Research And Application Of High Throughput Dna Extraction And Speed Breeding Techniques In Rice

Abract: Rice is the most important grain crop in China and the progress of its variety breeding is directly related to the food security of China.The combination of conventional breeding technology and molecular marker technology provides a strong support for new rice variety breeding and accelerates the process of rice breeding.However,rice is a typical short-light and temperature-loving crop.In most regions of China,it can only be planted for1-2 generations a year,while it usually takes 8-10 generations for 5-6 years to cultivate a new rice variety,which undoubtedly limits the efficiency of new rice variety breeding.For molecular markers,the rapid and efficient extraction of a large amount of DNA is a key link to improve the application efficiency of molecular markers.However,currently the commonly used method is to cut the leaves of plants and extract DNA,which is costly,inefficient and prone to errors that cannot be matched by a single plant and DNA sample.Therefore,the research of accelerated breeding program and high-throughput DNA extraction technology suitable for rice has important application value for improving the breeding efficiency of new rice varieties.Aiming at the above problems,this study proposed to capture rice root tissues in batches with 96-well plates and extract DNA efficiently,so as to overcome the limitations of traditional DNA extraction methods.In addition,in this study,indica rice Huahang 39 and Nipponbare were used as materials to study the effects of different densities and different periods of light treatment on the growth and development of rice under full liquid culture conditions,so as to find a suitable technical process of accelerating rice breeding.The main research results are as follows:1.This study developed a "double-layer growth plate" device suitable for rice seed growth and batch extraction of root tissues,so as to realize the acquisition of rice tissue samples from sowing to root tissues in 96-well plates.Compared with the traditional method of blade shearing,this method only takes about 10 min to obtain 96 sample tissues,while the method of blade shearing takes about 30 min.In addition,the sample organization obtained by this method is exactly corresponding to the individual source,no additional sample marking is required,and the success rate is 100 %.2.In this study,improved TRIS-EDTA method was developed to extract rice root tissues,and the efficiency of this method to extract DNA was analyzed.Compared with CTAB method and magnetic bead method,the differences between DNA extracted by modified TRIS-EDTA method were small,with an average concentration of 92.54ng/ul.The average value of DNA A260/280 was 1.74,with high DNA purity.The time required is only 1/20 of the traditional CTAB method.GS3 gene primer amplification was used to extract DNA.The amplification products were bright and clear,the fragment size was consistent with the expectation,and the stability was high,which could meet the requirements of molecular marker analysis.3.In this study,a liquid culture method suitable for rice during the whole growth period was developed to improve the efficiency of rice cultivation.In this study,an improved rice nutrient solution was proposed to replace NH4NO3 with Ca(NO3)2· NH4NO3·10H2O to provide nitrogen source.The foam plate was used to locate the seedlings and suspend them on the liquid medium to provide sufficient space for root development.The effect of planting density on agronomic traits was further compared,and it was found that the seed setting rate and thousand-grain weight were not highly correlated with planting density,and the planting density of 1-2 plants /cm2 was an appropriate density for the whole liquid culture.4.Under the condition of liquid culture during the whole growth period,this study found that different illumination time had important effects on rice growth period and agronomic characters.After treatment with 16 h light +8h darkness for 10 d and then transferred to 10 h light +14h darkness,the whole growth period could be shortened by50%,and there was no adverse effect on agronomic traits.The trend of indica rice and japonica rice was consistent.The method proposed in this experiment to control the growth and development of rice can achieve 4 generations of breeding per year from transplanting to harvest.Compared with the natural breeding process(2 generations/year),the efficiency can be improved by about 50%,significantly accelerating the process of rice breeding.In summary,the method for obtaining DNA from rice root tissues developed in this study overcomes the disadvantages of large workload and time-consuming and laborious acquisition of traditional DNA samples,and can effectively serve rice molecular breeding and mutant genotype screening.The proposed nutrient solution culture and light control during the whole growth period can improve the rice planting efficiency,shorten the rice growth period and accelerate the rice genetic process.The results have important practical value for improving the breeding efficiency of new rice varieties.