Xinhong Chen

TaGS5‐3A, a grain size gene selected during wheat improvement for larger kernel and yield

Grain size is a dominant component of grain weight in cereals. Earlier studies have shown that OsGS5 plays a major role in regulating both grain size and weight in rice via promotion of cell division. In this study, we isolated TaGS5 homoeologues in wheat and mapped them on chromosomes 3A, 3B and 3D. Temporal and spatial expression analysis showed that TaGS5 homoeologues were preferentially expressed in young spikes and developing grains. Two alleles of TaGS5-3A, TaGS5-3A-T and TaGS5-3A-G were identified in wheat accessions, and a functional marker was developed to discriminate them. Association analysis revealed that TaGS5-3A-T was significantly correlated with larger grain size and higher thousand kernel weight. Biochemical assays showed that TaGS5-3A-T possesses a higher enzymatic activity than TaGS5-3A-G. Transgenic rice lines overexpressing TaGS5-3A-T also exhibited larger grain size and higher thousand kernel weight than TaGS5-3A-G lines, and the transcript levels of cell cycle-related genes in TaGS5-3A-T lines were higher than those in TaGS5-3A-G lines. Furthermore, systematic evolution analysis in diploid, tetraploid and hexaploid wheat showed that TaGS5-3A underwent strong artificial selection during wheat polyploidization events and the frequency changes of two alleles demonstrated that TaGS5-3A-T was favoured in global modern wheat cultivars. These results suggest that TaGS5-3A is a positive regulator of grain size and its favoured allele TaGS5-3A-T exhibits a larger potential application in wheat high-yield breeding.

Isolation and Characterization of a Psathyrostachys huashanica Keng 6Ns Chromosome Addition in Common Wheat

The development of alien addition lines is important for transferring useful genes from exotic species into common wheat. A hybrid of common wheat cv. 7182 (2n = 6x = 42, AABBDD) and Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) via embryo culture produced the novel intergeneric disomic addition line 59-11. The seed morphology of 59-11 resembled the parent 7182 and it exhibited extreme agronomic characteristics, i.e., twin stable spikelets, fertile florets, and multi-kernel clusters. Furthermore, 59-11 produced plump kernels with a high seed-setting percentage during the advanced maturation stage. The line was screened based on genomic in situ hybridization, EST-SSR, EST-STS, and gliadin to identify P. huashanica chromatin in the wheat background. The chromosome number and configuration of 59-11 was 2n = 44 = 22 II and we confirmed the 6Ns disomic chromosome additions based on A-PAGE analysis and molecular markers. The results suggested that the production of twin spikelets and multiple kernels per spike in the wheat-P. huashanica addition line was related to homologous group 6 in the wheat chromosome. This is the first report of the introduction of improved spike traits into common wheat from the alien species P. huashanica and it opens up the possibility of increasing the wheat yield based on this enlarged gene pool.

A yield-associated gene TaCWI, in wheat: its function, selection and evolution in global breeding revealed by haplotype analysis

Key messageWheat anther-specific invertase genes were haplotyped in wheat. Strong allelic selection occurred during wheat polyploidization, domestication and breeding because of their association with yield traits.AbstractPlant invertase hydrolyzes sucrose into glucose and fructose. Cell wall invertase (CWI), one of the three types of invertase, is essential for plant development. Based on isolated TaCWI genes from chromosomes 4A, 5B and 5D, two SNPs were detected in the promoter region of TaCWI-4A, and four SNPs and two Indels were present in the TaCWI-5D gene. No polymorphism was detected in TaCWI-5B coding or promoter regions. CAPS markers caps4A and caps5D were developed to discriminate haplotypes of TaCWI-4A and TaCWI-5D. Marker/trait association analysis indicated that Hap-5D-C at TaCWI-5D was significantly associated with higher thousand kernel weight (TKW) in 348 Chinese modern cultivars grown in multiple environments. Geographic distributions and changes over time of favored haplotypes showed that Hap-5D-C was the most frequent haplotype in modern cultivars and was strongly positively selected in six major wheat production regions worldwide. However, selection for haplotypes at TaCWI-4A was not so evident, possibly due to balancing effects of the two haplotypes on TKW and grain number per spike (GN). In rainfed production regions, Hap-4A-C was favored because it brought more seeds, but in well irrigated conditions, Hap-4A-T was favored in modern breeding because of higher TKW. Evolutionary analysis among wheat and its relatives showed that genetic diversity of TaCWI genes on chromosomes 4A and 5D declined dramatically in progression from the diploid level to modern polyploid cultivars. There was strong allelic selection during polyploidization, domestication and breeding.

Developmental Changes in Composition and Morphology of Cuticular Waxes on Leaves and Spikes of Glossy and Glaucous Wheat (Triticum aestivum L.).

The glossy varieties (A14 and Jing 2001) and glaucous varieties (Fanmai 5 and Shanken 99) of wheat (Triticum aestivum L.) were selected for evaluation of developmental changes in the composition and morphology of cuticular waxes on leaves and spikes. The results provide us with two different wax development patterns between leaf and spike. The general accumulation trend of the total wax load on leaf and spike surfaces is first to increase and then decrease during the development growth period, but these changes were caused by different compound classes between leaf and spike. Developmental changes of leaf waxes were mainly the result of variations in composition of alcohols and alkanes. In addition, diketones were the third important contributor to the leaf wax changes in the glaucous varieties. Alkanes and diketones were the two major compound classes that caused the developmental changes of spike waxes. For leaf waxes, β- and OH-β-diketones were first detected in flag leaves from 200-day-old plants, and the amounts of β- and OH-β-diketones were significantly higher in glaucous varieties compared with glossy varieties. In spike waxes, β-diketone existed in all varieties, but OH-β-diketone was detectable only in the glaucous varieties. Unexpectedly, the glaucous variety Fanmai 5 yielded large amounts of OH-β-diketone. There was a significant shift in the chain length distribution of alkanes between early stage leaf and flag leaf. Unlike C28 alcohol being the dominant chain length in leaf waxes, the dominant alcohol chain length of spikes was C24 or C26 depending on varieties. Epicuticular wax crystals on wheat leaf and glume were comprised of platelets and tubules, and the crystal morphology changed constantly throughout plant growth, especially the abaxial leaf crystals. Moreover, our results suggested that platelets and tubules on glume surfaces could be formed rapidly within a few days.

Small RNA and Degradome Sequencing Reveal Complex Roles of miRNAs and Their Targets in Developing Wheat Grains

Plant microRNAs (miRNAs) have been shown to play critical roles in plant development. In this study, we employed small RNA combined with degradome sequencing to survey development-related miRNAs and their validated targets during wheat grain development. A total of 186 known miRNAs and 37 novel miRNAs were identified in four small RNA libraries. Moreover, a miRNA-like long hairpin locus was first identified to produce 21~22-nt phased siRNAs that act in trans to cleave target mRNAs. A comparison of the miRNAomes revealed that 55 miRNA families were differentially expressed during the grain development. Predicted and validated targets of these development-related miRNAs are involved in different cellular responses and metabolic processes including cell proliferation, auxin signaling, nutrient metabolism and gene expression. This study provides insight into the complex roles of miRNAs and their targets in regulating wheat grain development.

Development and characterization of a Psathyrostachys huashanica Keng 7Ns chromosome addition line with leaf rust resistance.

The aim of this study was to characterize a Triticum aestivum-Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) disomic addition line 2-1-6-3. Individual line 2-1-6-3 plants were analyzed using cytological, genomic in situ hybridization (GISH), EST-SSR, and EST-STS techniques. The alien addition line 2-1-6-3 was shown to have two P. huashanica chromosomes, with a meiotic configuration of 2n = 44 = 22 II. We tested 55 EST-SSR and 336 EST-STS primer pairs that mapped onto seven different wheat chromosomes using DNA from parents and the P. huashanica addition line. One EST-SSR and nine EST-STS primer pairs indicated that the additional chromosome of P. huashanica belonged to homoeologous group 7, the diagnostic fragments of five EST-STS markers (BE404955, BE591127, BE637663, BF482781 and CD452422) were cloned, sequenced and compared. The results showed that the amplified polymorphic bands of P. huashanica and disomic addition line 2-1-6-3 shared 100% sequence identity, which was designated as the 7Ns disomic addition line. Disomic addition line 2-1-6-3 was evaluated to test the leaf rust resistance of adult stages in the field. We found that one pair of the 7Ns genome chromosomes carried new leaf rust resistance gene(s). Moreover, wheat line 2-1-6-3 had a superior numbers of florets and grains per spike, which were associated with the introgression of the paired P. huashanica chromosomes. These high levels of disease resistance and stable, excellent agronomic traits suggest that this line could be utilized as a novel donor in wheat breeding programs.

Cytogenetic and Molecular Marker-Based Characterization of a Wheat-Psathyrostachys huashanica Keng 2Ns(2D) Substitution Line

Wheat-Psathyrostachys huashanica Keng substitution line 16-6, which possessed superior numbers of florets and kernels per spike, was developed from a cross between common wheat cv. 7182 and P. huashanica via embryo culture. This line was characterized based on cytological, genomic in situ hybridization (GISH), simple sequence repeat (SSR), fluorescent in situ hybridization (FISH), expressed sequence tag-sequence-tagged site (EST-STS), and morphological analyses. Line 16-6 contained 21 bivalents during meiosis, while GISH studies detected an alien chromosome pair substitution. Sixteen microsatellite markers for chromosome arms 2DS and 2DL of hexaploid wheat and FISH using the probe pAs1 indicated that chromosome 2D was missing. P. huashanica 2Ns(2D) chromosome introgression was confirmed using 780 EST-STS multiple-loci markers that covered all seven homoeologous groups and 14 EST-STS markers associated with homoeologous group 2 of wheat. After inoculation using mixed races of stripe rust (CYR31, CYR32, and SY11-14) in the adult stages, line 16-6 exhibited resistance, which was derived from the donor species. Compared with the control, the spike length, number of spikelets per spike, and kernel number per spike were significantly higher.

A sucrose:fructan-6-fructosyltransferase (6-SFT) gene from Psathyrostachys huashanica confers abiotic stress tolerance in tobacco.

Fructans are accessible carbohydrate reserves in various plant species, which possess many physiological functions including anti-oxidation, stabilizing subcellular structures, and osmotic adjustment. In addition, fructans may play important roles in stress tolerance in plant species. In this study, we isolated a Psathyrostachys huashanica (2n=2x=14, NsNs) sucrose:fructan-6-fructosyltransferase (Ph-6-SFT) using homologous cloning and genomic walking. Sequencing and gene structure analysis showed that Ph-6-SFT contains four exons and three introns, with a transcript of 2207 bp. Sequence analysis indicated that the coding sequence of Ph-6-SFT is 1851 bp long and it encodes 616 amino acids, where the structure shares high similarity with 6-SFTs from other plants. Furthermore, Ph-6-SFT was transferred into tobacco (Nicotiana tabacum L.) cv. W38 via Agrobacterium-mediated transformation. Compared with the wild-type plants, the transgenic tobacco plants exhibited a much higher tolerance of drought, cold, and high salinity. In all conditions, physiological studies showed that the tolerance of transgenic plants was associated with the accumulation of carbohydrate and proline, but reductions in malondialdehyde. Our results suggest that the 6-SFT gene from P. huashanica enhanced stress tolerance in tobacco plants and it may be applied as a genetic tool for improving stress tolerance in other crops.

Cytogenetic and molecular identification of a wheat-Leymus mollis alien multiple substitution line from octoploid Tritileymus x Triticum durum

Leymus mollis (Trin.) Pilger (NsNsXmXm, 2n = 28), a wild relative of common wheat, possesses many traits that are potentially valuable for wheat improvement. In order to exploit and utilize the useful genes of L. mollis, we developed a multiple alien substitution line, 10DM50, from the progenies of octoploid Tritileymus M842-16 x Triticum durum cv. D4286. Genomic in situ hybridization analysis of mitosis and meiosis (metaphase I), using labeled total DNA of Psathyrostachys huashanica as probe, showed that the substitution line 10DM50 was a cytogenetically stable alien substitution line with 36 chromosomes from wheat and three pairs of Ns genome chromosomes from L. mollis. Simple sequence repeat analysis showed that the chromosomes 3D, 6D, and 7D were absent in 10DM50. Expressed sequence tag-sequence tagged sites analysis showed that new chromatin from 3Ns, 6Ns, and 7Ns of L. mollis were detected in 10DM50. We deduced that the substitution line 10DM50 was a multiple alien substitution line with the 3D, 6D, and 7D chromosomes replaced by 3Ns, 6Ns, and 7Ns from L. mollis. 10DM50 showed high resistance to leaf rust and significantly improved spike length, spikes per plant, and kernels per spike, which are correlated with higher wheat yield. These results suggest that line 10DM50 could be used as intermediate material for transferring desirable traits from L. mollis into common wheat in breeding programs.

A novel SCAR marker for detecting Psathyrostachys huashanica Keng chromatin introduced in wheat.

In this study, we cloned and sequenced a 938-base pair polymorphic band, pHs27, in the tightly linked random amplified polymorphic DNA marker OPU10 and converted it into a sequence-characterized amplified region (SCAR) marker referred to as RHS141, which was specific for the Ns genome of Psathyrostachys huashanica. A GenBank basic local alignment search tool search showed that the sequence of pHs27 had no primary sequence homology with known sequences, and Southern blotting confirmed this result. This SCAR marker was used to detect Ns genome chromatin in wheat, and it was successfully amplified in P. huashanica itself, a complete set of wheat-P. huashanica disomic addition lines (1Ns-7Ns), and undetermined homoeologous group addition lines. This SCAR marker will be a powerful tool for the marker-assisted selection of P. huashanica chromosome(s) in a wheat background, and it should also allow wheat breeders to screen for the excellent traits found in P. huashanica chromatin.