Studies of new EST-SSRs derived from Gossypium barbadense

Existing cotton EST-SSR markers are mostly derived from Gossypium arboreum and Gossypium hir-sutum, but EST-SSR markers from Gossypium barbadense are scarce. One hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G. barbadense cv. Pima3-79. Among the SSRs, trinucleotide AAG appeared at a high frequency of 11.76%. 36 accessions (consisting of 13 diploids of the A genome, 11 diploids of the D genome and 12 allotetraploids of the AD genome) were employed to test new EST-SSRs. 76 EST-SSRs were successfully amplified, and 313 polymorphic fragments were yielded, with an average of 4.11 fragments per primer pair. The PIC ranged from 0.17 to 0.95 with an average of 0.53. Based on Jaccard’s genetic similarity coefficient, these 36 accessions were clustered into three groups. 21 EST-SSRs exhibited polymorphisms in BC1 population ((Emian22 × Pima3-79) × Emian22), 24 polymor- phic loci were generated, while 22 of the 24 polymorphic loci were integrated with our interspecific BC1 backbone genetic linkage map, and anchored in 12 chromosomes. This study effectively proved that EST-SSRs from G. barbadense are valuable for genetic diversity analysis and genetic mapping.     

Fine mapping of the red plant gene R1 in upland cotton(Gossypium hirsutum)

Sub 16 is a substitution line with G. hirsutum cv. TM-1 genetic background except that the 16th chromosome (Chr. 16) is replaced by the corresponding homozygous chromosome of G. barbadense cv. 3-79, and T586 is a G. hirsutum multiple gene marker line with 8 dominant mutation genes. The R ₁ gene for anthocyanin pigmentation was tagged in Chr. 16 in T586. The objective of this research was to screen SSR markers tightly linked with R ₁ by using the F₂ segregating population containing 1259 plants derived from the cross of Sub 16 and T586 and the backbone genetic linkage map from G. hirsutum×G. barbadense BC₁ newly updated by our laboratory. Genetic analysis suggested that the segregation ratio of red plants in the F₂ population fit Mendelian 1:2:1 inheritance, confirming that the red plant trait was controlled by an incomplete dominance gene. Preliminary mapping of R ₁ was conducted using 237 randomLy selected F₂ individuals and JoinMap v3.0 software. Then, a fine map of R1 was constructed using the F₂ segregating population containing 1259 plants, and R ₁ was located between NAU4956 and NAU6752, with only 0.49 cM to the nearest maker loci (NAU6752). These results provided a foundation for map-based cloning of R ₁ and further development of cotton cultivars with red fibers by transgenic technology. Supported by National Natural Science Foundation of China (Grant No. 30730067) and Programme of Introducing Talents of Discipline to Universities (Grant No. B08025)     

Development of <Emphasis Type="Italic">Gossypium barbadense</Emphasis> chromosome segment substitution lines in the genetic standard line TM-1 of <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

Chromosome segment substitution lines （CSSL） consist of a battery of near-isogenic lines that have been developed and cover the entire genome of some crops. With the exception of one homozygous chromosome segment transferred from a donor parent, the remaining genome of each CSSL line is the same as the recipient parent. It is an ideal material for genome research and particularly QTL mapping. In the present study, we first developed one set of CSSL lines using G hirsutum acc. TM-1 （the genetic standard）, as the recipient parent and G barbadense cv. Hai7124 as the donor parent using molecular assistedlselection in BC5S1-3 generations. The CSSL consisted of 330 different lines, in which 1-4 different lines had the same or overlapping substituted segments. The genetic length of the substituted segments covered 5271.9 cM with an average segment distance of 10.9 cM, 1.5 times the total genetic length of Upland cotton （3514.6 cM）. The substituted segments of each line varied in length, ranging from 3.5 cM for the shortest segment to 23.2 cM in the longest segment. Our CSSL have not yet to cover the entire tetraploid cotton genome, due to the absence of some donor parent interval segments.     

Fine mapping of the awn gene on chromosome 4 in rice by association and linkage analyses

Awnness is a key trait in rice domestication, yet no studies have been conducted on fine mapping or association mapping of the rice awn gene. In this study, we investigated the awnness and genotype of a core collection of 303 cultivated rice varieties and a BC5F2 segregating population of 200 individuals. Combining association and linkage analyses, we mapped the awnness related genes to chromosome 4. Primary association analysis using 24 SSR markers revealed five loci significantly associated with awnness on chromosome 4. The associated markers cover previously identified regions. Fine association mapping was conducted using another 29 markers within a 4-Mb region, covering the associated marker in34, which is close to the awn gene Awn4.1. Seven associated markers were revealed, distributed over an 870-kb region. Combining the fine association mapping and linkage analysis of awnness in the 200 BC5F2 segregating population, we finally identified a 330-kb region as the candidate region for Awn4.1. The results indicate that combining association mapping and linkage mapping provides an efficient and precise approach to both genome-wide mapping and fine mapping of rice genes.     

Modified fiber qualities of the transgenic cotton expressing a silkworm fibroin gene

A silkworm gene for fibroin was introduced into the upland cotton WC line by Agrobacterium-mediated transformation. PCR detection for fibroin, nptII and gus genes, Kanamycin (Km)-resistance analysis and GUS-histochemical assay were conducted on 30 regenerated plants from 9 callus lines, and 17 positive plants were obtained by these 5 screening methods. By Km-resistance analysis and PCR for fibroin, 6 homozygous lines in T₃ were obtained. Southern blot and Northern bolt demonstrated that the fibroin gene was inserted into the genome of these 6 lines, stably inherited and expressed. Compared to the control, the surface structure of mature fiber in the 6 lines was significantly distorted and an increased number of convolution was observed by scanning electron microscopy (SEM). Fiber quality traits analysis indicated that fiber elongation of the 6 homozygous lines was all increased and fiber strength of 3 lines was enhanced. These results indicated that fibroin expression influenced cotton fiber structure and quality, suggesting that fibroin has great potential for improving cotton fiber quality by genetic engineering. Supported by National High Technology Research and Development Program of China (Grant No. 2006AA100105), Science & Technology Pillar Program of Jiangsu Province (Grant No. BE2008310) and Programme of Introducing Talents of Discipline to Universities (Grant No. B08025) Contributed equally to this work     

Construction and initial analysis of five Fosmid libraries of mitochondrial genomes of cotton （Gossypium）

Cotton （Gossypium） is an important crop providing textile fiber and edible oil. To gain the insights into mechanism of the cyto- plasmic male sterility （CMS） inheritance, we constructed five fosmid libraries of mitochondrial genomes from mitotype of G. harknessii Brandegee. （one CMS line and its restorer）, mitotype of G. hirsutum L. （one CMS line and its maintainer）, and G. bar- badense L. The numbers of the clones in these libraries ranged from 1152 to 2016 with an average insert size of 36.2 to 38.4 kb, equivalent to 70-119.3 mitogenomes. The libraries were screened with 28 markers derived from the conservative sequences and yielded 22, 19, 26, 21, and 23 positive clones, respectively. These positive clones were used to construct the physical map of G. harknessii Brandegee. CMS line and G. barbadense L. mitogenomes that shared six syntenis regions. A total of 30 genes in nine clusters showed conservative and had high similarity with those in the mitochondrial genomes of cotton, Carica papaya, Cucur- bita pepo and Nicotiana tabacum. Further investigation indicated that gene rrn26 had two copies in all five cotton mitogenomes, while genes atpl, rrn5 and rrn18 had two copies only in G. barbadense L. The positive clones and physical map are considered being useful resources in cotton genomics research.     

The cytogenetics and fiber properties of an allohexaploid betweenGossypium hirsutum andG. somalense

Cytogenetics and fiber properties were studied on the Gossypium hirsutum×G. somalense F₂ hybrid and later generations. The cytological analyses of pollen mother cells (PMCs) were made at meiotic metaphase Ⅰ. The results indicated that the hybrid was a new allohexaploid, its chromosome number determined as 2n=6x=78, and genome group as 2[(AD)₁E₂]. Chromosomal configurations of the hexaploid averaged 0.15Ⅰ+ 38.72Ⅱ+ 0.11Ⅲ + 0.02Ⅳ per cell, 85.09% PMCs having 39 bivalents, and only 11.84% PMCs having 1 to 2 multivalents, indicating that heterogenetic recombinations of chromosomes occurs between (AD)₁ and E₂, nevertheless, the frequency is lower. The hexaploids are self-fertile and the progenies remain the hexaploids, whose fibers are light brown and have higher strength by measurement of HVI 900 system. The fiber strength increases 42% than that of upland cotton variety. Therefore, it is possible for the hexaploid to be an important germplasm resource to improve fiber strength of cotton.     

Characterization, development and exploitation of EST-derived microsatellites in Gossypium raimondii Ulbrich

COTTON IS AN IMPORTANT GLOBAL CASH CROP. IN THE RECENTYEARS, MOLECULAR MARKER TECHNOLOGY HAS BEEN WIDELY APPLIED TO SUCH STUDIES ON COTTON AS GENETIC MAP- PING[1―4], VARIETY PURITY DETECTION[5], GENETIC DIVERSITY ANALYSIS[6], MOLECULAR MARKER-ASSISTED BR…     

Observation of fiber ultrastructure of Ligon lintless mutant in upland cotton during fiber elongation

Lintless mutant is a super-short fiber mutanl in upland cotton only 4-8 mm in fiber length and also named Ligon cotton controlled by one dominant gene Li1. Fiber ultrastructure of the mutant (Li1) and its its wild (li1) in siti and in vitro was observed under an electron microscope to understand its cytological characteristics during the fiber cell elongation. The resulls showed that the mutant fiber in situ had thinner cytoplasm, more small vacuoles, less mitochondria, Golgi apparatus and endoplasmic reticula, and there were more starch granules which were free or packed in the amyloplast beside the cell wall than thai of wild type. It was indicated that scarcity of functional organelles and disability of transformation from starch to sugar might be associated with the tact that the mutant fiber cell was aborted too early to elongate into normal length. Mutant ovule in some media containing GA3 could produce a kind of huge callus that grew faster than normal ovules. The callus was covered with many white, loose, and semitransparent fiber-like cells that apt lo get off from ovule. These fiber-like cells were multicellular fibers generated by cell division and had black dots just like pigment glands in the stem and leaf of cotton. There were lots of micro-tubes beside cytoplasm membrane uf the multiecllular fiber, which were thought to be primary preparation for second wall deposition of multicellular fiber. It was indicated that GA3 might induce the expression of gene(s) that kept inactive in the field condition and then stimulate the original fiber cell in vitro to undergo divisionagain.     

QTL mapping of resistance to sheath blight in maize（Zea mays L.）

Maize sheath blight (Rhizoctonia Solani) is a widely occurring fungus disease with great harm to corn-pro- ducing regions in the world. The first happening of sheath blight in China was reported in Jilin Province as early as in 1966[1]. Since the 1970s, the enlargement of corn- growing regions, the application of maize hybrids, the increasing use of fertilizers, especially the nitrogenous fertilizer, and a higher growth-density, all have caused a quick spread of sheath blight, the occurring …     

Breeding,introgression and inheritance of delayed gland morphogenesis trait from<Emphasis Type="Italic">Gosspium bickii</Emphasis> into upland cotton germplasm

Cotton is a valuable economic crop. The cottonseedafter ginning is made up of fuzz, kernel and hull, amongwhich kernel constitutes about 50% to the seed weight.Cottonseed kernel of G. hirsutum contains about 40% ofprotein and more than 35% of cottonseed oil, which obvi-ously is a potential rich source of high quality protein andedible oil supplement[1,2]. However, the utilization of cot-tonseed is limited by the presence of gossypol and its de-rivatives in seeds of ordinary glanded cotton cult…     

Cloning of fiber-specific cDNAs and their structural variations in 4 fiber mutants

A mRNA preferentially expressed in cotton fiber was cloned from fiber total RNA of normal upland cotton TM-1 (wild-type) by using RT-PCR and corresponding cDNA (signed as TM-E6) was sequenced. TM-E6 gene had no intron and contained an open reading frame of 771 bp long, and might encode a peptide of 246 amino acids. Other 4 genes, Fl-E6, Li-E6, N-E6 and Bl-E6, which were homologous to TM-E6 gene, were also isolated from 4 fiber mutants of Fiberless Xu-zhou 142, Ligon lintless, Naked seed and Brown lint, respectively. Sequence analysis of each of these mutant genes revealed many variations in structure and nucleotide composition of gene when compared with the sequence of TM-E6 gene. (ⅰ) There was a changeable repetitive segment in which GGCTCA (Gly-Ser) was repeated 3—5 times between the 82nd and the 93rd codons in different mutant genes. Since the change of Gly-Ser repetitive segment occurred not only in the mutants but also in the wild-type cotton, the repeat frequency might not be associated with the mutation of fiber characteristics. (ⅱ) Among the 4 mutant genes, the percentage of changed codons was 7.05% in Fl-E6, 4.98% in Li-E6, and 4.15% in N-E6 and Bl-E6. It seems that the percentage of changed codons in E6 sequence was positively correlated to the degree of fiber morphological variation. (ⅲ) E6 polypeptides of two long-fiberless mutants (Fiberless Xuzhou 142 and Ligon lintless) contained high similar (99.4%) variation in the region of 1—174 amino acids from N-terminus, and those of short-fiberless mutants (Fiberless Xuzhou and naked seed) revealed identical variation in the region of 116th—220th amino acids. It also seems that there was a parallel relation between E6 protein variation and fiber phenotype mutation. (ⅳ) Li-E6 and Bl-E6 genes also expressed at low level in seed coat besides at high level in fiber.     

Construction of a linkage map and QTL mapping for fiber quality traits in upland cotton (Gossypium hirsutum L.)

With the development in spinning technology, the improvement of cotton fiber quality is becoming more and more important. The main objective of this research was to construct a high-density genetic linkage map to facilitate marker assisted selection for fiber quality traits in upland cotton (Gossypium hirsutum L.). A genetic linkage map comprising 421 loci and covering 3814.3 cM, accounting for approximately 73.35% of the cotton genome, was constructed using an F2 population derived from cross GX1135 (P 1 )×GX100-2 (P 2 ). Forty-four of 49 linkage groups were assigned to the 26 chromosomes. Fiber quality traits were investigated in F2 population sampled from individuals, and in F2:3 , and F2:4 generations sampled by lines from two sites and one respectively, and each followed a randomized complete block design with two replications. Thirty-nine quantitative trait loci were detected for five fiber quality traits with data from single environments (separate analysis each): 12 for fiber length, five for fiber uniformity, nine for fiber strength, seven for fiber elongation, and six for fiber micronaire, whereas 15 QTLs were found in combined analysis (data from means of different environments in F2:3 generation). Among these QTLs, qFL-chr5-2 and qFL-chr14-2 for fiber length were detected simultaneously in three generations (four environments) and verified further by combined analysis, and these QTLs should be useful for marker assisted selection to improve fiber quality in upland cotton.     

Inheritance of the delayed gland morphogenesis trait in Australian wild species ofGossypium

Five Australian wild cotton species with the delayed gland morphogenesis trait, as well as G. arboreum, G. davidsonii and four different gland genotypes of G. hirsutum, Gl₂Gl₂Gl₃Gl₃, Gl₂Gl₂gl₃gl₃, gl₂gl₂Gl₃Gl₃, and gl₂gl₂gl₃gl₃, were used in this experiment and 10 interspecific hybrids were obtained by the crossing among them. According to the gland expression on the seeds and plants of the interspecific hybrids, the inheritance of the delayed gland morphogenesis trait of Australian wild cotton species was opened out as follows: (ⅰ) the inheritance of the delayed gland morphogenesis trait was almost the same among the 5 Australian wild cotton species, and the gene or genes which controlled this trait may be located in the same loci. (ⅱ) The glandless seed trait of the Australian wild cotton species was dominant over the glanded seed trait of G. arboreum, a genome A species, and the seeds of interspecific hybrid F₁ between them were glandless. However, it was recessive over the glanded character of genome D species, G.davidsonii, and their F₁ was a typical glanded one. (ⅲ) The glandless seed trait of the Australian wild cotton species was recessive or incomplete dominant over the glanded cotton but dominant over the glandless cotton of G. hirsutum, and the glandless genes (gl₂gl₂gl₃gl₃) of upland cotton had great weakening effect on the glanded plant trait of the Australian wild cotton species on the other hand. For the two main glanded genes of upland cotton, the delayed gland morphogenesis trait of the Australian wild cotton species was dominant epistatic over glandless genes, gl₂gl₂gl₃gl₃, and one of the glanded genes, Gl₂Gl₂, but was recessive epistatic over the other glanded gene, Gl₃Gl₃. Therefore, it is much convenient to use Gl₂Gl₂gl₃gl₃ as the upland cotton parent in the interspecific hybridization and backcrossing afterward, in order to produce the upland cotton germplasm with glandless seeds and glanded plant trait.     

SCFP,a novel fiber-specific promoter in cotton

To investigate the expression pattern of GhSCFP which was isolated from cotton fiber cDNA library, a 1006 bp upstream fragment of the gene was cloned by chromosome walking and fused to GUSand GFP respectively. Histochemical GUS and GFP fluorescence analysis revealed that the expression of the report genes driven by the promoter sequence was detectable only in outer layer cells during the seed development in the transgentic tobaccos. In transgenic cotton, strong GUS activity was observed in spherical protrusions on 0 dpa （days post anthesis） ovule surface, and in the 2-36 dpa fiber cells, while no GUS signals were detected in the root, leaves, stem, corolla, anther and stigma. Our data demonstrated that GhSCFP upstream sequence is a cotton fiber-specific promoter and this promoter will be useful in the molecular research on fiber cell development and in cotton fiber improvements by genetic modification.     

Identification of RAPD marker linked with fertility-restoring gene of cytoplasmic male sterile lines in upland cotton

Bulked segregant analysis was employed to construct two mixed DNA pools to screen the RAPd marker linked with the fertility-restoring gene(Rf _i) of upland cotton. A total of 425 arbitrary 10-mer oligonucleotide primers were screened on two DNA pools, bulked male fertile and sterile DNAs isolated from BC₃ segregating population of (0-613-2R X Simian No. 3). Three primers produced repeatable polymorphisms between the paired bulks and their parents. DNA was extracted and amplified with these three primers for 92 plants of (Zhong 12A-1 × 0-613-2R)F₂. Based on the male fertility scoring and RAPD amplification, it is found that one RAPD marker fragment designated OPV-15₃₀₀ was linked with the fertility-restoring gene (Rf₁) with a recombination value of 13.0±2.57%.     

天然棕色棉纤维发育过程中生化物质变化规律的研究

目的:比较棕色棉和白色棉纤维发育过程中生化物质含量的差异,分析棕色棉纤维色素合成与纤维发育的关系,为棕色棉育种提供理论依据。方法:以白色棉泗棉3号为对照,测定4种棕色棉各发育阶段纤维中主要生化物质含量及其动态变化规律,分析了棕色棉纤维各发育时期生化物质含量与棕色棉纤维色素含量相关关系。结果:棕色棉纤维40DPA含水率、10DPA还原糖含量低于白色棉,30-40DPA还原糖含量高于白色棉。白色棉20DPA可溶性蛋白质出现高峰期,棕色棉相应的蛋白质高峰期出现在25DPA,棕色棉纤维生长的各阶段纤维素的含量均低于白色棉;棕色棉和白色棉,含水率、还原性糖、可溶性蛋白质的含量都随着棉纤维发育不断降低,而纤维素含量不断增加。成熟棉纤维色素含量与10DPA还原性糖含量、30-40DPA纤维素含量负相关达到显著水平(p<0.05),与35DPA还原性糖含量正相关达到极显著水平(p<0.01),与15DPA纤维素含量负相关达到极显著水平(p<0.01)。结论:棕色棉纤维的各发育阶段的生化物质含量与白色棉表现明显的差异,但是二者在动态变化规律上表现一致;棕色棉色素合成与棉纤维发育过程中生化物质组成关系密切。     

Influence of Characteristics on Bending Strength of Layered Steel Fiber Reinforced Concrete

The influence of two main characteristics of steel fiber, the aspect ratio （Df） and volume fraction （ρf）, on the bending strength of Layered Steel Fiber Reinforced Concrete （LSFRC） is investigated by using orthogonal test. Via the variance analysis on the experimental results and trend analysis on the two characteristics, Df is found significantly related to the bending strength of LSFRC. The influence ratio is 63.3%. The bending strength of LSFRC increases if Df increases, makes better when Df reaches 100. ρf has ordinary influence on the bending strength of LSFRC. The influence ratio is 29.2%. Other characteristics, such as the shape of steel fiber and the mix proportion, have less influence. The best ρf contributing to the bending strength of LSFRC is 1.5 %. If ρf is greater than 1.5%, it has negative influence on the bending strength of LSFRC. So, ρf makes a limited contribution to the bending strength of LSFRC.     

Gene expression profiling related to powdery mildew resistance in wheat with the method of suppression subtractive hybridization

“Bainong 3217 × Mardler” BC₅F₄ wheat line at the initial stage of inoculation with powdery mildew pathogen (Erysiphe graminis DC) was used to construct a suppression subtractive hybridization (SSH) cDNA library. Totally 760 ESTs were obtained through sequencing. Similarity analysis of ESTs based on BLASTn and BLASTx with the sequences in GenBank, in combination with macroarray differential screening, revealed that 199 ESTs of 65 kinds were known to be functionally disease resistance related. Based on the gene expression profiling in the present study, it is postulated that salicylic acid (SA) and MAP-related signal transduction pathways were involved in powdery mildew resistance in wheat. System acquired resistance genes were predominant in terms of kinds and quantity. With the initiation of cell defense reaction, the genes conferring anti-oxidation substances were largely expressed and thus cell protection mechanism was activated. Much evidence revealed that phenylpropanes metabolic pathway was involved in phytoalexin synthesis in wheat powdery mildew resistance. Genes conferring some enzymes of structural modification of cell walls and proteinase inhibitors inhibiting pathogen growth were also detected. The genes controlling a few proteinases (mainly cysteine proteinase) had a considerable redundancy of expression.