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Creating Novel Glutinous Rice Germplasms by Editing Wx Gene via CRISPR/Cas9 Technology
The Wx gene,which controls the synthesis of amylose in rice endosperm,is the major gene that determines rice eating and cooking quality(ECQ). It can significantly improve rice ECQ by moderately reducing amylose content(AC)of endosperm. Editing the C-terminal of Wx encoded GBSSI enzyme to fine-tune its enzyme activity and grain AC is expected to further improve rice ECQ. By analyzing the functional domain of GBSSI through bioinformatics websites,two target sites,T1 and T2(located in the 12th and 13th exons of the Wx gene,respectively),were subjected for CRISPR/Cas9-medicated editing. The homozygous mutants were validated by PCR and Sanger sequencing,followed by quantifying the apparent amylose content,gel-permation chromatography,western blot and qRT-PCR. A total of 8 homozygous lines,C1-C8,with retained major domains of GBSSI were obtained. Among which,the 518-550/551 codons were shifted in the predicted protein in C2 and C3 lines and the predicted protein in C1,C4-C8 were shifted after codon 551,517 or 518. The apparent amylose content of C1-C8 was significantly reduced from 16.79% to 4.44%-3.69%,but significantly higher than that of near-isogenic line(NIL)carrying the conventional wx allele(AAC=2.92%). GPC results showed that there was no amylose synthesis in the selected mutants,but their chain length distribution of Ap2 was longer than that in NIL wx. The qRT-PCR and western blot suggested no significant difference on transcript of Wx gene but obvious reduction of GBSSI accumulation in the developing seeds in mutants. No GBSSI accumulation was detected in all the homozygous lines except for C2 and C3. As a result,this study generated glutinous rice lines with fine-tuned starch fine structure to conventional glutinous rice by CRISPR strategy,and provided evidence of the 518-551 amino acids on the formation of GBSSI enzyme activity. These results provide a reference for further analyzing the structure of GBSSI protein to achieve its precise editing.
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Flowing Rock
Evaluation on Phenotypic Variation of Rice Bean Germplasm collected from Hunan Province
In order to find out the geographical distribution characteristics of the collected agricultural biological germplasm resources in Guizhou,the external geographical environment and the internal spatial performance of the collected resources were analyzed with the methods of spatial statistical analysis and diversity analysis. Firstly,the characteristics of elevation,temperature,precipitation and other basic geographic environment of germplasm resources are determined by spatial superposition and statistics. Secondly,the spatial distribution,the orientation and difference as well as heterosexuality,density and richness of germplasm resources are analyzed using the optimizing hotspot analysis,the geographic distribution center analysis and spatial diversity analysis. The results show that the interval of elevation and meteorological factors in the area where the resources are located is large,but most of them are distributed in relatively concentrated geographical environment. 68.7% of the germplasm resources were found to be distributed between 600 and 1400 m of altitude,65.1% were distributed between 15 and 17 degrees centigrade of average annual temperature,and 61.7% were distributed from 1250 to 1350 mm of annual precipitation. In addition,the 21 selected counties can basically reflect the spatial distribution characteristics of agricultural biological resources in Guizhou from the perspective of geographic space. Germplasm resources of different topics are cross-distributed,with the exception of edible fungi concentrated in the southwest. The resources of food crops,economical crops,vegetables,fruit trees and medicinal plants,which were provided by 14 ethnic group,are relatively evenly distributed in the province. Among them,the Miao ethnic group provided the highest amount(37%)of resources,followed by the Han(16%) and Buyi(15%)ethnic groups. The proportion of resources provided is less than 1% of the total. Thus,this study described the distribution status and characteristics of agricultural biological resources in Guizhou Province, which will provide reference for the formulation of relevant policies for effective protection and efficient utilization of biological resources in Guizhou Province.
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Flowing Rock
Collection and Preliminary Study of Wild Rice Genetic Resources in Southeast and South Asia
The development and utilization of wild rice genetic resources are increasingly important given the fact that the genetic resources in cultivated rice (Oryza sativa L.) gene pool are insufficient to sustain the requirement in modern rice breeding. Although China hosts the world's largest collection of cultivated rice germplasm accessions, a large-proportion of stocks were domestically collected plus the alien wild germplasm accounting for only 10%. The wild rice germplasm resources, which are locally adapted to the diversified climates and habitats in countries from South and Southeast Asia, represented the high potential value in genetic development and breeding in China. In this paper, via summarizing the field surveys and the collections of wild rice (Oryza rufipogon and O. nivara) in 10 South and Southeast Asian countries from 2009 to 2019, 1166 samples from 66 populations were collected.Considering the ecotypes of Oryza rufipogon and O. nivara as well as environmental conditions in South and Southeast Asiancountries, we proposed suggestions for future collection of wild rice resources in this region.
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Flowing Rock
Genetic Diversity Analysis of Wheat Cultivars/Lines Derived from Wheat Cultivar Zhoumai 22
Zhoumai 22 is not only the major cultivar in China, but also has become an important breeding parent in Huang-huai river valley winter wheat region. In order to reveal the genetic diversity and contributions of Zhoumai 22 to its derivatives and provide a reference for parent selection, we analyzed 106 derived varieties (lines) of Zhoumai 22 by genealogy and selected its earlier derived 80 varieties (lines) for SNP genotyping using the Affymetrix 55K SNP chip. The results showed that the 106 derivative lines of Zhoumai 22 involved 82 direct parents, and the main ones used as the opponent parents were Bainong AK58、Zhoumai 24, Zhoumai 18, Luomai 21, Zhoumai 16, Zhoumai 19, etc. These parents were directly or indirectly derived from Zhou 8425B and Zhoumai 9 and belonged to the "Zhoumai" consanguinity family. Of the A、B、D genomes, the B genome has the largest number of polymorphic loci, the D genome has the least number of polymorphic loci, but the ratio of polymorphism SNP to raw SNP number of A and D genomes is basically the same. Genetic similarity between Zhoumai 22 and 80 derived varieties (lines) ranged 0.392~0.856 with an average of 0.685, while genetic similarity among the derived lines varied from 0.381 to 0.991 with an average of 0.676. There were some genetic diversity differences among Zhoumai 22 and its derivatives. The 81 varieties were divided into 8 subpopulations, of which the I、II、III and IV subpopulations had richer genetic diversity and the V、VI、VII and VIII subpopulations was insufficient on genetic diversity. Through the polymerization of high quality protein subunits and differential selection, the new wheat varieties with high yield and multi-resistance and high quality characteristics were bred, such as Zhoumai 26, Cunmai 8, Zhoumai 36, Cunmai 11 and so on.
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