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Somatic hybridization via PEG (Polyethylene 6000)-mediated protoplast fusion was achieved between two different wheat culture lines (Triticum aestivum L., "Jinan"177, T1 and T2) and Setaria italica (L.) P. Beauv. The T1 recipient originated from non-regenerable long-term cell suspensions, while T2 was derived from embryogenic calli with a high regeneration capacity. Donor protoplasts were obtained from embryogenic calli of S. italica (S) (with low regeneration capacity) irradiated with different doses of ultraviolet light. Twenty-three putative hybrid cell lines were produced in fusion combinations with the donor protoplasts treated with UV light for 30 s (combination I) and 1 min (combination II), but only one (from combination II) differentiated into green plants. Three cell lines from combination I and five cell lines from combination II possessed the nuclear genomes of T1, T2, and S. italica as revealed by cytological, isozyme, RAPD, and 5S rDNA spacer sequence analyses. Genomic in situ hybridization (GISH) analysis showed that most hybrid cell lines had 22-36 wheat chromosomes, 0-2 S. italica chromosomes, and 1-6 wheat - S. italica recombinant chromosomes, whereas the regenerable cell line had 44-56 wheat chromosomes and 3-6 recombinant chromosomes, but no intact S. italica chromosomes. RFLP analysis of organellar DNA revealed that mitochondrial and chloroplast DNA of both parents coexisted in all hybrid cell lines and recombined in most hybrid cell lines. These results indicate that the regeneration of hybrid plants involves not only the integration of S. italica nuclear and organellar DNA, but also the genome complementation of T1 and T2.

Background Dasypyrum villosum is an important wild species of wheat ( Triticum aestivum L.) and harbors many desirable genes that can be used to improve various traits of wheat. Compared with other D. villosum accessions, D. villosum#4 still remains less studied. In particular, chromosomes of D. villosum#4 except 6V#4 have not been introduced into wheat by addition or substitution and translocation, which is an essential step to identify and apply the alien desired genes. RNA-seq technology can generate large amounts of transcriptome sequences and accelerate the development of chromosome-specific molecular markers and assisted selection of alien chromosome line. Results We obtained the transcriptome of D. villosum#4 via a high-throughput sequencing technique, and then developed 76 markers specific to each chromosome arm of D. villosum#4 based on the bioinformatic analysis of the transcriptome data. The D. villosum#4 sequences containing the specific DNA markers were expected to be involved in different genes, among which most had functions in metabolic processes. Consequently, we mapped these newly developed molecular markers to the homologous chromosome of barley and obtained the chromosome localization of these markers on barley genome. Then we analyzed the collinearity of these markers among D. villosum , wheat, and barley. In succession, we identified six types of T. aestivum - D. villosum#4 alien chromosome lines which had one or more than one D. villosum#4 chromosome in the cross and backcross BC 3 F 5 populations between T. durum – D. villosum#4 amphidiploid TH3 and wheat cv. Wan7107 by employing the selected specific markers, some of which were further confirmed to be translocation or addition lines by genomic in situ hybridization (GISH). Conclusion Seventy-six PCR markers specific to chromosomes of D. villosum#4 based on transcriptome data were developed in the current study and their collinearity among D. villosum , wheat, and barley were carried out. Six types of Triticum aestivum - D. villosum#4 alien chromosome lines were identified by using 12 developed markers and some of which were further confirmed by GISH. These novel T. aestivum - D. villosum#4 chromosome lines have great potential to be used for the introduction of desirable genes from D. villosum#4 into wheat by chromosomal translocation to breed new wheat varieties. Electronic su...

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Bisexual fertile diploid androgenetic individuals (A(0)) (2n=100) were formed by androgenesis. In this way, the diploid spermatozoa from male allotetraploid hybrids (AT) (4n=200) of red crucian carp (Carassius auratus red var.) (female) x common carp (Cyprinus carpio L.) (male) were used to fertilize the UV-treated haploid eggs of goldfish (Carassius auratus), and living androgenetic diploid fish were developed. The A(0) became sexually mature at the age of 2 years, and they fertilized with each other to form their offspring (A(1)). In this study, we observed the chromosomal number, gonadal structure and appearance of A(1) fish. The results are as follows: (1) In A(1), there were 85% tetraploids (A(1)-4n), 10% triploids (A(1)-3n) and 5% diploids (A(1)-2n), suggesting that diploid A(0) could produce diploid gametes. It was concluded that the formation of diploid gametes generated from diploid A(0) was probably related to the mechanism of pre-meiotic endoreduplication. (2) Among A(1), only A(1)-4n possessed normal ovaries and testes. The mature males of A1-4n produced white semen. Under the electron microscope, the head of diploid sperm generated by A(1)-4n was bigger than that of haploid sperm generated by red crucian carp. In the testes of the A(1)-4n, there were many mature normal spermatozoa with a head bearing plasma membrane and a tail having the typical structure of "9+2" microtubules. Between the head and the tail, there were some mitochondria. The ovaries of A(1)-4n developed well and mainly contained II, III and IV-stage oocytes. The IV-stage oocytes were surrounded by inner and outer follicular cells. The micropyle was observed on the oolemma of follicular cells. There were abundant yolks and plenty of endoplasmic reticulum in the cytoplasm of IV-stage oocytes. Because A(1)-2n and A(1)-3n were distant crossing diploid hybrids and triploid hybrids respectively, they possessed abnormal gonads, and no mature semen and eggs were observed. (3) Compared with allotetraploids, the A(1)-4n fish not only had advantages such as fast growth rate and strong resistibility but also showed some new good performances such as high ratio of body width to body length, smaller heads and shorter tails. These results indicated that androgenesis could produce bisexual fertile tetraploids and improve the shape of allotetraploid hybrids as well, which will be of great significance in both the cell genetics research and fish breeding.

We characterized a wheat-Psathyrostachys huashanica derived line 3-6-4-1 based on genomic in situ hybridization (GISH), molecular marker analysis, and agronomic trait evaluations. The GISH investigations showed that the 3-6-4-1 contained 42 wheat chromosomes and a pair of P. huashanica chromosomes. The homoeologous relationships of the introduced P. huashanica chromosomes were determined using EST-STS multiple loci markers from seven wheat homoeologous groups in the parents and the addition line. Twelve EST-STS markers located on the homoeologous group 2 chromosomes of wheat amplified polymorphic bands in 3-6-4-1, which were unique to P. huashanica. An introduced Ns chromosome pair that belonged to homoeologous group 2 was identified using chromosome-specific markers. Inoculation with isolates of the stripe rust pathotypes, CYR31, CYR32, and SY11-14, and mixed races (CYR31, CYR32, and SY11-14) in the seeding and adult stage, respectively, showed that 3-6-4-1 was generally resistant to stripe rust, which was probably attributable to its P. huashanica parent. We also compared a complete set of wheat-P. huashanica disomic addition lines (1Ns-7Ns, 2n = 44 = 22II) to assess their agronomic traits and morphological characteristics, which showed that 3-6-4-1 had improved spike traits compared with its parents. The P. huashanica 2Ns chromosome-specific molecular markers in 3-6-4-1 could be useful for marker-assisted selection in breeding programs to combat stripe rust. This line can be used as a donor source to introduce novel excellent genes from P. huashanica into wheat to widen its genetic diversity, thereby providing new germplasms for wheat breeding.

We isolated a wheat germplasm line, 22-2, which was derived from common wheat (Triticum aestivum '7182') and Psathyrostachys huashanica 'Keng' (2n = 2x = 14, NsNs). Genomic composition and homoeologous relationships of 22-2 was analyzed using cytology, genomic in situ hybridization (GISH), EST-SSR, and EST-STS to characterize the alien chromatin in the transfer line. The cytological investigations showed that the chromosome number and configuration were 2n = 44 = 22 II. Mitotic and meiotic GISH using P. huashanica genomic DNA as the probe indicated that 22-2 contained a pair of P. huashanica chromosomes. The genomic affinities of the introduced P. huashanica chromosomes were determined by EST-SSR and EST-STS using multiple-loci markers from seven wheat homoeologous groups between the parents and addition line. One EST-SSR and 17 EST-STS markers, which were located on the homoeologous group 3 chromosomes of wheat, amplified polymorphic bands in 22-2 that were unique to P. huashanica. Thus, these markers suggested that the introduced Ns chromosome pair belonged to homoeologous group 3, so we designated 22-2 as a 3Ns disomic addition line. Based on disease reaction to mixed races (CYR31, CYR32, and Shuiyuan14) of stripe rust in the adult stages, 22-2 was found to have high resistance to stripe rust, which was possibly derived from its P. huashanica parent. Consequently, the new disomic addition line 22-2 could be a valuable donor source for wheat improvement depending on the excellent agronomic traits, especially, the introduction of novel disease resistance genes into wheat during breeding programs.

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