Studies On Genetic Mechanism Of Ramie Male-sterility And Its Application

Ramie (Boehmeria. nivea), is an important fiber crops. As our national treasure, it is also called "China grass". Breeding scientists pay a lot of attention to Ramie male sterility since it has been found. However, there are not only few documents and little applications, but also little known of genetic mechanism on Ramie male sterility. To accelerate Ramie breeding process, it is very necessary for us to study Ramie male sterility roundly. In this dissertation, many studies on male-sterility in Ramie were applied, such as genetic analysis, the characteristics in physiology and biochemistry and cytology investigation, NAD7 gene clone and analysis, molecular markers construction and heterosis utilizing. The main results showed as follows:1. Ramie male sterility phenotype identificationCompared with normally plants, the male sterility plants mainly display on the male flower bud: the male flower bud is thin, small, un-crack, the anther is withered, the contents are very few and without pollen; non-color change with I₂-KI dyes. The female flower opens when the male flower bud withered and gradually falls off. The female flower growth is normal.2. Ramie male sterility genetic analysisGenetic analysis of generations of 6 crossed combinations showed that the male sterility character of the male sterility line C26 were controlled by one pair of recessive nuclear genes and cytoplasm gene. So C26, gene type T (rr) ,belong to CMS Fertility. While the gene type of the restoring line B8 was F (Rr).3. Ramie male sterility material metabolismIn the same developmental stage, the male-sterile line soluble sugar content is higher than its fertility restoring line. The distinct difference was observed in the big flower bud stage: male-sterile line C26 and C4 respective is its fertility restoring line 1.91 and 2.59 times. Along with the flower bud growth, the male-sterile line male flower bud starch content don't changes obviously, but the fertility restoring line accumulates starch steadily: in flower bud stage, fertility restoring line B16 starch content high 72.8% compared to male-sterile line C4, the big flower bud stage, fertility restoring line B8 achieve 79.6% compared to male-sterile line C26. Along with flower bud growth, regardless of the male-sterile line or the fertility restoring line, the soluble protein descends gradually. But with respect to the contents, it displays not consistently between materials: male-sterile line C26 is lower than its restoring line B8, but male-sterile line C4 is higher than its restoring line B16. The free proline content is consistent in the starch content change. In the male-sterile plants and common plants of F1, all material displays the same as its parents, except in the protein content: fertile plant is higher than sterile.4. Ramie male sterility isozyme analysisThe male sterility characteristic bands have not been observed in the POD electrophoresis. The only difference is the band intensity: the restoring line stronger than the male-sterile line. Therein to Rf 0.59 band in restoring line is stronger than that of male-sterile line in all stages. With respect to SOD, the isozyme bands have not been examined in various times of the male flower bud growth but only in the functional leaves. The mainly difference between male-sterile line and restoring line exist in the band intensity, the same as POD. The POD, SOD activities mensurating results showed that the restoring line stronger than the male-sterile line (fertile plants stronger than sterile plants in F1).5. Ramie male sterility paraffin sectionThe paraffin section results indicated that Ramie male sterility occurs in each stage from the spore cell forms to the tetrad, in which male-sterile line C4 main abortion thoroughly in growth early time because of having no spore original cell differentiation, while the male-sterile line C26 main abortion time in meiosis process. The male-sterile line C26 abortion form mainly has: the microspore mother cell cannot develop into meiosis normally, because of the extrusion from the huge tapetum. When the tapetum disintegrates gradually later, the microspore mother cell has still not entered meiosis until disintegrated. Moreover, the phenomenon that canaliculus bundle degenerate suddenly has been found in the experiment.6. Cloning and analysis of NADH dehydrogenase subunit 7 gene fragmentA cDNA fragment of Ramie NAD7 gene was obtained by RT-PCR with special primers, which were designed according to the conserved sequences of NAD7 gene. The cDNA fragment had the sequence length of 969 bp, encoding 322 amino acids. Analysis of sequence alignment and phylogenetic tree revealed that NAD7 gene of Ramie shared in 98% identity with those of Brassica napus and Arabidopsis thaliana. The results also showed that the NAD7 gene of Ramie is an ancestral type in plant.7. Genetic relation analysis of Ramie cultivars and their parents from Sichuan with ISSR21 ISSR primers, which were screened from 100 ISSR primers, were used to analyze and identify the relationship of 8 Ramie with ISSR technology. PCR amplification showed that 86 clear and repeatable bands were obtained in all materials, of which 71 bands appeared to be polymorphic (82.5%). Genetic distance and the cluster analysis indicated that these materials were divided into 2 groups, and the value of genetic distance was ranged from 0.48 to 0.90. Moreover, it was also found that the genetic relationships between hybrids and their parents were not equal, the male parent transfer more genetic loci to their descendants, which indicated that hybrids inherited more genetic information from the male parent.8. Study on the Molecular Markers Linked to Male Sterile GeneA specific DNA segment was amplified only in the fertile cultivars but not in the sterile cultivars using the ISSR primer U835. And the ISSR-marker was converted into SCAR marker that can be used to molecular marker-assisted selection of Ramie breeding.9. Heterosis utilizing of Ramie male sterilityUsing the male-sterile breeding materials, the combination of hybrid Ramie male sterility, the combination of hybrid heterosis and separation variation were carried out. The results indicated that heterosis of Ramie male sterile line is quite obvious. Ramie heterosis for the use of CMS not only provides a scientific basi, a new channel of Ramie hybrid and Ramie breeding theory. But also reduce costs, increase farmers income, solve the problems of production, increase Ramie production market competitiveness and resilience of its social and economic efficiency has been remarkable.