Analysis of F1 hybrid and BC1 monosomic alien addition line plants from Brassica oleracea × Sinapis alba by GISH

Sterile and semi-fertile F₁ plants were obtained by intergeneric sexual hybridization between paternal Brassica oleracea var. alboglabra (genome CC, 2n=18) and maternal Sinapis alba (genome SS, 2n=24), BC₁ plants were obtained by backcrossing between paternal B. oleracea and maternal semi-fertile F₁ plants. Genomic in situ hybridization (GISH) combined with dual-colour fluorescence in situ hybridization (dcFISH) showed that sterile F₁ plants contained 21 chromosomes consisting of one B. oleracea chromosome set and one S. alba chromosome set, belonging to expected hybrids, and semi-fertile F₁ plants contained 30 chromosomes consisting of two B. oleracea chromosome sets and one S. alba chromosome set. It is obvious that the semi-fertile F₁ plants belong to unexpected hybrids. 1―3 trivalents were detected at meiotic metaphase I of semi-fertile F₁ pollen mother cells (PMCs). Different separation ratios of S chromosomes were detected at anaphase I. A monosomic alien addition line (MAAL) was identified by GISH-dcFISH from BC₁ plants; it contained 19 chromosomes consisting of 18 C chromosomes and 1 S chromosome. At meiotic metaphase I, 9 divalents from B. oleracea and one univalent from S. alba could be detected. Sometimes, one putative C-S trivalent could also be detected. The achievement of B. oleracea-S. alba monosomic alien addition lines lays a foundation for gene introgression, location and cloning.     

Development and characterization of interspecific hybrids between <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Subscript>O. latifolia</Subscript> by <Subscript>in situ</Subscript> hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization （GISH） and fluorescent in situ hybridization （FISH） analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase Ⅰ of the hybrid pollen mother cell, poor chromosome pairing was identified and most of the chromosomes were univalent, which resulted in complete male sterility in the hybrid.     

Development and characterization of interspecific hybrids between Oryza sativa and O. latifolia by in situ hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase I of the hybrid pollen mother cell, poor chro- mosome pairing was identified and most of the chromosomes were univalent, which resulted in com- plete male sterility in the hybrid.     

Development of Triticum aestivum-Leymus racemosus ditelosomic substitution line 7Lr#1S（7A） with resistance to wheat scab and its meiotic behavior analysis

Leymus racemosus is highly resistant to wheat scab （Fusarum head bright）. The transfer of scab resistant gene from L. racemosus to Triticum aestivum is of great significance for broadening the base of wheat resistance. In the present study, the pollen of T. aestivum-L, racemosus monosomic addition line with scab resistance was treated by irradiation with 1200 R ^60Co-γ-rays prior to pollinating to emasculated wheat cv. Mianyang 85-45. Nine plants with a telocentric chromosome 7Lr#1S were observed in M1, and one ditelosomic substitution line 7Lr#1S was selected from selfcrossing progenies and confirmed by chromosome C-banding and GISH. Furthermore, a co-dominant EST-SSR marker CINAU 31 was employed to identify this substitution line. A pair of chromosome 7A of common wheat were found to be replaced by a pair of telocentric chromosome 7Lr#1S, and further investigation showed that chromosome configuration of the substitution line at MI of PMCs after GISH was 17.50Ⅱ^w ＋ 2.19Ⅱ^w ＋ 0.42Ⅱ^7Lr#1S ＋ 1.08 Ⅰ^7Lr#1S ＋ 0.69 Ⅰ^w. Two telocentric chromosomes paired as a bivalent in 59.7% of PMCs. Abnormal chromosome behaviors of telocentric chromosomes were observed in part of PMCs at anaphase I and telophase I, including the moving of two telocentric chromosomes to the same pole, lagging and earlier separation of their sister chromatid. All these abnormal behaviors can be grouped into three distinct types of tetrads according to different numbers of 7Lr#1S in their daughter cells and various micronucleus in some tetrads. However, due to the high transmission frequency of the female and male gametes with a 7Lr#1S, 84% of the selfcrossing progeny plants had ditelosomic substitution. The substitution line showed high resistance to wheat scab in a successive two-year test both in the greenhouse and field; hence, the line will be particularly valuable for alien gene mapping, small fragment translocation induction and telosomic cytological behavior analysis.     

Development of Triticum aestivum-Leymus racemosus ditelosomic substitution line 7Lr#1S(7A) with resistance to wheat scab and its meiotic behavior analysis

Leymus racemosus is highly resistant to wheat scab (Fusarum head bright). The transfer of scab resistant gene from L. racemosus to Triticum aestivum is of great significance for broadening the base of wheat resistance. In the present study, the pollen of T. aestivum-L. racemosus monosomic addition line with scab resistance was treated by irradiation with 1200 R 60Co-γ-rays prior to pollinating to emasculated wheat cv. Mianyang 85-45. Nine plants with a telocentric chromosome 7Lr#1S were observed in M1, and one ditelosomic substitution line 7Lr#1S was selected from selfcrossing progenies and confirmed by chromosome C-banding and GISH. Furthermore, a co-dominant EST-SSR marker CINAU 31 was employed to identify this substitution line. A pair of chromosome 7A of common wheat were found to be replaced by a pair of telocentric chromosome 7Lr#1S, and further investigation showed that chromosome configuration of the substitution line at MI of PMCs after GISH was 17.50○II W 2.19 IIW 0.42II7Lr#1S 1.08 I7Lr#1S 0.69 IW. Two telocentric chromosomes paired as a bivalent in 59.7% of PMCs. Abnormal chromosome behaviors of telocentric chromosomes were observed in part of PMCs at anaphase I and telophase I, including the moving of two telocentric chromosomes to the same pole, lagging and earlier separation of their sister chromatid. All these abnormal behaviors can be grouped into three distinct types of tetrads according to different numbers of 7Lr#1S in their daughter cells and various micronucleus in some tetrads. However, due to the high transmission frequency of the female and male gametes with a 7Lr#1S, 84% of the selfcrossing progeny plants had ditelosomic substitution. The substitution line showed high resistance to wheat scab in a successive two-year test both in the greenhouse and field; hence, the line will be particularly valuable for alien gene mapping, small fragment translocation induction and telosomic cytological behavior analysis.     

含抗条锈病基因和黑麦碱基因(sec-1)的小麦-黑麦小片段易位系的鉴定

An elite wheat line 1R734 was selected from the selfed progeny lines(BC-2F-8)of the monosomic 1R addition lines between common wheat cultivar Mianyang 11 and an inbred rye line “baili”,with good resistance to stripe rust.APAGE(acid-polyacrylamide gel electrophoresis)analyses showed that the rye secalin bands were present in 1R734. However, genomic in situ hybridization(GISH)provided that the terminal regions of the short arm of a pair of wheat chromosomes showed yellowgreen fluorescent signals. It has been concluded that a small segment of rye chromosome 1R carrying the gene(s)for resistance to stripe rust and secalin(sec-1)has been transferred to wheat line 1R734. Besides, the use of monosomic addition lines for transferring the small segments of alien chromosomes into wheat is an effective approach.     

Isolation and identification of the three rice monotelosomics

In order to obtain rice monotelosomic, the progeny of 24 telotrisomics, derived from an indica rice variety, Zhongxian 3037, were screened. The variants that differed morphologically from the diploids and the original primary trisomics as well as the telotrisomics were collected for cytological identification. The variants with 24 chromosomes were selected according to the prometaphase chromosomes. From these variants, three monotelosomies with one chromosome arm deletion in each were verified by fluorescence in situ hybridization （FISH） using a rice centromeric BAC clone of 17p22 as a marker probe. The three monotelosomics were derived from telotrisomic 1S, 4L and 11L, respectively. Further identification was conducted on the prometaphase or pachytene chromosomes of the three variants, which were probed with the same centromeric BAC clone together with the corresponding chromosome arm specific makers, a0059H02 （on the short arm of chromosome 1）, a0034E24 （on the long arm of chromosome 4）, and a0071H11 （on the long arm of chromosome 11）. The results indicated that the telocentric chromosomes in the three monotelosom. ics were derived from their respective corresponding telotrisomics. According to the telocentric chromosomes of the variants, they were monotelosomic 1S （one long arm of chromosome 1 was lost）, monotelosomic 4L （one short arm of chromosome 4 was lost） and monotelosomic 11L （one short arm of chromosome 11 was lost）, respectively.     

Hybrid plant regeneration from interfamilial somatic hybridization between grapevine (vitia vinifera L.) and Red Thorowax (Bupleurum scorzonerifolium willd)

The protoplasts of Red Thorowax ( Bupleurum scorzonerifolium) irradiated by ultraviolet light (UV) at an intensity of 260μW/cm2 for 0, 1,2 and 3 min respectively were fused with that of grapevine ( Vitia vinifera). The regenerated 19 clones, every one derived from a single fused cell, were identified as hybrids by phenotype, isozyme, chromosome and 5S rDNA spacer region analysis. The results reveal that all of them are somatic hybrids. 11 hybrid calli including asymmetric and symmetric products regenerated somatic embryos and young leaves after 5 months of culture, of which 4 hybrid cell lines derived from asymmetric fusion regenerated plants with roots after 8-10 months of culture. Inspection of chromosome showed that regeneration of whole plant was related to the decrease of chromosome number. Identification of 5S rDNA spacer region of the plants confirmed that they were interfamilial hybrid plants.     

Quantitative trait loci （QTLs） for quality traits related to protein and starch in wheat

Quality traits in wheat （Triticum aestirum L.） were studied by quantitative trait locus （QTL） analysis in a recombinant inbred line （RIL） population, a set of 131 lines derived from Chuan 35050 × Shannong 483 cross （ChSh）. Grains from RILs were assayed for 21 quality traits related to protein and starch. A total of 35 putative QTLs for 19 traits with a single QTL explaining 7.99-40.52% of phenotypic variations were detected on 10 chromosomes, 1D, 2A, 2D, 3B, 3D, 5A, 6A, 6B, 6D, and 7B. The additive effects of 30 QTLs were positive, contributed by Chuan 35050, the remaining 5 QTLs were negative with the additive effect contributed by Shannong 483. For protein traits, 15 QTLs were obtained and most of them were located on chromosomes 1 D, 3B and 6D, while 20 QTLs for starch traits were detected and most of them were located on chromosomes 3D, 6B and 7B. Only 7 QTLs for protein and starch traits were co-located in three regions on chromosomes 1D, 2A and 2D. These protein and starch trait QTLs showed a distinct distribution pattern in certain regions and chromosomes. Twenty-two QTLs were clustered in 6 regions of 5 chromosomes. Two QTL clusters for protein traits were located on chromosomes 1D and 3B, respectively, three clusters for starch traits on chromosomes 3D, 6B and 7B, and one cluster including protein and starch traits on chromosome 1D.     

Generating of rice OsCENH3-GFP transgenic plants and their genetic applications

In order to investigate rice functional centromeres, OsCENH3-GFP chimeric gene was constructed and transformed into the indica rice variety, Zhongxian 3037, mediated by Agrobacturium. The integration of the exogenous genes in the transgenic plants was confirmed by PCR and Southern blotting. The transgenic plants grow normally during their whole life time, just like Zhongxian 3037. No significant defects were detected in either mitosis or meiosis of the transgenic plants. The overlapping of GFP signals and anti-CENH3 foci in both mitotic and meiotic cells from T0 and T1 generation plants indicated that GFP had been successfully fused with CENH3, so the GFP signals can well represent the CENH3 locations on each chromosome. To evaluate the applicability of the transgenic plants to other genetic studies, fluorescence in situ hybridization (FISH) using rice centromeric tandem repetitive sequence CentO as the probe was conducted on the zygotene chromosomes of pollen mother cells (PMCs). It has been revealed that the GFP signals are overlapping with CentO FISH signals, showing that CentO is one of the key elements constituting rice functional centromeres. Immunofluorescent staining using anti-α-tublin antibody and anti-PAIR2 antibody on the chromosomes during mitosis and meiosis stages of the transgenic plants further reveals that OsCENH3-GFP transgenic plants can be widely used for studying rice molecular biology, especially for tagging functional centromeres in both living cells and tissues.     

Generating of rice OsCENH3-GFP transgenic plants and their genetic applications

In order to investigate rice functional centromeres, OsCENH3-GFP chimeric gene was constructed and transformed into the indica rice variety, Zhongxian 3037, mediated by Agrobacturium. The integration of the exogenous genes in the transgenic plants was confirmed by PCR and Southern blotting. The transgenic plants grow normally during their whole life time, just like Zhongxian 3037. No significant defects were detected in either mitosis or meiosis of the transgenic plants. The overlapping of GFP signals and anti-CENH3 foci in both mitotic and meiotic cells from T0 and T1 generation plants indicated that GFP had been successfully fused with CENH3, so the GFP signals can well represent the CENH3 locations on each chromosome. To evaluate the applicability of the transgenic plants to other genetic studies, fluorescence in situ hybridization （FISH） using rice centromeric tandem repetitive sequence CentO as the probe was conducted on the zygotene chromosomes of pollen mother cells （PMCs）. It has been revealed that the GFP signals are overlapping with CentO FISH signals, showing that CentO is one of the key elements constituting rice functional centromeres. Immunofluorescent staining using anti-o-tublin antibody and anti-PAIR2 antibody on the chromosomes during mitosis and meiosis stages of the transgenic plants further reveals that OsCENH3-GFP transgenic plants can be widely used for studying rice molecular biology, especially for tagging functional centromeres in both living cells and tissues.     

Microdissection of chromosome 7B of common wheat and cloning of low-copy specific DNA sequences

The 7B chromosome of common wheat was microdissected from pollen mother cells of the 7B monosomic line of common wheat cv. Chinese Spring (CS). After proteinase K and DNA topoisomerase Ⅰ treatments, the isolated chromosomes were subjected to 1-3 rounds of DOP-PCR amplification, which produced continuous DNA fragments ranging from 150 to 700 bp. Ge-nomic Southern hybridization confirmed that the PCR products were originated from the wheat genome. Cloning of portion ( > 200 bp) of the 3rd round DOP-PCR products (50 μL) could generate about 20 000 recombinant clones. Characterization of 50 randomly chosen clones indicated that 21 clones produced discrete PCR products with the size of 240-600 bp. Dot-blot hybridization showed that among the 21 clones, 11 ( ～ 55%) were of low-copy nature while 10 ( ～ 45%) were repetitive. Southern hybridization with the complete set of the CS "nullisomic-tetrasomic (NT)" lines demonstrated that all the 6 low-copy clones were specific to either chromosome 7B or the 7th homoeologous group, whereas the 3 arbitrarily chosen repetitive clones were non-specific, disperse sequences.     

Knob-associated tandem repeats on mitotic chromosomes in maize,Zea diploperennis and their hybrids

Knob-associated tandem repeats, 180-bp repeats and TR-1 elements, together with 45S rDNA were located on mitotic chromosomes of Zea diploperennis (DP),maize inbred line F102 and their hybrid. In DP, knobs on the short arm of chromosomes 1 and 4 and on the long arm ofthe chromosomes 4 and 5 are composed predominantly of the 180-bp repeats. In addition, 180-bp repeats existed together with TR-1 elements were also detected on the short arm ofchromosomes 2 and 5 and on the long arm of the chromosomes 2, 6, 7, 8 and 9. In maize inbred line F102, 180-bp repeats were present in chromosomes 7S and one homologue of chromosomes 8L. TR-1 elements appeared on satellite of chromosome 6 and no detectable hybridization site co-located with 180-bp repeats was observed in maize F102.Polymorphism of size, number, and distribution of 180-bp and TR-1 signals were revealed among different chromosomes in these two species and heteromorphism existed between some homologous chromosomes in the same species.Using these excellent landmarks, the interspecific hybrid of maize and DP were identified. The results suggest that comparative analysis of 180-bp repeats and TR-1 elements may help understand the genome organization and the evolution in Zea.     

Morphological and molecular characterization of two G. somalense monosomic alien addition lines （MAALs）

Two G. somalense monosomic alien addition lines were identified from the derived backcross progenies of allohexaploid between G. hirsutum and G. somalense through cytological and morphological observation. Furthermore, the alien addition chromosome was identified and distinguishedby RAPD analysis. A total of 160 RAPD primers were usedfor PCR amplification. Primer SBSG11 could produce a specific molecular marker (600 bp) for monosomic alien addition line Ⅰ (MAAL Ⅰ ). Primer SBSC03 could produce aspecific molecular marker (700 bp) for monosomic alien ad-dition line Ⅱ (MAAL Ⅱ). SBSE07 and SBSE08 could re-spectively produce common molecular marker for mono-somic alien addition lines Ⅰ and Ⅱ. G. somalense alienaddition lines could be important for cotton improvement.     

Distribution of highly repeated DNA sequences in Haynaldia villosa and its application in the identification of alien chromatin

Haynaldia villosa (L.) is a wild relative species of common wheat that possesses many beneficial genes that can be used for wheat improvement. The accurate detection of H. villosa chromosomes in the genetic background of wheat is critical for transferring its beneficial genes to common wheat by chromosome engineering. The aim of the present study was to investigate the distribution patterns of two repeated DNA sequences, pSc119.2 and pAs1, as well as two rDNA multigene family sequences, 45S rDNA and 5S rDNA, in the individual chromosomes of H. villosa for the future precise identification of alien chromatin in germplasm development and breeding programs. A set of common wheat-H. villosa disomic addition 1V-7V lines was used to determine these specific signals on individual chromosomes of H. villosa. The results showed that two rDNA probes, pTa71 (45S rDNA) and pTa794 (5S rDNA), were located on 1VS and 5VS, respectively, and the signal could be discriminated exclusively in the common wheat background as effective markers of 1VS and 5VS. Furthermore, all seven chromosomes of H. villosa could be distinguished clearly by fluorescence in situ hybridization using pSc119.2 and pAs1 as probes in combination. The utilization of these cytogenetic markers of repetitive sequences, combined with other molecular markers sometimes, will make it possible for a precise identification of alien chromosomes with high efficiency.     

Breeding and molecular cytogenetic identification of wheat-Thinopyrum intermedium addition lines resistant to powdery mildew

Wheat-related species Th. intermedium was used to cross with common wheat Yannong 15. In the self progenies of the hybrid, two addition lines, Ⅱ-1-7-1 and Ⅱ-3-3-2, stable in cytology, were developed by cytology and powdery mildew resistance identification. Their chromosome number were 2n = 44 and formed 22 bivalents at PMC MI. In F₁ of the two addition lines crossing with Yannong 15, there appeared about one univalent at PMC MI, respectively. Resistance identification in greenhouse and field using the No. 15 and mixed strains of E. gramnis f. sp. tritici showed that they were immune to powdery mildew. Chromosome number and resistance identification using the F² single plants of the addition line crossing with Yannong 15 indicated that the resistant gene was located on the alien chromosomes. In situ hybridization using St and E genomic DNA as probe showed that the added chromosome in the two addition lines probably came from the E genome of Th. intermedium, which indicated that a pair of E genome chromosomes carried a new resistant gene to powdery mildew.     

Centromere inactivation in a dicentric rice chromosome during sexual reproduction

T0135 is a variant selected from the progeny of a rice line telotrisomic for the short arm of chromosome 11 （2n＋IIS＇）. Fluores- cent in situ hybridization （FISH） results indicated that T0135 contained two telocentric chromosomes, which have two centro- mere-specific molecular markers （5S rDNA） for chromosome 11; thus T0135 is a newly-described rice chromosome variant with two dicentric chromosomes, named 22＋11L-＋11L＇＋I IS.11S-＋I 1S-11S. （22 represents the 22 chromosomes excluding chromo- some 11 in the rice genome, ＂-＂ represents the centromere）. To investigate the genetic stability of the rice dicentric chromosomes during sexual reproduction, we observed the chromosome types in the progeny. Ninety-four percent of the progeny had the same chromosome type as the parental line. This result indicates that the dicentric chromosomes are mostly stable during mitosis and meiosis. Immunofluorescence analysis for centromere specific histone H3 （CENH3） revealed that only one centromere is active and the other centromere is inactivated in the rice dicentric chromosomes.     

Mouse Karyotype Obtained by Combining DAPI Staining with Image Analysis

In this study, mitotic metaphase chromosomes in mouse were identified by a new chromosome fluorescence banding technique combining DAPI staining with image analysis. Clear 4＇, 6-diamidino-2-phenylindole （DAPI） multiple bands like （J-hands could be produced in mouse. The Meta- Morph software was then used to generate linescans of pixel intensity for the banded chromosomes from short arm to long arm. These linescans were sufficient not only to identify each individual chromosome but also analyze the physical sites of bands in chromosome. Based on the results, the clear and accurate karyotype of mouse metaphase chromosomes was established. The technique is therefore considered to he a new method for cytological studies of mouse.     

QTL analysis of flag leaf in barley （Hordeum vulgate L.） for morphological traits and chlorophyll content

To understand genetic patterns of the morphological and physiological traits in flag leaf of barley, a double haploid （DH） population derived from the parents Yerong and Franklin was used to determine quantitative trait loci （QTL） controlling length, width, length/width, and chlorophyll content of flag leaves. A total of 9 QTLs showing significantly additive effect were detected in 8 intervals on 5 chromosomes. The variation of individual QTL ranged from 1.9% to 20.2%. For chlorophyll content expressed as SPAD value, 4 QTLs were identified on chromosomes 2H, 3H and 6H; for leaf length and width, 2 QTLs located on chromosomes 5H and 7H, and 2 QTLs located on chromosome 5H were detected; and for length/width, I QTL was detected on chromosome 7H. The identification of these QTLs associated with the properties of flag leaf is useful for barley improvement in breeding programs.     

FISH of 5S rDNA and telomeric (TTAGGG) n repeats in normal and translocated populations of the frog Quasipaa boulengeri (Anura, Ranidae)

Quasipaa boulengeri,a spiny frog,is widely distributed in the low mountain regions,around Sichuan Basin.Our previous study revealed five karyotypes,caused by a translocation,that are randomly distributed throughout different populations.5S rDNA and telomere sequence(TTAGGG) n are potential good markers for chromosome identification and karyological evolution.In this study,we examined the sequences of 14 populations using fluorescence in situ hybridization(FISH) to detect if there is any variation between karyologically normal and translocated populations.5S rDNA loci were located at the same position on chromosomes 1 in 7 translocated populations.In two of the seven normal populations,5S rDNA also occurred on chromosome 5 in addition to chromosome 1.Our findings further indicate that the 5S rDNA on No.1 most likely represents the ancestral condition,while the minor loci represent the derived state.Signal density variations of the 5S rDNA were observed beteween homologous chromosomes or sister chromatids of pair 1 in both normal and translocated populations.Telomere sequences were identically located on all ends of the 26 chromosomes in seven rearranged populations,however,no ITSs were observed on the translocated chromosomes 1 and 6.Two of the six normal populations were found to contain ITSs which indicates that populations with translocation events diverged prior to those with ITSs rearrangements.In the KKS and BF populations,the ITSs of chromosome 3 are not always found on both homologues.Inter-chromosomal signal strength of telomeric sequences commonly differs within all populations.