Jixin Zhao

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.

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.

An intronless sucrose:fructan-6-fructosyltransferase (6-SFT) gene from Dasypyrum villosum enhances abiotic tolerance in tobacco

Fructans play vital roles in enhancing plant abiotic stress tolerance by reducing oxidative damage, stabilizing cell membranes, improving the osmotic adjustment capacity, and lowering the freezing point. In this study, a sucrose: fructan-6-fructosyltransferase (6-SFT) gene involved in the synthesis of fructans was isolated from Dasypyrum villosum, Dv-6-SFT, using genomic walking and reverse transcription (RT)-PCR. Alignment of the cDNA sequence with its genomic counterpart showed that no introns were present in the Dv-6-SFT gene, and thus it differs from all other plant 6-SFTs that have been cloned previously. Sequence analysis showed that the cDNA of the Dv-6-SFT sequence comprised 2 175 bp with a 1 863 bp open reading frame, and its deduced protein comprised 620 amino acids with a predicted molecular mass of 68.47 kDa. The Dv-6-SFT gene was transferred into tobacco (Nicotiana tabacum L.) cv. W38 viaAgrobacterium-mediated transformation. The screened plants were tested by PCR and semi-quantitative RT-PCR, and the transgenic plants were evaluated under drought, cold, and salt stresses. The Dv-6-SFT transgenic tobacco plants had higher resistance to drought, cold, and salt stress than the non-transgenic plants. Further analysis showed that the transgenic plant expressing Dv-6-SFT had increased content of saccharides and proline, but reduced content of malondialdehyde in leaves. The results of this study demonstrate that the Dv-6-SFT gene is a potential candidate for conferring abiotic stress tolerance in plants and it could be used in crop improvement breeding programs.

Development of 5Ns chromosome-specific SCAR markers for utilization in future wheat breeding programs

In previous studies, we developed a wheat-Psathyrostachys huashanica Keng disomic addition line 3-8-10-2, which exhibited high stripe rust resistance and could be used as a donor source for introducing novel disease resistance gene(s) into wheat in future breeding programs. It was identified using cytology, genomic in situ hybridization (GISH), EST-SSR, EST-STS and morphological analyses. However, these techniques are not suitable for breeding programs that require the rapid screening of large numbers of genotypes because they are highly technical and time-consuming. In this study, three Ns genome-specific SCAR markers were developed via random amplified polymorphic DNA (RAPD) markers. These SCAR markers were further validated using a complete set of wheat-P. huashanica disomic addition lines, which segregated the 5Ns disomic addition line individuals. Our results indicated that the SCAR markers associated with the 5Ns chromosome of P. huashanica and they provide a low cost, high efficiency, alternative tool for screening 5Ns chromosomes in a wheat background. These newly developed SCAR markers that species-specificity of the markers was proved by analysis of a wide range of cereal species, and specific for 5Ns chromosome, which should be useful in marker-assisted selection for wheat breeders who want to screen genotypes that may contain 5Ns chromatin.

Molecular cytogenetic identification of a novel 1AL.1RS translocation line with powdery mildew resistance

A wheat germplasm line 13-2-2 with resistance to powdery mildew was isolated; this line was derived from common wheat cv. W770B and rye, Secale cereale L. (2n = 2x = 14, RR). The line was characterized based on cytological, genomic in situ hybridization (GISH), sequence-characterized amplified region (SCAR), and simple sequence repeat (SSR) analyses. The mitotic and meiotic investigations showed that the chromosome number and configuration of 13-2-2 were 2n = 42 = 21 II. GISH using rye genomic DNA as a probe detected a pair of R genome chromosome arms with strong hybridization signals in 13-2-2. Three 1RS chromosome-specific SCAR markers amplified 1RS specific bands in line 13-2-2. We screened 320 SSR primer pairs on the long or short arms from seven wheat homoeologous groups in the translocation line and parents. However, only three 1AS primers could not be amplified in line 13-2-2, whereas the others were amplified. Thus, these markers suggested that the line 13-2-2 was 1AL.1RS translocation line. Line 13-2-2 was immune to powdery mildew after inoculation with Blumeria graminis f. sp. tritici isolates E05 and E07 during the adult plant stages. In contrast, the maternal parent W770B, Kavkaz with Pm8, and Amigo with Pm17 were heavily infected with spores and had reaction response scores of susceptible. Thus, the new wheat-rye 1AL.1RS translocation line with resistance to powdery mildew could be a new and valuable donor source for wheat improvement. The molecular markers developed in this study might also be useful tools for marker-assisted selection.