Innovation Of Rice Germplasm By Transferring Alien DNA Of Wild Species And Research Of The Molecular Mechanisms

Germplasm innovation with distant species is the frontier of plant genetics and breeding. Exogenous DNA transformation methods, a series of genetic manipulation technologies, were firstly invented and initiated by Chinese scientists. Previous studies showed that transferring genomic DNA of wild species into cultivated species with these methods could cultivate abundant variants, and improve the agronomic traits of the recipients comprehensively. However, the molecular mechanisms underlying exogenous DNA transformation methods, such as the full picture of the phenotypic variations and genetic variations of variants, the transfer and integration of alien DNA fragments, and the molecular mechanisms underlying the phenotypic variations, were unclear, scientists and breeders are puzzled and confused to these methods.In this paper, we carried out a lot of related researches of germplasm innovation with "spike-stalk injection method (SIM)", the patterns of its heredity and segregation, and the underlying mechanisms. The genomic DNA of different wild specieswere introduced into different cultivated rice for cultivating new rice germplasms, conducted the studies of the patterns of heredity and segregation in low-generation variant lines and the molecular mechanisms underlying "spike-stalk injection method" in high-generation variant lines. The main contents are stated as following:1. The genomic DNA of maize, sorghum, Echinochloa crusgalli, Arabidopsis thaliana and three kinds of wild rice were introduced into RH78and9311with "spike-stalk injection method", and had selected a lot of rice variants with significantly phenotypic variations in plant height, yield-related characters, and leaf, and so on; the variation rate ranged from0.7%o to4.4%o. Additionally, analyzing the genetic diversity between variants and recipients by SSR (simple sequence repeat) and indel (insertion and deletion) markers confirmed that the genetic diversity between variants and recipient ranged from0.88%to32.93%, and the existence of "mutation hotspot". 2. Whole-genome deep re-sequencing of the variant line ERV1and its recipient RH78, RAD(Restriction-site associated DNA tags) sequencing of216individuals, QTL(quantitative trait locus) association analysis of the yield-related characters and comparatively genomic analysis founded that:(1) the phenotypic characters of the variants, especially the yield-related characters, such as1000-grain weight, grain number per spike and seed setting rate, were improved significantly compared to the recipient rice;(2)5,518homozygous SNPs and449,726heterozygous SNPs were detected between the recipient RH78and the variant ERV1; meanwhile, the distribution of these SNPs in ERV1chromosomes was uneven;(3)14,955non-synonymous SNPs in the coding sequencing regions (CDS) of7,325ERVlgenes were detected in ERV1;(4)13QTL or genes closely-linked to heading data, plant height, spike length, effective tiller number, seed setting rate and100-grains weight were mapped, which could explain4.8%-10.5%of the phenotypic variances;(5) The genomic homozygosity of50%D2individuals reached up to90%, with a mean of88.2%, and the mean heterozygosity was1.57×10-4;3. Investigation and comparison of the phenotypic traits showed a wide range of morphological and physiological differences between the high-generation variant lines YVB and its donor V20B, especially of the improvement of YVB grain quality; Whole-genome sequencing and comparative genomic analysis among YVB, V20B and the donor O.minuta were conducted, and identified310,500SNPs (single-nucleo-tide polymorphisms),44,305small indels (insertions and deletions) and7,771SVs (structure variations) between YVB and V20B, additionally,12O.minuta-specific DNA fragments and/or genes in the YVB genome were discovered and validated; QTL association analysis of100individuals of the BC1F5population crossed between YVB and V20B were performed, and founded that the sequence variations in Wx and DEP1genes may explain the phenotypic differences of gel consistency and grain length between YVB and V20B. In addition, A QTL region with high LOD value in the YVB chromosome5ranging from4.3to5.1CM were identified, and this region was closely related to the YVB variation of1000-grains weight, grain width, head rice weight and chalkiness area, the sequence variations of the cloned gene GS5and GW5in this region may explain the variations of grain quality...