Tissue expression pattern show that both TaCKX2.1 and TaCKX2.2 genes are highly expressed in young spikes and culms of wheat. The detailed spatial expression pattern of TaCKX2.1 were further conducted by in situ hybridization and promoter-fused GUS expression in Arabidopsis experiments. A collection of 12 typical common wheat varieties exhibiting grain number per spike ranging from 31 to 139 were used for the transcription abundance detection of two TaCKX2 genes. A significantly positive correlation between expression level of two TaCKX2 genes and grain number per spike suggests that TaCKX2.1 and TaCKX2.2 on wheat chromosome 3DS may play an important role in wheat spike morphogenesis.

Genetic diversity between ex situ and in situ samples of Agropyron cristatum (L.) Gaertn. based on simple sequence repeat molecular markers

Fifty-five populations of Agropyron cristatum (L.) Gaertn., both ex situ conserved and in situ collected and originating from northern China, were analysed using 29 polymorphic simple sequence repeat (SSR) primers mapped on the wheat genome. The study showed ex situ and in situ populations had different numbers of alleles and allelic frequencies in 17 of the 29 primer pairs. The SSR markers revealed significant genetic differences between gene bank-conserved and in situ recollected populations from similar ecogeographical regions. AMOVA analysis indicated 15% of the total variation resided between groups, and 85% resided within groups. According to a dendrogram generated by unweighted pair group method with arithmetic average cluster analysis based on Nei’s genetic distance matrix and principal coordinate analysis, recollected and gene bank-conserved populations from the same regions were distinguished. The results indicate that both in situ and ex situ conservation play complementary roles in the conservation of A. cristatum.

Genome origins in Leymus (Poaceae: Triticeae): evidence of maternal and paternal progenitors and implications for reticulate evolution

The polyploid Leymus species have the Ns nuclear genomes evolved from Psathyrostachys and the Xm nuclear genomes from unknown origins. Currently, little is known about the cytoplasmic genomes of Leymus, and the species of Psathyrostachys that transferred the Ns genome to Leymus remain elusive. To determine the origins of the plastid and nuclear genome for Leymus species, sequences of two chloroplast genes, rpoA and rbcL, and partial sequences of the nuclear gene DMC1 of 15 Leymus species were phylogenetically analyzed with those of 71 accessions belonging to 19 monogenomic genera in Triticeae. Both rpoA and rbcL sequences revealed that Psathyrostachys juncea, P. lanuginosa, and P. stoloniformis had close phylogenetic relationships with the Eurasian and one North American Leymus accessions, L. cinereus. Three American Leymus species, L. erianthus, L. triticoides, and L. innovatus had different maternal origins from Eurasian Leymus species. DMC1 sequences of Leymus were grouped into several phylogenetically distant clades indicating that either Ns or Xm originated from different lineages. These results suggest multiple contributions to the chloroplast as well as nuclear genomes in Leymus species. The comprehensive data indicate reticulate evolution in polyploid Leymus.

On the use of SSR markers for the genetic characterization of the Agropyron cristatum (L.) Gaertn. in Northern China

Genetic diversity of 69 populations of Agropyron cristatum (L.) Gaertn. originated from various regions of northern China was analyzed using 29 polymorphic microsatellite primers that were mapped on the wheat genome. The number of polymorphic bands ranged from 2 (Xgwm285, Xgwm43, Xgwm291, and Xgwm257) to 27 (Xgwm314) with an average of 10.480. The highest genetic diversity value was detected in the populations from Xinjiang Province (0.735), and the lowest was observed in populations from Qinghai Province (0.553). The proportion of diversity among and within regions was 16.9% and 83.1% of the total variation, respectively. According to the dendrogram generated by UPGMA cluster analysis based on Nei’s genetic distance matrix, all the populations of A. cristatum were distinctly clarified. At the Nei’s distance of 0.62, the populations were divided into 6 groups. The phenogram indicated that populations from similar ecogeographical regions were clustered together. The principal coordinate analysis showed that the populations from Inner Mongolia were more closely related to each other, and were less variable than the populations from Xinjiang Province.

Isolation and application of P genome-specific DNA sequences of Agropyron Gaertn. in Triticeae

AbstractMain conclusionDifferent types of P genome sequences and markers were developed, which could be used to analyze the evolution of P genome in Triticeae and identify precisely wheat-A. cristatumintrogression lines. P genome of Agropyron Gaertn. plays an important role in Triticeae and could provide many desirable genes conferring high yield, disease resistance, and stress tolerance for wheat genetic improvement. Therefore, it is significant to develop specific sequences and functional markers of P genome. In this study, 126 sequences were isolated from the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) products of microdissected chromosome 6PS. Forty-eight sequences were identified as P genome-specific sequences by dot-blot hybridization and DNA sequences analysis. Among these sequences, 22 displayed the characteristics of retrotransposons, nine and one displayed the characteristics of DNA transposons and tandem repetitive sequence, respectively. Fourteen of 48 sequences were determined to distribute on different regions of P genome chromosomes by fluorescence in situ hybridization, and the distributing regions were as following: all over P genome chromosomes, centromeres, pericentromeric regions, distal regions, and terminal regions. We compared the P genome sequences with other genome sequences of Triticeae and found that the similar sequences of the P genome sequences were widespread in Triticeae, but differentiation occurred to various extents. Additionally, thirty-four molecular markers were developed from the P genome sequences, which could be used for analyzing the evolutionary relationship among 16 genomes of 18 species in Triticeae and identifying P genome chromatin in wheat-A. cristatum introgression lines. These results will not only facilitate the study of structure and evolution of P genome chromosomes, but also provide a rapid detecting tool for effective utilization of desirable genes of P genome in wheat improvement.

A proteomic study of spike development inhibition in bread wheat

Spike development in wheat is a complicated development process and determines the wheat propagation and survival. We report herein a proteomic study on the bread wheat mutant strain 5660M underlying spike development inhibition. A total of 121 differentially expressed proteins, which were involved in cold stress response, protein folding and assembly, cell‐cycle regulation, scavenging of ROS, and the autonomous pathway were identified using MS/MS and database searching. We found that cold responsive proteins were highly expressed in the mutant in contrast to those expressed in the wild‐type line. Particularly, the autonomous pathway protein FVE, which modulates flowering, was dramatically downregulated and closely related to the spike development inhibition phenotype of 5660M. A quantitative RT‐PCR study demonstrated that the transcription of the FVE and other six genes in the autonomous pathway and downstream flowering regulators were all markedly downregulated. The results indicate that spike development of 5660M cannot complete the floral transition. FVE might play an important role in the spikes development of the wheat. Our results provide the theory basis for studying floral development and transition in the reproductive growth period, and further analysis of wheat yield formation.