Studies On Cytology, Endogenous And Molecular Markers Between Male And Female Plants Of Asparagus Officinalis L.

The dioecious species Asparagus officinalis L.(Liliaceae, Asparagus)is an important crop plant which is cultivated in many countries world wide. Compared to the female plants that bear seeds, male plants produce thicker, larger spears, have a higher yield capacity, are more tolerant to diseases and exhibit greater longevity. However, the males and female plant cannot be differentiated from even at the flowering stage. Moreove, A. officinalis is a impotant model species in researching of sex determination mechanism of dioecious. But, genome information and the genetic distance between the markers and the locus is not short enough for marker assisted selection, map-based cloning of the sex gene and understanding of sex determination mechanism.In this paper,the differences among female and male A. officinalis were studied from the analysis of cytology, endogenous polyamine, endogenous hormone, RAPD and AFLP markers of genomic DNA. This would to develop codominant sex-linked molecular markers in order to distinguish male, female plants at the early seedling stage. This would also benefit the cloning of sex-determining genes and the understanding of corresponding mechanism at the molecular level. The results were showed as follows:1. The cytology research of A. officinalis sex chromosome. The karyotype, anther mother cell meiosis, chromosome Geimsa-band and DAPI band were analysed using the general method. The result shows that karyotype formula is 2n=2x=20=10m+10sm. The male plant has a pair of heteromorphic sex chromosome XY and the female has XX. The karyotype belongs to"2B"type, which is original species. More chromatin were showed in X and Y chromosome. X and Y chromosome were showed more frequently in G-band and DAPI band, it is imply that the sex chromosome contain heterochromatin rich.2. Comparative studies on flower differentiation of endogenous product. The development process of male and female flower of A. officinalis were obvserved by compared the changes of endogenous hormones, polyamine, and amino acid content at four distinct developmental stage (vegetative, transition, meiosisi and mature flower, respectively). The results showed that endogenous product change was different at the development process of both male and female flower. Endogenous products were all involved in the differentiation and development of flower organ of Aspargus. Total content of amino acid in male plant was higher than female. But the changes of basic amino acid and neutral amino acid were not different between male and female four development stages.In male plant, carbonitride contents changes was not obviously during development process. The results showed that C/N reduce gradually with floral development in male plant, it is suggest that lower C/N was beneficial to male flower differentiation. But changes of C/N was not distinct in female plants .Except the IAA, the contents of GA3, ZRs and ABA showed significant different between male and female during the process of flower development in Aspargus. And this different appeared after monosexual floral differentiation. It is imply that endogenous hormones were not concerned with sex determination, though involved in sex differentiation.Changes of the endogenous polyamine contents during the floral sex differentiation of Aspargus were studied. The results showed that, in contrast to spermine, it was siginificant fluctuate of changes of putrescine and spermindine contents during monosexual floral differerntiation. Endogenous spermine content in female flower from begin to differentiation to before mature were higher remarkably than those in male flower. It is suggest that high level of endogenous spermine content was favorable to female floral development. The total content of polyamine raise gradually with flower development in male plant, but the highest level appear begin flower differentiation and depress hereafter in male plant.3. The random amplified polymorphic DNA(RAPD) technique was employed with the comparative study of the sex difference in A. officinalis. A total of 100 primers were tested with the same PCR cycling procedures. Female-associated fragment with a length of 928bp and 1178bp were amplified by primer S368. These fragments were isolated, cloned and sequenced. These fragments were AT rich in sequence and AT content was 62% and 64%, respectively. Both markers proved to be unique, since no sequence, either at nucleotide or deduced amino acid sequence, with significant homology to these markers. The marker S368928 were converted into sequence-characterized amplified region (SCAR) marker, which would be efficient to identify the different sex of A. officinalis Southern analysis indicated that hybridization signals have appeared in all male and female randomly selected individuals. Moreover, S368928 exists in the genome by high, middle and low copy, which suggest that it was a repetitive DNA sequence. FISH results showed that S368928 the signals were obvserved on some DAPI band on the X and Y chromosome and two pairs of autosomes. The X and Y chromosome and a pair of autosome have the weak signals near the centromere, the other pair of autosome have the strong signal on the terminal region of chromosome arm.3. The Amplified fragment length polymorphism (AFLP) technique was employed with the comparative study of the sex difference in A. officinalis. A total of 72 primer combinations were tested with the AFLP procedures. Male-linked fragment with a length of 555bp was amplified by primer combination E-AAG + M-CAT. The fragment was isolated, cloned and sequenced. The marker was AT rich in sequence and AT content was 59% and proved to be unique, since no sequence, either at nucleotide or deduced amino acid sequence, with significant homology to this marker. The marker was named MLDA555 (Male-linked DNA from Aspargus). The marker MLDA555 was converted into sequence-characterized amplified region (SCAR) marker. Corresponding to our expectations, the SCAR primer was able to amplify a single DNA band (523bp) and the SCAR marker was efficient in the identification of male individuals. Southern results showed that hybridization signals only have appeared all male plants, the female absence of signals. In male genome, MLDA555 exists by low copy repetitive DNA sequence. FISH analysis indicated that hybridization signals only have appeared all male plants, the female absence of signals. One weak signal was obvserved on the Y chromosome.