Recent advances in identification of male specificity determinant and its function in S-RNase-mediated gametophytic self-incompatibility

S-RNase-mediated gametophytic self-incompatibility （GSI） is controlled by a multiallelic S-locus at which two separate genes, the female （pistil） and male （pollen） specificity determinants, are tightly linked. This review described both the identification of pollen specific F-box genes, SLF/SFBs, in Antirrhinum, Petunia and Prunus species and the demonstration of SLF/SFB as pollen determinant together with their functions in GSI response. Recent studies of how the pollen determinant functions in pollination reaction revealed that pollen determinant interacted with S-RNases in a non-allele-specific manner. It targeted all of the non-self S-RNases for ubiquitination through a functional SCF complex and subsequent degradation via 26S proteasome pathway in compatible reaction. It allows pollen tube to reach into the embryo sac and to finish double fertilization. In incompatible response, the intact self S-RNases were left to function as a cytotoxin that degrades self-pollen tube RNA, resulting in the cessation of pollen tube growth.

GAMETOPHYTIC FACTOR 1,Involved in Pre-mRNA Splicing,Is Essential for Megagametogenesis and Embryogenesis in Arabidopsis

RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of function of GAMETOPHYTIC FACTOR 1 （GFA1）; a gene likely involved in RNA biogenesis in Arabidopsis. The mutant is featured in the phenotype of diminished female gametophyte development at stage FG5 and is associated with the arrest of early embryo development in Arabidopsis. Bioinformatics data showed that homologs of gene GFA1 in yeast and human encode putative U5 snRNPspecific proteins required for pre-mRNA splicing. Furthermore, the result of yeast two-hybrid assay indicated that GFA1 physically interacted with AtBrr2 and AtPrp8, the putative U5 snRNP components, of Arabidopsis. This investigation suggests that GFA1 is involved in mRNA biogenesis through interaction with AtBrr2 and AtPrp8 and functions in megagametogenesis and embryogenesis in plant.

Evolutionary Genomics of Plant Gametophytic Selection

It has long been recognized that natural selection during the haploid gametophytic phase of the plant life cycle may have widespread importance for rates of evolution and the maintenance of genetic variation.Recent theoretical advances have further highlighted the significance of gametophytic selection for diverse evolutionary processes.Genomic approaches offer exciting opportunities to address key questions about the extent and effects of gametophytic selection on plant evolution and adaptation.Here,we review the progress and prospects for integrating functional and evolutionary genomics to test theoretical predictions,and to examine the importance of gametophytic selection on genetic diversity and rates of evolution.There is growing evidence that selection during the gametophyte phase of the plant life cycle has important effects on both gene and genome evolution and is likely to have important pleiotropic effects on the sporophyte.We discuss the opportunities to integrate comparative population genomics,genome-wide association studies,and experimental approaches to further distinguish how differential selection in the two phases of the plant life cycle contributes to genetic diversity and adaptive evolution.

Pollen Mother Cell Miosis and Male Gametophyte Development of Pumpkin

[Objective] Pollen mother cell miosis and male gametophyte development of pumpkin were observed in this study, to provide some cytological basis for pumpkin anther or microspore culture. [Method] Ehrlich＇s hematoxylin staining-methyl salicylate clearing technique was used for observation and research of the variation of cell structure and chromosomal behavior during pollen mother cell miosis and male gametophyte development of ‘Tianhong＇ pumpkin. [Result] The meiosis in pollen moth- er cells of pumpkin was simultaneous cytokinesis. In the process of nuclear division, nuclear membrane and nucleolus of pumpkin pollen mother cells gradually disappeared in the metaphase I and reappeared in telophase I , phragmoplast formed between the two generated crescent-shaped nuclei without cell wall, the phragmoplast gradually disappeared in the metaphase II and reappeared in telophase II. Phragmoplast spread outward from the center of spindle during the second division was connected with that formed on the central interface of two nuclei during the first division, cell wall of microspores generated from periphery to center. Most of the tetrads contained four sub-cells while a few contained extra small cells. During the period of uniuclete microspore at periphery, the single nucleolus split into 2-3 or more small nucleoli, mature pollen grain was two-celled. Mononucleate pollen cells were mostly appeared in the flower buds with length of 1.0-2.0 cm, which could be used as an important indicator to collect materials for anther or microspore culture. [Conclusion] This study laid the foundation for research of the cytogenetics of pumpkin.

Assessing the Germplasm of Laminaria (Phaeophyceae) with Random Amplified PoLymorphic DNA (RAPD) Method

Eighteen gametophytes includingL. japonica, L. ochotensisandL. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains ofLaminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species ofLaminaria.

Cryopreservation of Gametophytes of Laminaria japonica (Phaeophyta) Using Encapsulation-Dehydration with Two-Step Cooling Method

Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen using encapsulation-dehydration with two-step cooling method. Gametophytes cultured at 10℃ and under continuous irradiance of 30 μmol m^-2 s^-1 for 3 weeks were encapsulated in calcium alginate beads. The beads were dehydrated in 0.4 molLl sucrose prepared with seawater for 6 h, desiccated in an incubator set at 10℃ and 70% relative humidity for 4 h, pre-frozen at either -40℃ or -60℃ for 30 min, and stored in liquid nitrogen for 〉24 h. As high as 43% of survival rate was observed when gametophytes were thawed by placing the beads in 40℃ seawater and re-hydrated in 0.05 molL^-1 citrate sodium prepared using 30‰ NaCl 7 d later. More cells of male gametophytes survived the whole procedure in comparison with female gametophytes. The cells of gametophytes surviving the preservation were able to grow asexually and produce morphologically normal sporophytes.

INTRASPECIFIC CROSSINGS OF UNDARIA PINNATIFIDA (HARV.) SUR.-A POSSIBLE TIME-SAVING WAY OF STRAIN SELECTION

Unicellular gametophytes of Undaria pinnatifida (Harv.) Sur. were isolated in Qingdao, P. R. China in April 1993 and in Tokushima, southem Japan in March 1995. Diferent intraspecific crossings by using unicellular male and female gametophytes were successfully undertaken in Sept. of 1995 in Qingdao.Sporophytes were transplanted to two different locations for open an cultivation. One was at Zhanqiao (ZQ) Bay where the water current was slower than that of another location - Taipingjiao(TPJ). A total of218 adult sporophytes were harvested on January 12 and 18 from TPJ and ZQ repectively. For eacncombination, 10 sporophytes un cultivated. Analysis of the mprpholgical characteristics of adult spprophytes indicated that the longest length between two bases of the serration of pinnate bldes (W2) is a morphological characteristic that can be transferred from the parent plant to the next generations regardless of environmental variations. There was evidence that W2 was apparently determined by sex-linked factors, i.e, by male parental grametophyte.. Sporophytes from certain crossing combinations showed more vigorous growth than those from other crossing combinations. It is therrfore possible to select grametophyte strains which can be used as parental gametophytes for the seeding production of sporophytes with more vigorous growth within shorter cultivation period. The morphology of hybrids from a Qingdao strain and a Tokushima cultivated strain resembled that of both parental plants in frond feaures (wrinkled or smooth) and W2. Sporophyll formation also varied with strains. The fact that adult sporophytes resulting from the same crossing combinations have identical morphological characteristics under the same environmental conditions indicates the possibility of a new way to select strains which are expected to be ideal for commerical production by purposely selecting, propagating, and seeding unicellular gametophytes for sporeing production through freeliving techniques of gametophytes.

Prediction of Suitable Habitat for Lycophytes and Ferns in Northeast China: A Case Study on Athyrium brevifrons

Suitable habitat is vital for the survival and restoration of a species.Understanding the suitable habitat range for lycophytes and ferns is prerequisite for effective species resource conservation and recovery efforts.In this study, we took Athyrium brevifrons as an example, predicted its suitable habitat using a Maxent model with 67 occurrence data and nine environmental variables in Northeast China.The area under the curve(AUC) value of independent test data, as well as the comparison with specimen county areal distribution of A.brevifrons exhibited excellent predictive performance.The type of environmental variables showed that precipitation contributed the most to the distribution prediction, followed by temperature and topography.Percentage contribution and permutation importance both indicated that precipitation of driest quarter(Bio17) was the key factor in determining the natural distribution of A.brevifrons, the reason could be proved by the fern gametophyte biology.The analysis of high habitat suitability areas also showed the habitat preference of A.brevifrons: comparatively more precipitation and less fluctuation in the driest quarter.Changbai Mountains, covering almost all the high and medium habitat suitability areas, provide the best ecological conditions for the survival of A.brevifrons, and should be considered as priority areas for protection and restoration of the wild resource.The potential habitat suitability distribution map could provide a reference for the sustainable development and utilisation of A.brevifrons resource, and Maxent modelling could be valuable for conservation management planning for lycophytes and ferns in Northeast China.

Parentage Analysis of Dongfang No.2, a Hybrid of a Female Gametophyte Clone of Laminaria japonica (Laminariales, Phaeophyta) and a Male Clone of L. longissima

The cultivation of the first filial generation of monoploid gametophyte clones of different Laminaria species (hybrid Laminaria) is an effective way of utilizing heterozygous vigor (heterosis). A female gametophyte clone of L. japonica and a male gametophyte clone of L. longissima were hybridized, generating Dongfang No.2 hybrid Laminaria. The parentage of this hybrid Laminaria was determined using AFLP of total DNA, SNP of the ITS region of ribosomal RNA transcription unit and microsatellite DNA variation at two loci. In addition to 167 AFLP bands shared by Dongfang No.2, L. japonica and L. longissima, Dongfang No.2 hybrid Laminaria shared another 70 and 55 bands with L. japonica and L. longissima, respectively, which were obviously more than 11 bands shared by L. japonica and L. longissima. Dongfang No.2 held both ‘T’ and ‘C’ at position 847 of the ITS region, while ‘T’ at this position was specific for L. japonica and ‘C’ for L. longissima, respectively. Dongfang No.2 also held the microsatellite DNA alleles of two parents together at two microsatellite DNA marker loci. These observations clearly proved that Dongfang No.2 is a true hybrid of L. japonica and L. longissiuma. Unfortunately, the origin of the chloroplast of Dongfang No.2 was not determined based on the variation of RuBisCo spacer. More sequence variants of both ITS region and RuBisCo spacer were identified in Dongfang No.2 and most of them did not exist in either L. japonica or L. longissima. The unexpected variants may be due to the mutation of ga- metophyte clones occurring during their vegetative amplification.

A bacterial pathogen infecting gametophytes of Saccharina japonica(Laminariales,Phaeophyceae)

A newly identified bacterial disease of kelp(Saccharina japonica) gametophytes was found in clone cultures.It is characterized by swollen gametophyte cells in the early period of infection followed by filamentous fading.An alginolytic marine bacterium referred to as A-1 was isolated from the diseased gametophytes.On the basis of 16S rDNA sequencing and morphological,physiological and biochemical characteristics,the bacterium was identified as a strain of the genus Alteromonas.By testing Koch's postulates,Alteromonas sp.A-1 was further confirmed as the pathogen.The infection process was also investigated using both scanning electron and light microscopy.

Early development of Costaria costata(C.Agardh) Saunders and cultivation trials

Costaria costata(C.Agardh) Saunders is one of common kelps distributed in many coastal areas worldwide;however,in China,no reports have been made on cultivation of the genus.To investigate potential cultivation of the species in the northern part of China,trials on isolation and preservation of the gametophytes were conducted using C.costata from Korea;growth and development of the gametophytes were observed.We showed that at 10±1°C,60 μmol m-2s-1 and 12:12 h(L:D),freshly released zoospores settled down within 1 hour,and then developed into the primary cell during the following 2 days.After a vegetative growth phase lasting 6-8 days,female gametophytes became 3-4 times larger in diameter than that of the primary cell,but still remained at a unicellular stage,while male gametophytes divided into 4-10 cells with only a slight change in size.Fertilization occurred within 10 days after the zoospores were released from the sporangia,and the apical and basal tissues of the juvenile sporophyte divided and differentiated into the blade and stipe.Temperature and irradiance influenced gametophytic vegetative growth and developmental patterns.Generally,low irradiance(15 μmol m-2s-1 and 30 μmol m-2s-1) was unfavorable to the induction of fertility,but it enhanced female gametophyte division.The optimal conditions for vegetative growth were 15°C and 30 μmol m-2s-1.After transplantation of the juvenile seedlings and after eight months cultivation,the harvested mature blade reached 194 cm in length and 32.7 cm in width.Our study proves that it is feasible to implement propagation and large scale cultivation of C.costata in northern China.

Effects of blue light on gametophyte development of Laminaria japonica (Laminariales, Phaeophyta)

Laminaria gametophyte was greatly influenced by light in its growth and development. Using light-emitting diodes (LED) as blue and red light sources, we analyzed the light effect on gametophytes devel- opment of Laminaria japonica Aresch. The gametophytes were obtained from zoospores collected in April, May, July, 2003 and September, 2004. We found that the growth of gametophytes was stimulated by increasing inten- sity of blue light (BL) and red light (RL) illumination, of which BL was obviously stronger than that of RL. The fertilization of gametophytes depended largely on BL, and only sufficient BL illumination could take the repro- ductive effect. In addition, we noticed that there was a significant difference in light responses for gametophytes developed from zoospore collected in different times. For zoospores released in April, under BL1 (73.90 μmol photons/m·s), the unicellular female gametophytes and multi-cellular male gametophytes produced eggs and sperms respectively, and further developed towards sporophytes. However, for gametophytes developed in May, July or September, they became multi-cellular and never formed oogonia or antheridia. It is believed that the Laminaria sporangium maturation stage could affect the gametophytes reaction to BL under laboratory culture conditions. Therefore, cryptochrome- or phototropin-like BL photoreceptors is probably involved in BL-induced development of Laminaria gametophytes.

EFFECTS OF ULTRASONIC TREATMENT ON FEMALE GAMETOPHYTES OF LAMINARIA JAPONICA (PHAEOPHYTA)

This study on the effects of ultrasonic treatment on female gametophytes of Laminariajaponica showed that:1. Ultrasonic treatment had shortening effect on filaments of female gametophytes. Within certainperiod of time, the average length of filamentous female gametophytes was shortened.2. Ultrasonic treatment had emptying effect on cells. The number of empty cells increased with timeof treatment. Ultrasonic treatment had harmful effect on cells.3. Ultrasonic treatment could break down cell walls. The combination of frequency of 20 kHz, out-put of 15 W, 40 s and 60 s of treatment was best for this purpose. After ultrasonic treatment, the regner-ation of female gametophytes into sporophytes was effected. Female gametophytes could not recover aftertoo long period of treatment.

Zoospore-derived monoecious gametophytes in Undaria pinnatifida(Phaeophyceae)

The annual life cycle of the brown seaweed Undariapinnatifida （Harvey） Suringer comprises a macroscopic diploid sporophyte stage and a microscopic haploid gametophyte stage. In 2011, an unusual zoospore-derived monoecious gametophyte isolate （designated as line 10-5-3） of U. pinnatifida was observed. To understand this phenomenon, a comprehensive screening of eighty-two previously identified male gametophyte cultures, isolated from three randomly selected cultivars （lines 10, 7, and 5） was performed. Thirty-six of the isolates developed both antheridia and oogonia on the same filamentous fragment in a standard gametogenesis test （SGT： 18℃, 60 umol photons/（m2.s））. Selfing of the monoecious gametophyte or crossing it with a normal male gametophyte both gave rise to morphologically normal sporophytic offspring. However, crossing resulted in a much higher fertilization rate （89.7%）. The hybrid and selfed sporophytic offspring were grown to maturity in flow tanks at an ambient temperature of 10-18℃ over a period of 69 days. Active zoospores were released from both types of mature sporophylls. The majority of these developed into male gametophytes, while 15%-20% developed into the observed monoecious structures on the same filament. Using PCR amplification it was found that all the monoecious gametophyte isolates and the sporophytic offspring resulting from the selfing and crossing lacked the femalelinked microsatellite sequence （a part of the locus Up-AC-2A8, GenBank accession No. AY738602.1）, indicating their male nature. U.pinnatifida is an invasive species in some regions and the implications of the above findings for this species in nature are briefly discussed.

Study on the breeding technology of heterosis seedling of Laminaria and new combinations

After continuous breeding gametophyte of Laminaria, then breaking protonema, heterosis seedling is got by bilineal hybridization. The result shows that the weight-increasing rate of female and male gametophyte clone cells are 14.03% and 13.87%, after 16 d crossbreeding, the rate of ovulation is up to100%; the length of juvenile sporophyte after 40 d breeding is 2-3 cm. By farming on the sea, a hybrid combination is primarily screened.

Identification of a Putative Tetrasporophyte-Specific Gene in Gracilaria lemaneiformis(Gracilariales,Rhodophyte)

A putative tetrasporophyte-specific gene,designated as SSH466(GenBank accession No. DQ019223) ,was one of the genes identified in this work using suppression subtractive hybridization(SSH) method in Gracilaria lemaneiformis. The full length of the gene was obtained using SMART RACE strategy. Sequence analysis revealed that the gene had 1 019 nucleotides,including an open reading frame of 498 nucleotides encoding 166 amino acid residues,158 nucleotides of 5' untranslated region and 363 nucleotides of 3' non-coding region. Protein motif and secondary structure prediction showed that there existed a transmembrane domain with a unique β-sheet. Thus,SSH466 protein might be a cross-membrane protein. Sequence homology search in the public GenBank databases did not reveal any significant match with SSH466. Virtual Northern blot analysis confirmed that it was a tetrasporophyte-specific gene.

Laminaria gametophyte clone culture and its application in sporeling cultivation

In China, the total annual production of cultured Laminaria japonica reached half million tons in dry weight there few years. The routine sporeling culture technique conducted in the greenhouse took at least three and half months. In such a case, sometimes the sporelings died within a few days due to destructive diseases. In Order to overcome the mentioned problems, a new sporeling culture technique, the clone technique, is developed. The method includes three stead: (1) Gametophyte clone culture. The spores and the gametophytes are cloned in flasks under favorable environments. (2) Sporeling cul ture. Male and female clones are crushed and spread onto a frame to allow the gametophytes to attach to the substrata. The frames are cultured in tanks, and the sporophytes reach 1 cm in length within one and a half months. (3) Outgrowing of the plant. The frames are put in the open sea when seawater temperature decreased to 20℃. After one month, the sporelings are large enough to be transplanted. It is concluded that the clone technique has the following advantages: (1)Large amount of clones can be produced in a short period of time. (2) Clone seeding method makes it free from the biological rhythm, one can seed the plant anytime all the year round. (3) It takes only one and a half months to complete the process of sporeling cultivation in the greenhouse. At present, this technique is used in the breeding of new strains of Laminaria.

Morphologic characters and element content during development of Pinus tabuliformis seeds

An embryo classification system for Pinus tabuliformis Carr. was established by time-tracing sam- pling and observation of zygotic embryos. The zygotic embryos were divided into nine stages. Key elements of the zygotic embryo and female gametophyte （FG） tissue of P. tabuliformis were analyzed, using inductively coupled plasma-emission spectroscopy. Several elements--includ- ing aluminum, iron, sodium, and copper--are found in both embryo and FG tissue. Boron, phosphorus, magnesium,zinc, and calcium are also required for zygotic embryo development and therefore accumulated. Manganese is selectively excluded from the embryo. The zygotic embryo development needs a low-sodium and high-potassium nutrition proportion. The results of elemental analysis from zygotic embryos and FGs can provide the mineral targets for optimizing the formulation of culture medium for somatic embryogenesis.

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.

Florogenesis and Female Gametophyte Development in <i>Allium cepa</i>L. cv. Krishnapuram

Florogenesis is one of the most complicated and interesting processes in the nature. This process involves developmental, physiological and molecular events leading to transformation from vegetative to reproductive phase for optimal seed production and the continuation of species. The basic knowledge about flowering processes, male and female sexual systems support basic and applied research and breeding programs. Most of the onion varieties from India are short day varieties, more diverse than other exotic germplasm and useful as a source of new alleles for supporting breeding programs. The present investigation was focused to study for the first time florogenesis process by scanning electron study for the first time florogenesis process by scanning electron microscopy (SEM) and development of female gametophytes by light microscopy in order to acquire basic knowledge useful for optimizing in vitro process to produce gynogenic haploid to support and speed breeding program in short-day onion Allium cepa L. cv. Krishnapuram (KP) or Bangalore Rose. This study revealed that shoot primordium differentiated into inflorescence meristem in the month of December, while seeds were planted in the field in the September. The individual florets are preceded by a variying number of floral initials. The female gametophyte developed from chalazal side megaspore. The embryo sac development is a bisporic Allium type showing short-lived antipodals. The histological study suggests that the use of big or preanthesis flower buds with embryo sac for production of gynogenic haploids to support breeding program in onion cv. Krishnapuram (KP). However further studies are needed for confirmation of this observation.