Investigation of chromosomal genetic characteristics and identification of structural variation in the offspring of hexaploid triticale×hexaploid wheat.

Hexaploid triticale is an important genetic resource for genetic improvement of common wheat, which can broaden the genetic basis of wheat. In order to lay a foundation for the subsequent research and utilization of triticale germplasm materials, the chromosomal genetic characteristics of cross and backcross offspring of hexaploid triticale×hexaploid wheat were investigated in the process of transferring rye chromatin from hexaploid triticale to hexaploid wheat. Hybrid and backcross combinations were prepared with hexaploid triticale 16yin171 as the maternal parent and hexaploid wheat Chuanmai62 as the paternal parent. The chromosomes in root tip cells of F1, BC1F1 and BC1F2 plants were traced and identified non-denaturing florescence in situ hybridization (ND-FISH). The results indicated that the backcross setting rate of hybrid F1 was 2.61%. The transmission frequency of 2R chromosome was the highest in BC1F1 plants while the transmissibility of rye chromosome in BC1F2 plant was 6R>4R>2R, and the 5B-7B wheat translocation in BC1F2 plants showed severe segregation. A total of 24 structural variant chromosomes were observed both in BC1F1 and BC1F2 plants, including chromosome fragments, isochromosomes, translocations, and dicentric chromosomes. In addition, the seed length and 1000-grain weight of some BC1F2 plants were better than that of the hexaploid wheat parent Chuanmai 62. Therefore, multiple backcrosses should be adopted as far as possible to make the rapid recovery of group D chromosomes, ensuring the recovery of fertility in offspring, when hexaploid tritriale is used as a bridge to introduce rye genetic material into common wheat. At the same time, the potential application value of chromosomal structural variation materials should be also concerned.

QTL Mapping and Validation of Adult Plant Resistance to Stripe Rust in Chinese Wheat Landrace Humai 15.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a devastating foliar disease that affects common wheat and barley throughout the world. The reasonable deployment of adult plant resistance (APR) wheat varieties is one of the best methods for controlling this disease. Wheat landraces are valuable resources for identifying the genes/QTLs responsible for disease resistance. Humai 15 is a Chinese spring wheat landrace and it has exhibited adequate levels of APR to the prevalent Pst races in field environments for many years. In this study, a population of 177 recombinant inbred lines (RILs) was derived from Humai 15 × Mingxian 169. After screening based on a 90K chip array using 45 RILs and Kompetitive Allelic Specific PCR marker genotyping for the population of RILs, a major effect QTL in Humai 15 was located on the centromere of chromosome 2B, where it accounted for up to 47.2% of the phenotypic variation. Two other minor QTL genes from Humai 15 were located on chromosome arms 3BS and 4BL. The Yr18 gene was identified on chromosome arm 7DS in Mingxian 169.

[Identification of the 1RS-7DS.7DL wheat-rye small segment translocation lines].

Rye (Secale cereale L., RR) is a valuable genetic resource for the improvement of common wheat (Triticum aestivum L., AABBDD). Transferring alien rye genes into wheat by distant hybridization and automatic chromosome doubling is an important and efficient method to boost agronomic traits, disease resistance and widening the gene pool in wheat. In this study, an octoploid triticale CD-13 (AABBDDRR) was obtained via automatic chromosome doubling by crossing landrace Penganbaimaizi (T. aestivum L., AABBDD) and rye "Qinling rye" (S. cereale cv. Qinling, RR). GISH and FISH analyses indicated that CD-13 contained a 1RS-7DS.7DL wheat-rye small segment translocation chromosome. In order to transfer the 1RS-7DS small segment translocation into hexaploid wheat, 58 lines of the F5 inbred population from the cross CD-13 x Chuanmai 42 were screened for rye chromosome segments by GISH and FISH analyses. The results showed that 13 lines contained the 1RS-7DS.7DL small segment translocation chromosome by reciprocal translocation between 1RS and 7DS. These translocation lines carrying 1RS small rye alien segment were tested for the translocation breakpoints and the presence of a storage protein locus Sec-1. The Sec-1 locus was absent in the line 811, a stable 1RS-7DS.7DL small segment translocation line. The translocation breakpoint of 1RS-7DS.7DL of this line was located in the interval of IB267-IAG95 around the telomere of 1RS chromosome. Thousand-kernel weight of the line 811 was much higher than the parent CD-13, but not significantly different from Chuanmai 42. This indicated that 1RS-7DS.7DL small segment translocation had no negative effect on thousand-kernel weight in the genetic background of Chuanmai 42. The line with 1RS-7DS.7DL translocation chromosomes can be used as a new genetic material for further studies of valuable genes and their genetic effect on 1RS small segment.

Molecular and Cytogenetic Characterization of a Powdery Mildew-Resistant Wheat-Aegilops mutica Partial Amphiploid and Addition Line.

Aegilops mutica Boiss., a diploid species (2n = 2x = 14, TT), has been rarely studied before. In this research, a hexaploid wheat (cv. Chinese Spring)-Ae. mutica partial amphiploid and a wheat-Ae. mutica addition line were characterized by chromosome karyotyping, FISH using oligonucleotides Oligo-pTa535-1, Oligo-pSc119.2-1, and (GAA)8 as probes, and EST-based molecular markers. The results showed that the partial amphiploid strain consisted of 20 pairs of wheat chromosomes and 7 pairs of Ae. mutica chromosomes, with both wheat 7B chromosomes missing. EST-based molecular marker data suggested that the wheat-Ae. mutica addition line carries the 7T chromosome. Resistance tests indicated that both the partial amphiploid and the 7T addition line were highly resistant to powdery mildew, whereas the wheat control line Chinese Spring was highly susceptible, indicating the presence of a potentially new powdery mildew resistance gene on the Ae. mutica 7T chromosome. The karyotype, FISH patterns, and molecular markers can now be used to identify Ae. mutica chromatin in a wheat background, and the 7T addition could be used as a new powdery mildew resistance source for wheat breeding.

A novel wheat-Dasypyrum breviaristatum substitution line with stripe rust resistance.

The introduction of genetic variation from wild and cultivated Triticeae species has been a long-standing approach for wheat improvement. Dasypyrum breviaristatum species harbor novel and agronomically important genes for resistance against multi-fungal diseases. The development of new wheat-D. breviaristatum introgression lines offers chances for the identification of stripe rust resistance gene(s). A wheat line, D11-5, was selected from a cross between wheat line MY11 and wheat-D. breviaristatum partial amphiploid TDH-2. It was characterized by FISH and PCR-based molecular markers. Chromosome counting revealed that the D11-5 line shows a hexaploid set of 2n = 6x = 42 chromosomes. FISH analysis using the Dasypyrum repetitive sequence pDb12H as a probe demonstrated that D11-5 contained a pair of D. breviaristatum chromosomes, while FISH with wheat D-genomic repetitive sequences revealed that the chromosome 2D was absent in D11-5. The functional molecular markers confirmed that the introduced D. breviaristatum chromosomes belong to the homoeologous group 2, indicating that D11-5 was a 2V(b) (2D) disomic substitution line. Field resistance showed that the introduced D. breviaristatum chromosomes 2V(b) were responsible for the stripe rust resistance at the adult plant stage. FISH, C-banding, and PCR-based molecular marker analysis indicated that the chromosome 2V(b) of D. breviaristatum was completely different from the chromosome 2V of D. villosum. The identified wheat-D. breviaristatum chromosome substitution line D11-5 may be applied to produce agronomically desirable stripe rust resistance germplasm.

Characterization and phylogenetic analysis of α-gliadin gene sequences reveals significant genomic divergence in Triticeae species.

Although the unique properties of wheat α-gliadin gene family are well characterized, little is known about the evolution and genomic divergence of α-gliadin gene family within the Triticeae. We isolated a total of 203 α-gliadin gene sequences from 11 representative diploid and polyploid Triticeae species, and found 108 sequences putatively functional. Our results indicate that α-gliadin genes may have possibly originated from wild Secale species, where the sequences contain the shortest repetitive domains and display minimum variation. A miniature inverted-repeat transposable element insertion is reported for the first time in α-gliadin gene sequence of Thinopyrum intermedium in this study, indicating that the transposable element might have contributed to the diversification of α-gliadin genes family among Triticeae genomes. The phylogenetic analyses revealed that the α-gliadin gene sequences of Dasypyrum, Australopyrum, Lophopyrum, Eremopyrum and Pseudoroengeria species have amplified several times. A search for four typical toxic epitopes for celiac disease within the Triticeae α-gliadin gene sequences showed that the α-gliadins of wild Secale, Australopyrum and Agropyron genomes lack all four epitopes, while other Triticeae species have accumulated these epitopes, suggesting that the evolution of these toxic epitopes sequences occurred during the course of speciation, domestication or polyploidization of Triticeae.

Transcriptional abundance is not the single force driving the evolution of bacterial proteins.

BACKGROUND: Despite rapid progress in understanding the mechanisms that shape the evolution of proteins, the relative importance of various factors remain to be elucidated. In this study, we have assessed the effects of 16 different biological features on the evolutionary rates (ERs) of protein-coding sequences in bacterial genomes. RESULTS: Our analysis of 18 bacterial species revealed new correlations between ERs and constraining factors. Previous studies have suggested that transcriptional abundance overwhelmingly constrains the evolution of yeast protein sequences. This transcriptional abundance leads to selection against misfolding or misinteractions. In this study we found that there was no single factor in determining the evolution of bacterial proteins. Not only transcriptional abundance (codon adaptation index and expression level), but also protein-protein associations (PPAs), essentiality (ESS), subcellular localization of cytoplasmic membrane (SLM), transmembrane helices (TMH) and hydropathicity score (HS) independently and significantly affected the ERs of bacterial proteins. In some species, PPA and ESS demonstrate higher correlations with ER than transcriptional abundance. CONCLUSIONS: Different forces drive the evolution of protein sequences in yeast and bacteria. In bacteria, the constraints are involved in avoiding a build-up of toxic molecules caused by misfolding/misinteraction (transcriptional abundance), while retaining important functions (ESS, PPA) and maintaining the cell membrane (SLM, TMH and HS). Each of these independently contributes to the variation in protein evolution.

[Progress on identification and analysis of DNase I hypersensitive sites in plant genomes].

Eukaryotes's gene expression and regulation relies on the interaction of their cis-acting elements and trans-acting factors. The cis-elements are specific DNA sequences in the genome and frequently located in the untranslated regions. The trans-acting factors are usually considered to be proteins that bind to the cis-acting sequences to regulate gene expression. It is well known that the cis-elements are always associated with DNase I hypersensitive site, which is a signature of open chromatin. The identification of all the functional cis-element using high-throughput method in plant genome has not been initiated in plant genomes. With the rapid achievement of genomics studies, an increasing number of plant genomes have been sequenced. Genome-wide identification of DNase I hypersensitive sites will be a considerably efficient method to locate cis-element in plants, which will provide a vital potential for further plant functional genomics. The present review is to reveal the recent progresses on identification and analysis of DNase I hypersensitive site in plant genomes.

Characterization of wheat: Secale africanum introgression lines reveals evolutionary aspects of chromosome 1R in rye.

Wild Secale species, Secale africanum Stapf., serve as a valuable source for increasing the diversity of cultivated rye (Secale cereale L.) and provide novel genes for wheat improvement. New wheat - S. africanum chromosome 1R(afr) addition, 1R(afr)(1D) substitution, 1BL.1R(afr)S and 1DS.1R(afr)L translocation, and 1R(afr)L monotelocentric addition lines were identified by chromosome banding and in situ hybridization. Disease resistance screening revealed that chromosome 1R(afr)S carries resistance gene(s) to new stripe rust races. Twenty-nine molecular markers were localized on S. africanum chromosome 1R(afr) by the wheat - S. africanum introgression lines. Twenty markers can also identically amplify other reported wheat - S. cereale chromosome 1R derivative lines, indicating that there is high conservation between the wild and cultivated Secale chromosome 1R. Nine markers displayed polymorphic amplification between S. africanum and S. cereale chromosome 1R(afr) derivatives. The comparison of the nucleotide sequences of these polymorphic markers suggested that gene duplication and sequence divergence may have occurred among Secale species during its evolution and domestication.

Diversity and evolution of four dispersed repetitive DNA sequences in the genus Secale.

We present the first characterization of 360 sequences in six species of the genus Secale of both cultivated and wild accessions. These include four distinct kinds of dispersed repetitive DNA sequences named pSc20H, pSc119.1, pSaO5(411), and pSaD15(940) belonging to the Revolver family. During the evolution of the genus Secale from wild to cultivated accessions, the pSaO5(411)-like sequences became shorter mainly because of the deletion of a trinucleotide tandem repeating unit, the pSc20H-like sequences displayed apparent homogenization in cultivated rye, and the second intron of Revolver became longer. In addition, the pSc20H-, pSc119.1-, and pSaO5(411)-like sequences cloned from wild rye and cultivated rye could be divided into two large clades. No single case of the four kinds of repetitive elements has been inherited by each Secale accession from a lone ancestor. It is reasonable to consider the vertical transmission of the four repetitive elements during the evolution of the genus Secale. The pSc20H- and pSaO5(411)-like sequences showed evolutionary elimination at specific chromosomal locations from wild species to cultivated species. These cases imply that different repetitive DNA sequences have played different roles in the chromosome development and genomic evolution of rye. The present study adds important information to the investigations dealing with characterization of dispersed repetitive elements in wild and cultivated rye.

Molecular characterization of a wheat -Thinopyrum ponticum partial amphiploid and its derived substitution line for resistance to stripe rust.

Stripe rust (caused by Puccinia striiformis) occurs annually in most wheat-growing areas of the world. Thinopyrum ponticum has provided novel rust resistance genes to protect wheat from this fungal disease. Wheat - Th. ponticum partial amphiploid line 7430 and a substitution line X005 developed from crosses between wheat and 7430 were resistant to stripe rust isolates from China. Genomic in situ hybridization (GISH) analysis using Pseudoroegneria spicata genomic DNA as a probe demonstrated that the partial amphiploid line 7430 contained ten J(s) and six J genome chromosomes, and line X005 had a pair of J(s)-chromosomes. Giemsa-C banding further revealed that both lines 7430 and X005 were absent of wheat chromosomes 6B. The EST based PCR confirmed that the introduced J(s) chromosomes belonging to linkage group 6, indicating that line X005 was a 6J(s)/6B substitution line. Both resistance observation and sequence characterized amplified region (SCAR) markers displayed that the introduced chromosomes 6J(s) were responsible for the stripe rust resistances. Therefore, lines 7430 and X005 can be used as a donor in wheat breeding for stripe rust resistance.

Characterization of a partial wheat-Thinopyrum intermedium amphiploid and its reaction to fungal diseases of wheat.

Partial amphiploids between wheat (Triticum aestivum L.) and Thinopyrum species play an important role in the transfer and use of traits from alien species. A wheat-Thinopyrum intermedium partial amphiploid, TAI8335, and its alien parent were characterized by a combination of genomic in situ hybridization (GISH) and cytological observations. Evidence from GISH indicated that the donor parent Th. intermedium possessed seven pairs of S, seven J(s) and 21 J chromosomes. Mitotic observation showed that the majority of TAI8335 plants had 56 chromosomes, but a few had 54 to 55, in some cases with two to three additional telochromosomes. The chromosomes in most pollen mother cells of plants with 2n = 56 formed 28 bivalents, averaging 27.12 in 223 cells, suggesting a basic cytological stability. Sequential GISH patterns using genomic Pseudoroegneria spicata and genomic Th. intermedium DNA as probes revealed that TAI8335 had fourteen chromosomes derived from Th. intermedium and its alien genome consisted of one pair of S-, three pairs of J(s) - and one pair of J-genome chromosomes as well as two translocated chromosome pairs, one being a Robertsonian translocation and another an intercalary translocation, both of which involved J and S genome. Two of the telochromosomes in the aneuploid plants originated from the J genome and one from wheat. Disease screening demonstrated this line was highly resistant to leaf rust, stem rust, stripe rust and powdery mildew. This study showed that the partial amphiploid TAI8335 appears to serve as a novel source for the transfer of resistance genes for multiple fungal pathogens to wheat.

[Development and application of a genome specific marker for Secale africanum].

Genome in situ hybridization (GISH) analysis of wheat-Secale africanum amphiploid revealed that the S. africanum genome displayed significant divergence to the Secale cereale genome. It is thus valuable to deploy genes from S. africanum. We performed the PCR analysis on S. africanum, wheat-S. afticanum amphiploid, T. eastivum cv. Anyuepaideng and other genetic stocks by 100 ISSR primers. A specific segment of 561 bp, named pSaUBC815561, was obtained from S. africanum using primer UBC815. This segment was not amplified from the control wheat lines. Primer UBC815 also am-plified fragments from wild species of genus Secale, including S. vavilovii, S. sylvestre, and other cultivated ryes. Based on the sequence of pSaUBC815561, a pair of special primers U815-F and U815-R was designed and was used to amplify the DNA of wheat related species in Triticeae aimed at validating the specificity of pSaUBC815561. PCR analysis indicated that this specific DNA fragment was amplified not only from a set of Chinese Spring wheat-Imperial rye chromosome addition lines but also from certain wheat-rye introgression lines. Therefore, pSaUBC815561 can be used as a specific marker for detection of chromosomes of Secale genome in wheat.

Discrimination of repetitive sequences polymorphism in Secale cereale by genomic in situ hybridization-banding.

Genomic in situ hybridization banding (GISH-banding), a technique slightly modified from conventional GISH, was used to probe the Chinese native rye (Secale cereale L.) DNA, and enabled us to visualize the individual rye chromosomes and create a universal reference karyotype of the S. cereale chromosome 1R to 7R. The GISH-banding approach used in the present study was able to discriminate S. cereale chromosomes or segments in the wheat (Triticum aestivum L.) background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S. cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye; whereas the GISH-banding pattern of Secale vavilovii was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of adenine-thymine-riched heterochromatin is discussed.

[Analysis of St-chromosome-containing triticeae polyploids using specific molecular markers].

In this study, random amplified polymorphic DNA (RAPD) analysis was performed on Pseudoroegneria spicata, Aegilops ventricosa, Secale cereale cv. Jingzhou Rye, Dasypyrum villosum and other 11 triticeae materials using 200 random 10-based primers. A 542 bp specific RAPD fragment (Accession No. DQ992032), named OPH11542, was obtained from Ps. Spicata. Based on the sequence of OPH11542, a pair of sequence characterized amplified region PCR (SCAR-PCR) primers was designed and used to amplify the materials of triticeae. The result demonstrated that 3 specific DNA segments of 542 bp, 742 bp (DQ992033)and 743 bp (EF014218) respectively, were obtained from St chromosome, however, these 3 segments were not appear in materials not contain St chromosome. Sequence similarity analysis revealed that these 3 segments were new repeated DNA sequences. SCAR-PCR was also performed on 15 materials containing St chromosome, and the result showed that OPH11742 or OPH11743 was always found in materials containing StY chromosome, whereas OPH11542 in materials containing StH chromosome. These results indicated that, chromosome recombination or modification often occurred in St chromosome in the course of combination of St and other chromosome to form a polypolid, and OPH11542, OPH11742 and OPH11743 could be used as molecular markers for the detection of St chromosome.

[Cloning and expression of Gymnadenia conopsea GcSec61beta gene encoding endosplasmic reticulum membrane translocation channel protein].

Protein translocation channel in endosplasmic reticulum (ER) of eukaryotes is composed of several subunits of Sec61 complex, which is essential for protein secretion. In the present study, we cloned a full-length cDNA fragment of 621 bp coding 107 amino acids from a psychrophile and endangered plant Gymnadenia conopsea, which grows in high land. Sequence analysis revealed that the gene was highly homologous to the member Sec61beta of ER protein transporter channel, which was thus designated as GcSec61beta. Phylogenetic tree shows that the GcSec61beta was closely related to the corresponding genes from Arabidopsis thaliana and Oryza sativa. Results of semi-quantitative RT-PCR showed that the expression of GcSec61beta was high both in leaves and the bud, and also induced by low temperature treatment. The sequence of the GcSec61beta was introduced into pET28a vector and transformed to E. coli strain BL21. The growth of E. coli was slowed down but the cold resistance was increased by the expression of GcSec61beta, which provides a new function of GcSec61beta protein.

[Identification and phylogenetic application of unique nucleotide sequence of nad7 intron2 in Rhodiola (Crassulaceae) species].

Genetic characterization of 9 populations of Rhodiola crenulata, R. fastigiata and R. sachalinensis (Crassulaceae) species from Sichuan and Jilin Provinces of China, was investigated using the conserved primer of nad7 intron 2. All PCR products about 800 bp long were shorter than other Crassulaceae plants, which were used as molecular markers to identify the Rhodiola species. The sequence of the products indicated that total exon of 53 bp and intron of 738 bp exhibit only 9 nucleotide variations. Blasting the nad7 sequences to GenBank and the phylogenetic analysis showed that the sequence of Rhodiola species was clusted independently, and the length was smaller than all the registered sequences of higher plants. The result suggests that the Rhiodola species had a unique sequence in this gene region, which might be related to the special growth condition.

[Variation of Rye-Specific repetitive DNA pSc119.1 among sister T1RS.1BL translocations].

Specific primers were designed according to the rye-specific repetitive sequence pSc119.1 and were used for polymerase chain reaction (PCR) analysis using the genomic DNA of two sets of sister T1RS.1BL translocations, CN12, CN17, CN18, 96 I 176-1 and 96 I 176-3 as templates. The results indicated that the target fragments were amplified from CN12, CN17, and CN18. A target and a non-target fragment were produced from 96 I 176-1. However, no products were obtained from 96 I 176-3. Southern blot analysis indicated that the elimination of pSc119.1 did not occur in line 96 I 176-3. Three target fragments were cloned from CN12, CN17, and CN18 respectively through recovering. For each target fragment, ten clones were selected randomly for sequencing. Variation of the sequence pSc119.1 was observed in all of the three wheat lines and line CN18 had the most obvious variation. Most of the 30 sequences had 94% or 95% similarity with the sequence pSc119.1 published and the variation of bases of these sequences. Most variations of most bases arose from transition, and a few of them were transversion. Furthermore, there was great coherence among these changed bases in type and site. The evolution process of progenies of wide hybrids may be continuous. For each set of sister 1RS.1BL translocation, difference of some traits was observed among the wheat lines or cultivars. The difference was probably related to the variation of the repetitive DNA. This research provides some useful information for studying on mechanism of epigenetics.

[Transfer of a rye small chromosomal segment with powdery mildew-resistant gene(s) into common wheat (Triticum aestivum L.)].

The advanced progeny lines (BC1F5) from the monosomic addition lines between common wheat cultivar Mianyang 11, which is highly susceptible to powdery mildew, and an inbred rye line R12 were analyzed for selection of wheat-rye translocations. Based on a rye-specific repetitive sequence of pSc20H, which spread over all chromosomes of rye but did not existed in wheat, a set of PCR primer was designed and used to identify the rye chromosome segments in wheat. From 300 of the BC1F5 progeny lines 70 were found to contain chromosome composition of rye. An advanced line, 96II691-830-98, originated from 6R monosomic addition line was observed to be immune to powdery mildew, different from its wheat parent Mianyang 11. A small segment of rye chromosome at telomere in a pair of wheat chromosome in the line was found by means of GISH. The results indicated that a small segment of rye chromosome 6R carrying the gene(s) for resistance to powdery mildew has been transferred into common wheat. In the progeny of monosomic addition lines a high frequency of wheat-alien species translocation with various segments of chromosomes could be found by application of both PCR and GISH technique.