Analysis Of Differentially Expressed Genes Between Male Sterile Line And Its Maintainer Line In Double-recessive Genic Male Sterile Cotton (Gossypium Hirsutum L.) During Anther Development

Genie male sterility (GMS) is an inherited trait that prevents the production of functional pollen, but maintains female fertility. It has been widely used in breeding programs for F1 hybrid seed production in cotton (Gossypium hirsutum L.). The remarkable advantages of GMS are complete sterility, wide sources of recovery, and ease of obtaining high-vigor combinations. Several sterile lines, e.g., Mian A1 and Zhongkang A, have been cultivated based on ms5ms6 double-recessive sterile line by means of cross-breeding and back crossing. Furthermore, using ms5ms6 male sterile line as female parents, some hybrid species with heterosis have been developed. However, there are still some difficulties in applying the GMS for hybrid seeds breeding, such as developing new resources of homozygous two-type lines and restores, and little is known about the molecular mechanism of GMS in cotton. So for better utilization of the GMS, it is necessary to do some further investigation of it. In this research, ms5ms6 double-recessive GMS two-type line (ms5ms6A, sterile line; ms5ms6B, maintainer line) was used to identify the genes that may be associated with male gametogenesis and to understand the molecular mechanism of GMS in cotton. cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to identify differentially expressed genes between ms5ms6A. and ms5ms6B at different stages during anther development. The main results and conclutions of the research are following:1. Cytological inerstigation between the microspores of ms5ms6A and ms5ms6B demonstrated the relationship of bud length and pollen developmental stage. The buds at the three pollen developmental stages, i.e., sporogenous cell stage, pollen mother cell stage, and pollen grain stage, were <3.0 mm, 3.0-4.6 mm, and >4.6 mm in length, respectively.2. cDNA-AFLP was used to identify the differentially expressed genes between sterile and fertile plants of ms5ms6 double-recessive GMS two-type line at three different stages, sporogenous cell stage, pollen mother cell stage, and pollen grain stage. Totally, 48 differentially expressed TDFs were identified between sterile line and fertile line, and those TDFs belonged to 11 expressed patterns were isolated. Reverse northern blotting analysis further demonstrated 17 of the 48 TDFs were positive.3. The 17 positive TDFs were selected for cloning and sequencing. Using BLAST program, 14 of the 17 sequenced fragments could find homology sequences in GenBank. Functional analysis indicated that their corresponding genes may participate in the processes of signal transduction, transcription, energy metabolism, and plant cell wall development. Those other 3 no homology sequences may be some novle genes which involved in male gametogenesis.4. In this study, the excited discovery was that the sequence 0603 was significantly homologous to the T cytoplasm male sterility restorer factor 2(RF2) of maize, and it may encode the male sterility restorer factor of cotton. Furthermore, this sequence could be detected in fertile anthers but silenced in sterile anthers, and its silence may cause pollen abortion. The expression pattern of sequence 0603 observed by Northern blotting was similar to that of cDNA-AFLP, and Northern blotting further confirmed the credibility of the results of cDNA-AFLP.