Genetic analysis,molecular tagging and mapping of the thermo-sensitive genic male-sterile gene (wtms1) in wheat

A thermo-sensitive genic male-sterile (TGMS) wheat line ( Triticum aestivum L.) BNY-S was obtained from the spontaneous mutant of BNY-F. Its fertility was decided by the temperature during the differentiation stage of the spikelets. BNY-S was completely sterile when the temperature was lower than 10 degrees C during the differentiation stage of the spikelets, but fertile when the temperature was higher than 10 degrees C. Genetic analysis indicated that the sterility of BNY-S was controlled by a single recessive gene, which was named as wtms1. An F(2) population, consisting of 3,000 individuals from the cross between BNY-S and Lankao 52-24, was used for genetic analysis and statistical analysis of the TGMS and, out of them, 158 sterile and 93 fertile extremes were present for molecular tagging and mapping of the wtms1 gene. SSR (simple sequence repeat) and AFLP (amplified fragment length polymorphism) techniques combined with BSA (bulked segregant analysis) were used to screen markers linked to the target gene. As a result, wtms1 was preliminarily mapped on chromosome 2B according to SSR analysis. In AFLP analysis, 14 polymorphic AFLP loci were identified with a linkage relation to the wtms1 gene. Then linkage analysis using the F(2) population showed that three of them, E: AAG/M: CTA(163), E: AGG/M: CTC(220) and E: ACA/M: CTA(160), were linked to the wtms1 gene relatively close to a genetic distance of 6.9 cM, 6.9 cM and 13.9 cM, respectively. Finally, the wtms1 gene was mapped between the SSR marker Xgwm 374 and the AFLP marker E: AAG/M: CTA(163) with the distance of 4.8 cM and 6.9 cM, respectively. A partial linkage map was constructed according the SSR and AFLP data.     

Leafy head2, which encodes a putative RNA-binding protein, regulates shoot development of rice

During vegetative development, higher plants continuously form new leaves in regular spatial and temporal patterns. Mutants with abnormal leaf developmental patterns not only provide a great insight into understanding the regulatory mechanism of plant architecture, but also enrich the ways to its modification by which crop yield could be improved. Here, we reported the characterization of the rice leafy-head2 （lhd2） mutant that exhibits shortened plastochron, dwarfism, reduced tiller number, and failure of phase transition from vegetative to reproductive growth. Anatomical and histological study revealed that the rapid emergence of leaves in lhd2 was resulted from the rapid initiation of leaf primordia whereas the reduced tiller number was a consequence of the suppression of the tiller bud outgrowth. The molecular and genetic analysis showed that LHD2 encodes a putative RNA binding protein with 67% similarity to maize TEl. Comparison of genome-scale expression profiles between wild-type and lhd2 plants suggested that LHD2 may regulate rice shoot development through KNOXand hormone-related genes. The similar phenotypes caused by LHD2 mutation and the conserved expression pattern of LHD2 indicated a conserved mechanism in controlling the temporal leaf initiation in grass.     

Integration of a barley (Hordeum vulgare) molecular linkage map with the position of genetic loci hosting 29 developmental mutants

The first step in positional gene cloning is the integration into available molecular maps of genetic loci for which mutant alleles exist. We report the placement of 29 barley developmental mutants on a restriction fragment length polymorphism-amplified fragment length polymorphism (RFLP-AFLP) map. The mapping procedure used homozygous mutant F(2) plants in an iterative process: once a mutant linked AFLP was found, primer combinations were successively selected to generate AFLP fragments more tightly linked to the mutant locus. The mutants considered were adp, als, aur-a1, aur-a2, br1, br2, bra-d7, cul3, cul5, cul15, cul16, den6, den8, dub1, hex-v3, hex-v4, int-c5, K, li, lig-a2, lk2, lk5, sld1, sld4, tr, trd, unc, uc2 and uz. The 29 mutant loci were linked to the closest molecular markers by distances ranging from 0 to 23 cM, with an average value of 3.8 cM away. Since the efficiency of the mapping procedure is a function of the density of molecular markers, the RFLP-AFLP map of Castiglioni et al was further integrated with new AFLPs using 87 doubled haploid lines derived from the barley cross Igri x Danilo. A total of 819 mapped AFLP marker loci are now available in the combined map.     

The integration of mutant loci affecting maize endosperm development in a dense genetic map using an AFLP-based procedure

In this paper, 10 mutations conditioning the appearance of defective, miniature or collapsed endosperm, but with normal sporophyte development, were considered. Homozygous mutant kernels have reduced grain weight, kernel size, density and, in some of these, higher than normal seed protein content. The mutant loci were integrated into a high-resolution genetic map in order to associate them to specific genes. We have placed 1167 AFLP markers on a consensus map using IBM2 as a backbone and reaching an average of 1 marker every 1.9 cM. We have identified AFLP markers linked to all individual mutant alleles. BSA was adopted to screen the largest possible number of primer combinations on homozygous F₃ mutant and wild type plants. The ten mutant loci are linked to the closest AFLP or SSR markers with distances ranging from 0 to 17.9 cM. The genes we have defined by the existence of mendelian mutants can now be considered good candidates for testing the association to QT loci. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pigment accumulation and functional activity of chloroplasts in common Pisum sativum L. mutants with low chlorophyll level (chlorotica)

Pea mutants chlorotica 2004 and 2014 with a low content of chlorophyll were studied. The mutant 2004 has light green leaves and stem, and the mutant 2014 has yellow green leaves and stem. They accumulate approximately 80 and 50% chlorophylls of the parent form of pea Torsdag cv. The content of carotene in carotenoids of the mutant 2004 was much lower, and the accumulation of lutein and violaxanthine was increased. The accumulation of all carotenoids in the mutant 2014 decreased almost proportionally to a decrease in the chlorophyll content. The rate of CO2 evolution in mutant chlorotica 2004 and 2014 was established to be lower. The quantum efficiency of photosynthesis in the mutants was 29-30% lower as compared to the control, and in hybrid plants it was 1.5-2-fold higher. It is assumed that the increase in the activity of the night-time respiration in gas exchange of chlorotica mutants and the drop of photosynthesis lead to a decrease in biomass increment. The results obtained allow us to conclude that the mutation of chlorotica 2004 and 2014 affects the genes controlling the formation and functioning of different components of the photosynthetic apparatus.     

拟南芥叶片数目变化突变体对营养生长时相转变的影响

拟南芥(Arabidopsis thaliana)的营养生长可以分为2个阶段: 幼龄期与成熟期。由幼龄期向成熟期的转变(VPC)与叶片的形态学特征、茎顶端分生组织(SAM)形状、远轴面表皮毛的出现以及SPL家族转录因子表达水平的变化相关。研究表明, 造成这种转变的信号来源于叶原基。该研究利用2种莲座叶数目改变了的突变体和对野生型切除叶片的方法, 分析了叶片数目对VPC的影响。结果表明, 莲座叶数目的减少推迟了VPC的发生; 而莲座叶数目增多突变体amp1-1并未使VPC的发生提前, 推测叶源信号的产生受到了光合作用的影响。     

The first genetic linkage map of Eucommia ulmoides

In accordance with pseudo-testcross strategy, the first genetic linkage map of Eucommia ulmoides Oliv. was constructed by an F₁ population of 122 plants using amplified fragment length polymorphism (AFLP) markers. A total of 22 AFLP primer combinations generated 363 polymorphic markers. We selected 289 markers segregating as 1:1 and used them for constructing the parent-specific linkage maps. Among the candidate markers, 127 markers were placed on the maternal map LF and 108 markers on the paternal map Q1. The maternal map LF spanned 1116.1 cM in 14 linkage groups with a mean map distance of 8.78 cM; the paternal map Q1 spanned 929.6 cM in 12 linkage groups with an average spacing of 8.61 cM. The estimated coverage of the genome through two methods was 78.5 and 73.9% for LF, and 76.8 and 71.2% for Q1, respectively. This map is the first linkage map of E. ulmoides and provides a basis for mapping quantitative-trait loci and breeding applications.     

Mapping of a Wheat Resistance Gene to Yellow Mosaic Disease by Amplified Fragment Length Polymorphism and Simple Sequence Repeat Markers

Wheat （Triticum aestivum L.） yellow mosaic virus （WYMV） is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted plants. In the present study, the amplified fragment length polymorphisms （AFLP） and simple sequence repeat （SSR） were used to identify the molecular linkages with the resistance gene against WYMV. Bulked segregant analysis was performed with an F2 population derived from the cross of cultivar Ningmai 9 （resistant） × cultivar Yangmai 10 （susceptible）. By screening among the resistant or susceptible parents, the F2 pools and the individuals in the F2 population with 64 combined selective AFLP primers （EcoRI/MseI） or 290 reported SSR primers, a polymorphic DNA segment （approximately 120 bp） was amplified using the primer pair E2/M5, and an SSR marker （approximately 180 bp） was located on wheat chromosome 2A using the primer Xgwm328. Analysis with MAPMAKER/Exp Version 3.0b （Whitehead institute for Biomedical Research, Cambridge, MA, USA） indicated that these two markers were dominantly associated with the resistance gene at distances of 5.4 cM or 17.6 cM, respectively. The resistance gene to WYMV derived from Ningmai 9, is temporarily named YmNM, and was mapped to wheat chromosome 2A.     

A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea

Many fungal pathogens invade plants by means of specialized infection structures called appressoria. In the rice (Oryza sativa) blast fungus Magnaporthe grisea, the pathogenicity mitogen-activated protein (MAP) kinase1 (PMK1) kinase is essential for appressorium formation and invasive growth. In this study, we functionally characterized the MST7 and MST11 genes of M. grisea that are homologous with the yeast MAP kinase kinase STE7 and MAP kinase kinase kinase STE11. Similar to the pmk1 mutant, the mst7 and mst11 deletion mutants were nonpathogenic and failed to form appressoria. When a dominant MST7 allele with S212D and T216E mutations was introduced into the mst7 or mst11 mutant, appressorium formation was restored in the resulting transformants. PMK1 phosphorylation also was detected in the vegetative hyphae and appressoria of transformants expressing the MST7(S212D T216E) allele. However, appressoria formed by these transformants failed to penetrate and infect rice leaves, indicating that constitutively active MST7 only partially rescued the defects of the mst7 and mst11 mutants. The intracellular cAMP level was reduced in transformants expressing the MST7(S212D T216E) allele. We also generated MST11 mutant alleles with the sterile alpha motif (SAM) and Ras-association (RA) domains deleted. Phenotype characterizations of the resulting transformants indicate that the SAM domain but not the RA domain is essential for the function of MST11. These data indicate that MST11, MST7, and PMK1 function as a MAP kinase cascade regulating infection-related morphogenesis in M. grisea. Although no direct interaction was detected between PMK1 and MST7 or MST11 in yeast two-hybrid assays, a homolog of yeast STE50 in M. grisea directly interacted with both MST7 and MST11 and may function as the adaptor protein for the MST11-MST7-PMK1 cascade.     

The rice FON1 gene controls vegetative and reproductive development by regulating shoot apical meristem size

Most plant organs develop from meristems. Rice FON1, which is an ortholog of Clv1, regulates stem cell proliferation and organ initiation. The point muta-tions, fon1-1 and fon1-2, disrupt meristem balance, resulting in alteration of floral organ numbers and the architecture of primary rachis branches. In this study, we identified two knockout alleles, fon1-3 and fon1-4, generated by T-DNA and Tos17 insertion, respectively. Unlike the previously isolated point mutants, the null mutants have alterations not only of the reproductive organs but also of vegetative tissues, producing fewer tillers and secondary rachis branches. The mutant plants are semi-dwarfs due to delayed leaf emergence, and leaf senescence is delayed. SEM analysis showed that the shoot apical meristems of fon1-3 mutants are enlarged. These results indicate that FON1 controls vegetative as well as reproductive development by regulating meristem size.     

Molecular mapping of a rice gene conditioning thermosensitive genic male sterility using AFLP, RFLP and SSR techniques

The discovery and application of the thermosensitive genic male sterility (TGMS) system has great potential for revolutionizing hybrid seed production technology in rice. Use of the TGMS system in two-line breeding is simple, inexpensive, efficient, and eliminates the limitations associated with the cytoplasmic-genetic male sterility (CMS) system. An F₂ population developed from a cross between a TGMS indica mutant, TGMS–VN1, and a fertile indica line, CH1, was used to identify molecular markers linked to the TGMS gene and to subsequently determine its chromosomal location on the linkage map of rice. Bulk segregant analysis was performed using the AFLP technique. From the survey of 200 AFLP primer combinations, four AFLP markers (E2/M5–600, E3/M16–400, E5/M12–600, and E5/M12–200) linked to the TGMS gene were identified. All the markers were linked to the gene in the coupling phase. All except E2/M5–200 were found to be low-copy sequences. However, the marker E5/M12–600 showed polymorphism in RFLP analysis and was closely linked to the TGMS gene at a distance of 3.3 cM. This marker was subsequently mapped on chromosome 2 using doubled-haploid mapping populations derived from the crosses IR64×Azucena and CT9993×IR62666, available at IRRI, Philippines, and Texas Tech University, respectively. Linkage of microsatellite marker RM27 with the TGMS gene further confirmed its location on chromosome 2. The closest marker, E5/M12–600, was sequenced so that a PCR marker can be developed for the marker-assisted transfer of this gene to different genetic backgrounds. The new TGMS gene is tentatively designated as tms4(t). Received: 13 July 1999 / Accepted: 27 July 1999     

Far1, a Negative Regulatory Locus Required for the Repression of the Nitrate Reductase Gene in Chlamydomonas Reinhardtii

In Chlamydomonas reinhardtii, the genes required for nitrate assimilation, including the gene encoding nitrate reductase (NIT1), are subject to repression by ammonia. To study the mechanism of ammonia repression, we employed two approaches to search for mutants with defective repression of NIT1 gene expression. (1) PF14, a gene required for flagellar function, was used as a reporter gene for expression from the NIT1 promoter. When introduced into a pf14 mutant host, the NIT1:PF14 chimeric construct produced a transformant (T10-10B) with a conditional swimming phenotype. Spontaneous mutants with defective ammonia repression of the NIT1 promoter were screened for by isolating cells that gained constitutive motility. (2) Insertional mutagenesis was performed, followed by screening for chlorate sensitivity in the presence of ammonia ion. One insertional mutant and six spontaneous mutants were allelic and defined a new gene, FAR1 (free from ammonia repression). FAR1 was mapped to Linkage Group I, 7.7 cM to the right of the centromere. The far1-1 mutant strain was used to clone DNA adjacent to the site of plasmid insertion, which was then used as a hybridization probe to clone the FAR1 gene from wild type.     

Heterochronic Effects of Teopod 2 on the Growth and Photosensitivity of the Maize Shoot

Teopod 2 (Tp2) is a semidominant mutation of maize that prolongs the expression of juvenile vegetative traits, increases the total number of leaves produced by the shoot, and transforms reproductive structures into vegetative ones. Here, we show that Tp2 prolongs the duration of vegetative growth without prolonging the overall duration of shoot growth. Mutant shoots produce leaves at the same rate as wild-type plants and continue to produce leaves after wild-type plants have initiated a tassel. Although Tp2/+ plants initiate a tassel later than their wild-type siblings, this mutant tassel ceases differentiation at the same time as, or shortly before, the primary meristem of a wild-type tassel completes its development. To investigate the relationship between the vegetative and reproductive development of the shoot, Tp2/+ and wild-type plants were exposed to floral inductive short day (SD) treatments at various stages of shoot growth. Tassel initiation in wild-type plants (which normally produced 18 to 19 leaves) was maximally sensitive to SD between plastochrons 15 and 16, whereas tassel branching was maximally sensitive to SD between plastochrons 15 and 18. Tassel initiation and tassel morphology in Tp2/+ plants (which normally produced 21 to 26 leaves) were both maximally sensitive to SD between plastochrons 15 and 18. Thus, the constitutive expression of a juvenile vegetative program in Tp2/+ plants does not significantly delay the reproductive maturation of the shoot.     

水稻生殖突变体的外观形态特征研究

通过对3种水稻生殖突变体材料的外观形态进行观察后发现,FM1突变体植株不能由营养生长转入生殖生长,一直保持营养生长状态;FM2突变体植株不能分化发育出小穗,只能在进入生殖生长后分化出穗枝梗;FM3突变体植株在生殖生长过程中不能分化出正常小穗,其疑花内部的雌蕊和雄蕊明显退化。已经证实这3种生殖突变性体植株都不能产生有性生殖后代,只能通过群体内的显性杂合体分离出隐性突变体植株。     

A Dominant Gene for Male Sterility in Salvia miltiorrhiza Bunge

A natural male sterile mutant of Salvia miltiorrhiza (Labiatae, Sh-B) was found during field survey in 2002. Our objective was to analyze its genetic mechanism for producing F1 hybrid seeds and to develop a molecular marker linked to male sterile gene for selection of a hybrid parent line. The segregation ratios of male sterile plants to fertile plants in the progenies of both testcross and backcross were 1:1 in continuous experiments conducted in 2006–2009. The male sterile Sh-B was heterozygous (Msms). The male sterile plants could capture most pollen (2 granule/cm²·24 h) with row ratio (female : male 2 : 1) within 45-cm distance and harvest the largest amount of 6495 g hybrid seeds per hectare. We also developed DNA markers linked to the male sterile gene in a segregating population using bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) techniques. The segregating population was subjected to BSA-AFLP with 128 primer combinations. One out of fourteen AFLP markers (E11/M4208) was identified as tightly linked to the dominant male sterile gene with a recombination frequency of 6.85% and at a distance of 6.89 cM. This marker could be converted to PCR-based assay for large-scale selection of fertile plants in MAS (marker-assisted selection) at the seedling stage. Blastn analysis indicated that the male sterile gene sequence showed higher identity with nucleotides in Arabidopsis chromosome 1–5, and was more likely to encode S-adenosylmethionine-dependent methyltransferase, in which DNA methylation regulated the development of plant gametogenesis.     

The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo.     

水稻生殖突变体的外观形态特征研究

通过对3种水稻生殖突变体材料的外观形态进行观察后发现,FM1突变体植株不能由营养生长转入生殖生长,一直保持营养生长状态;FM2突变体植株不能分化发育出小穗,只能在进入生殖生长后分化出穗枝梗;FM3突变体植株在生殖生长过程中不能分化出正常小穗,其颖花内的雌蕊和雄蕊明显退化。已经证实这3种生殖突变体植株都不能产生有性生殖后代,只能通过群体内的显性杂合体分离出隐性突变体植株。     

Hormone physiology of pea mutants prevented from flowering by mutations gi or veg1

The veg1 ( vegetative ) mutant in pea ( Pisum sativum L.) does not flower under any circumstances and gi ( gigas ) mutants remain vegetative under certain conditions. gi plants are deficient in production of floral stimulus, whereas veg1 plants lack a response to floral stimulus. During long days in particular, these non-flowering mutant plants eventually enter a stable compact phase characterised by a large reduction in internode length, small leaves and growth of lateral shoots from the upper-stem (aerial) nodes. The first-order laterals in turn produce second-order laterals and so on in a reiterative pattern. The apical bud is reduced in size but continues active growth. Endogenous hormone measurements and gibberellin application studies with gi-1 , gi-2 and veg1 plants indicate that a reduction in gibberellin and perhaps indole-3-acetic acid level may account, at least partially, for the compact aerial shoot phenotype. In the gi-1 mutant, the compact phenotype is rescued by transfer from a 24- to an 8-h photoperiod. We propose that in plants where flowering is prevented by a lack of floral stimulus or an inability to respond, the large reduction in photoperiod gene activity during long days may lead to a reduction in apical sink strength that is manifest in an altered hormone profile and weak apical dominance.