Genetics Research Of Polyploid Hybrid Synthesized By Maize And Related Species

Hybridization and polyploidy is an important driving force for formation and evolution of higher plants. Polyploidy can lead to nuclear genome fusion of plant, often bring it new character variation, and make it suit the selection pressure and development needs. Polyploid are widely distributed in nature, while long time of evolution and fuzzy genetic relationship limit it further research and application. The newly synthetic polyploid is attracted much attention by clarity parents and short evolution. The maize wild relatives of teosinte and tripsacum are important genetic material. Through distant hybridization and polyploidization can create new germplasm resources, which will give an important theoretical and practical significance in the genetic improvement and the synthetic polyploid of maize. In this study, on the basis of distant hybridization and somatic doubling, a series of polyploid hybrids which contained maize, teosinte and tripsacum were synthesized. Using anatomy, cytogenetics and molecular biology techniques to analyse the changes of phenotypc, chromosomes, genomes, sequences and progeny chromosomal composition in newly synthetic polyploid. The results are as follows:1 Using maize (MM20,2n=20M), tetraploid perennial teosinte (9475,2n=40P) and tetraploid tripsacum (TZ07,2n=72T) as parental material, a series of polyploids were synthesized. Namely, the autotetraploid (MM40,2n=40M) was generated by MM20 homologous doubling; MM30 (2n=30M) was produced by crossing between MM20 and MM40; allotriploid MP30(2n=10M+20P) and PM30(2n=10M+20P) was produced by MM20 and 9475; the allotetraploid MP40 (2n=10M+20P) and PM40 (2n=10M+20P) was produced by crossing between MM40 and 9475; Heterologous hexaploid of MTP74 (MMTTPP) was produced by crossing among maize,9475 and tripsacum, chromosome composition was 2n=20m+20p+34T; MTP94 (MMTTPPPP) was produced by crossing between MTP74 and 9475, chromosome composition was 2n=20m+40p+34T. The analysis showed that the chromosome number of MM30, MM40 and allotetraploid were varying in different individuals. Allopolyploid (MTP74 and MTP94) were obtained by way of 2n+n.The two allopolyploid had vigorous vegetative growth, perennial, and could be propagated by asexual reproduction, and their chromosome number did not change with the cultivation years increasing.2 By anatomical morphology and microscopic measurement techniques, the stoma, pollen, nucleus and DNA content of newly synthetic polyploid were analyzed. Stoma had "polyploidization" effect; the size of stoma was increased by ploidy, and also influenced by distant hybridization and different composition of cytoplasm. The size of pollen and nucleus were not correlation with distant hybridization, different ploids and different cytoplasm. DNA content increased with the ploids, but the loss of DNA content mainly correlated by distant hybridization and the influence of different cytoplasm. Correlation analysis among stoma, pollen, the nucleus, DNA content and chromosome number showed that the size of the stoma was significantly positively related to chromosome number of parents and the nucleus was significantly positively related to chromosome number of tripsacum. Pollen character was not correlation with the DNA content, but some characters were significantly positively related with maize chromosome, some characters had negatively correlated with tripsacum chromosome number.3 In order to better understand the three parents chromosome structure and polyploid mitosis and meiosis chromosome behavior, using genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) to comparative analysis the three-parental chromosome. The situ hybridization results showed that the homology of maize and 9475 chromosome was high, genomic hybridization with each other produced stronger signal; the homology of maize and tripsacum only exsited in heterochromatin region (knob); the homology of 9475 and tripsacum also exsited in heterochromatin region (knob), but only tripsacum signals can be hybridized to the chromosomes of 9475. Comprehensive analysis showed that tripsacum chromosome and the other two parental chromosomes only existed the homology in Knob area, and the homology of maize and tripsacum in knob area was highest.4 Using two-color GISH to analyse the mitosis chromosome behavior and distribution of MP40 and MTP74. The results showed that both allotetraploids had the stability mitotic and had no abnormal chromosomes, independent regional of three-parent chromosomes did not occurr in mitotic, and three-parent chromosomes mutual blended together.5 Using fuchsin staining and two-color GISH, the meiosis behavior and stability of newly synthesized polyploids were studied. The results showed that A and B sub-genome of maize and 9475 existed homoeologous chromosomes; B1 and B2 sub-genome of 9475 also existed homology; The chromosomes composition of tripsacum might be the same as the 9475 and the base of chromosome might be 9. The valents of polyploid analysis showed that the valent which contained maize and 9475 chromosomes was highest, and 10 chromosomes of A sub-genome in maize has homology with 20 chromosome of A sub-genome in 9475; Because of the knob existed in maize,9475 and tripsacum, there was small amount of valents which contained tripsacum and the other parents in MTP74 and MTP94.Analysis the correlation between polyploid meiosis chromosome disorder and fertility showed that there was no correlation between of them. Analysis the influencing factors on the meiosis of polyploids showed that polyploid meiosis stability was affected by ploidy, different cytoplasm and genome composition. Comparing the stability of autopolyploid and allopolyploid showed that the tability of autopolyploid was lower than that of allopolyploid. Comparing the stability of different parents in polyploid showed that, the stability of maize genome was lower than that of 9475 in all polyploids, due to the distant relationship to maize and 9475, the stability of tripsacum chromosomes was highest in MTP74 and MTP946 With newly synthesized polyploids and their original parents,7 materials were used as this research object. Using the amplification fragment length polymorphism (AFLP) technology to study the genome changes of polyploids. Screening 16 pairs of AFLP primers for amplification and 635 clear bands were obtained. The three parents had both specific bands and common bands. The specific bands of tripsacum were the most (102); 9475 at least (32). The common bands of maize and 9475 were 164, the common bands of maize and tripsacum were 64, the common bands tripsacum and 9475 were 32, the common bands of three parents were 154. Further clustering analysis of the three parents showed that maize was the highest homology with the 9475 in the genome primary structure and tripsacum was the highest homology with the 9475.Analysis the AFLP bands of the polyploid showed that polyploid inherited mainly parental bands, but all polyploid had some lost bands. There was no relationship between the loss of bands and the increase of ploidy, but loss of bands was related to the homology of the parents in the genome. Classifying the loss bands as special bands and common bands, the polyploid had a tendency to lost special bands. Classifying the loss bands as parental types, the polyploid had a tendency to lost maize type bands.7 Using monoclonal sequencing technology and FISH to analyse the sequences evolution of maize polyploids. Centc,45S and 5S were used as the probe to study the distribution characteristics of metaphase chromosome hybridization signals. The results showed that the centc signals were loss accompanied by ploid and exogenous parent increasing, on the contrary, the signal of 45S and 5S were not significantly change.ITS which existed in 45S sequence was chosen to further research. The ITS sequence variation analysis showed that the length and GC content of ITS sequenc which belonged to parent respectively changed little, ITS sequence variation was mainly reflected by point mutation, and mutation sites existed in all three parents, and affected by distant hybridization, ploid and cytoplasm. The variation of ITS sequence in polyploid had the parental tendency, of which ITS sequenc belonged to 9475 had the most mutation sites. Analysis the information sites of ITS sequence showed that the replacement of information sites were affected by different genome composition, ploids, cytoplasm as well as distant hybridization. Although the tripsacum ITS sequences had a small fraction in MTP74 and MTP94 group, it was still involved in the concerted evolution.8 Using 9475 to backcross the MTP74 and MTP94 respectively, two different groups were obtained, and the chromosome number and chromosome composition of two groups were analyzed by GISH. The results showed that, the MTP74 backcross population had 21 individuals, the range of chromosome number was 37-82, and three different reproductive strategies which contained haploid parthenogenesis (n), normal reproduction (n+n) and apomixes (2n+n) were in this group. The chromosome range of maize,9475 and tripsacum of n+n group was 7-13,26-33 and 4-20 with the average of 10.32,29.42 and 16.32 respectively. The MTP94 backcross population had 17 individuals, the range of chromosome number was 47-87, and three different reproductive strategies which contained haploid parthenogenesis (n), normal reproduction (n+n) and double fertilization reproductive (n+n+n) were in this group. The chromosome range of maize,9475 and tripsacum of n+n group was 8-16,30-41 and 14-18 with the average of 11.11,37.94 and 16.24 respectively.The chromosome numbers variation of two groups were analyzed, the results showed that the parental chromosome composition variation degree of the MTP94 group was higher than that of MTP74 group except tripsacum chromosome composition variation. Chromosome structure variation analysis showed that two groups existed chromosome translocation phenomenon, and the translocation frequency of MTP74 group and MTP94 group were 2.63% and 1.72% respectively, in additional, the translocation occurred mainly between the maize and 9475 chromosome. Correlation analysis between polyploids meiosis and chromosome variation showed that the polyploid meiosis stability and chromosome composition variation had significant positive correlation and allosynapsis of maize and 9475 had significant positive correlation with chromosome translocation of maize and 9475.