| The authenticity and genetic purity of seed is the most major indicator of seed quality testing. Testing of distinctness, uniformity and stability of new varieties of plants is the important content for examining the factors of application varieties and deciding if they can be granted the right of new varities in the office for the protection of new varities of plants, MOA. Rice is the major crop in our country. Hybrid rice has heterosis, high product, good resistance. There is almost half of rice area for planting hybrid rice. Our country is the biggest country for producing and consuming the seed of hybrid rice in the world. The number of rice to be applied variety protection is increasing every year. But the method for testing authenticity and genetic purity of seed and DUS testing for new varieties of plants is still the method of identification relying on planting in fields. It is long time for one growth cycle period and it costs much labor and occupies much area of field. The marker characters for testing are limit. This method can not meet the demand for seed market and new varieties authorization in time. Thus, there is important significance to study rapid, simple, accurate method of DNA molecular marker to identify rice varieties for testing hybrid seed quality and granting the new variety authorization. In order to rapidly, accurately identify the authenticity and genetic purity of hybrid rice seed in the area scope of Jiangsu Province by using SSR molecular marker, severl key problems need to be considered as the follows:rapid, high effective DNA extraction method is an bottleneck technology for identifying varieties by using molecular marker. Traditional DNA extraction method has much difficulty of technology, tedious operating procedure. These disadvantages are one of main problems which prevent it from popularization; The sterile character of female parent of two-line hybrid rice is influenced easiliy by illumination time and temperature. The genetic purity is seriously influenced by selfing of female parent because of different conditions of illumination and temperature in different years and different regions. But sterile line is fertile sometime during planting identification in Hainan. It brings difficulties to identification according morphological characters; It is the area where there are both Indica and Japonica hybrid and conventional rice in the area of middle and lower Yangtze river. There contantly are one kind of subspecies hybrid combination between Indica and Japonica named "Daqingke"which is generated by Indica sterile lines and Japonica male parents or Japonica sterile lines and Indica male parents in the producing seed of hybrid in this area. This kind of offtype has high height of plant and late heading date. The corporate image and production were seriously influenced if there was Daqingke in hybrid rice. But it is difficult to identify Daqingke because Daqingke may not have the characters of high height plant and late heading date while planting in Hainan for identification. Thus, it was suggested to be studied as the following four aspects:the first is to improve the method and procedure of DNA extraction and do research for the rapid, high effective and high quality DNA extraction method. The second was to analyze the polymorphism of 7 parents of 4 common two-line hybrid and one Japonica hybrid and use the polymorphic SSR markers to identify two-line hybrid. The third is to establish DNA fingerprint data of 33 Indica and Japonica parents of hybrid common used during recent years in production and analyze the genetic similarity coefficients in order to offer the basis for identifying hybrid seed. The fourth is to screen SSR markers for identifying Daqingke offtype among 55 hybrid combination of Indica and Japonica which were made by common Indica, Japonica sterile line and janponica and Indica male parents in this study after the major characters of plant height, heading date and seed setting rate were investigated. It offered the basis for identifying Daqingke which might generate in hybrid rice in this area. The major results are obstained as follows:1. A rapid, high effective and high quality DNA extraction improved method was applied in this study. Conventional DNA extraction method and improved DNA extraction method were both used in this study. In the procedure of improved DNA extraction method, the tissue of seedling leave were frozen and dried for 2-3 days in the vacuum freezing machine, then, was ground instead of using liquid nitrogen. There is one small steel ball in each tube. The tissue was ground by grinding meachine. Because each sample was ground in separate tube, it prevented the cross contamination from each other. The plates of 96 connected tubes were used instead of seperated tubes, multiple-pipettes instead of single pipette during the whole procedure and CTAB method were used to extract DNA for the method. It was verified to be a rapid, high effective and high quality DNA extraction method through many times testing and comparing. According to this method, one person can extract about 1000 DNA samples per day.2. Four two-lines hybrids could be identified by one group of four SSR primers. DNA polymorphism of five parents of four two-line hybrid rice with Peiai64S as female parent were analyzed by 52 pairs of SSR primers with one Japonica hybrid rice named 86you8 as the control. The results are as follows:46 SSR primers could amplify polymorphism among 7 parents.8 pairs of SSR primers which could amplify polymorphism among the parents of 86you8 could distinguish the 4 two-line combinations from the control. There were 34,32,31,30 and 14 pairs of SSR primers which could be used to distinguish the combinations (F1) and the parents of Liangyoupeijiu, Liangyou108, Peiai64S/E32, Liangyou122 and 86you8, respectively.16 SSR primers could distinguish all of the 4 two-line hybrid rice and their parents. RM206 and RM286 could distinguish the five combinations and their parents in this study. RM505 was tested to identify Liangyoupeijiu and it’s parents. The result showed that it could distinguish Liangyoupeijiu and it’s parents accurately. There were several kinds of methods to distinguish the 4 two-line combinations according to SSR polymorphism and specificity in this study. The four two-line combinations of Liangyoupeijiu, Liangyou108, Peiai64S/E32 and Liangyou122 could be distinguished by the group of four pairs of primers including RM13 (or RM206, RM286 and so on), RM224 (or RM337), RM234 (or RM252, RM505 and RM565) and RM25 (or RM217, RM248 and RM585).3. The fingerprint of 33 parents based on SSR markers was established in this study.6 primers could be used to identify Indica and Japonica. Genetic similarity and SSR fingerprint was studied by 84 pairs of primers distributed on 12 chromosomes in rice using 33 parents including 5 CMS lines of Japonica,4 CMS lines and 1TGMS line of Indica,14 Indica male parents,9 Japonica male parents in rice. Fifty four of the 84 SSR primers showed polymorphism among the 33 parents, accounting for 64.3% of the primers used. Genetic similarity coefficients among 33 parents ranged from 0.40-0.99. The 33 parents were classified three groups at the genetic similarity coefficient of 0.66. The first group included Peiai 64S and Gang 46A. The second group included 17 Indica parents and the third group included 14 Japonica parents. The sterile lines and fertile varieties could be classified into subgroups among Indica or Japonica, which was coincident with the groups and subgroups determined by their pedigree analysis. It futher suggested that SSR fingerprint could be used to identify varieties. All of the 33 parents could be distinguished each other by 18 pairs of the primers. RM264 could distinguish the 4 CMS lines (II-32A, XieqingzaoA, Gang46A and K17A) and the fertile varieties in Indica varieties used. RM432 could distinguish the 5 CMS lines and the fertile varieties in Japonica varieties used. The primers of RM6, RM13, RM16,RM240, RM247 and RM248 could distinguish Indica varieties and Japonica varieties. These 6 pairs of primers could be used to identify the off-type plants produced by CMS pollinated by Japonica in Indica rice seed producing field, or by CMS pollinated by Indica in Japonica rice seed producing field.4. Several SSR markers were discovered to identify Daqingke in this study. The major agronomy characters and the characters of SSR molecular marker of 55 hybrid combination of Indica and Japonica were analyzed. Daqingke was defined as the hybrids whose plant height was over 135 cm and heading date was over 121d in this study. The results were as follows:There is one Daqingke named 9522A/Zijianxian-1 among 7 hybrids which were based on 9522A as female parent and there are 8 Daqingke which included all 8 hybrids based on LiuqianxinA as female parent among 38 hybrids which were based on Japonica sterile lines as female parents and Indica as male parents.There were one Daqingke namedⅡ-32A/C57 among 4 hybrids which were based onⅡ-32A as female parent among 17 hybrids which were based on Indica sterile lines as female parents and Japonica as male parents. Others of 17 hybrids were non-Daqingke. Five SSR markers including RM9, RM283, RM429, RM515 and RM483 had significant positive correlation with plant height. RM9, RM283, RM429 and RM515 had extramarked positive correlation with plant height. Ten SSR markers including RM9, RM44, RM206, RM152, RM276, RM228, RM515, RM211, RM432 and RM454 had significant positive correlation with heading date.8 primers including RM9, RM44, RM206, RM152, RM515, RM211, RM432 and RM454 had extramarked positive correlation with heading date. Hybrids between subspecies of Indica and Japonica could be identified according to existence of both Indica special marker and Japonica special markers. Daqingke among subspecies hybrids based on 9522A as female parent could be identified by two primers of RM50 and RMll(or RM25、RM152、RM228、RM251、RM252、RM286,RM302、RM415). Daqingke among subspecies hybrids based onⅡ-32A as female parent could be identified by RM9 or RM218. All of subspecies hybrids based on LiuqianxinA were Daqingke. They could be identified by one of six primers such as RM9、RM152、RM279、RM413、RM415 and RM429 in order to distinguish LiuqianxinA and other Japonica sterile lines.New hybrid combination 3726A/Minghui 63 which had high seed setting rate (87.1%), moderate plant height (122 cm) and suitable heading date (109 d) was discovered by investigating three major agronomic traits of 55 subspecies hybrid rice in this study. It might be used in production after further verification. |
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