| Sugarcane varieties with high yield,high sugar,strong drought resistance and strong ratoon ability are important for the development of sugar industry in China.However,the narrow genetic background of the parents is one of the main limiting factors for moderb sugarcane breeding.It is the key to sugarcane breeding that strengthening the germplasm innovation and enriching the parent gene pool.Narenga porphyrocoma(Hance)Bor,a related wild plant of sugarcane,has elite traits such as drought resistance,disease resistance and strong ratoon ability.It is an important germplasm resource for genetic improvement of sugarcane.In current distant hybridization of sugarcane,the genetic efficiency of the wild type gene in the offspring,the variation of the genome and the genetic mechanism of the hybridization are neglected,which limits the utilization efficiency of the wild sugarcane germplasm.In this study,amplified fragment length polymorphism(AFLP)combined with capillary electrophoresis(CE)analysis was used to analyze the genetic diversity of Narenga porphyrocoma(Hance)Bor germplasm resources.The hybrid F1,BC1 and their parents were used as the materials to analyze the transmission of chromosomes in offspring.The specific loci of parents were analyzed by AFLP;DNA methylation levels and genetic pattern in F1,BC1 and their parents were detected using nethylation sensitive amplification polymorphism(MSAP)technology;drought related genes were explored in Narenga porphyrocoma(Hance)Bor using high-throughput sequencing technology.The results are as follows.1.The genetic diversity of Narenga porphyroconma(Hance)Bor germplasm collections was studied,and the molecular ID cards were established.Fifteen samples of Narenga porphyrocoma(Hance)Bor gemplasm materials,collected from different areas of Guangxi,were used for analyses of AFLP and CE.The total bands,polymorphic bands and percentage of polymorphic bands(PPB)were counted.The genetic similarity was used for UPGMA(Unweighted Pair Group Method Analysis)and PCA(Principal Component Analysis),and the molecular identification cards were constructed for the 15 accessions of Narenga porphyrocoma(Hance)Bor gemplasm.A total of 2208 bands were amplified with 25 pairs of AFLP primers,of which 1914 were polymorphic and the percentage of polymorphic bands was 87.11%.The genetic similar coefficients of the 15 accessions of Narenga porphyrocoma(Hance)Bor germplasm ranged from 0.656 to 0.878,and the cluster analysis based on UPGMA showed that these germplasm accessions were divided into three major groups at the similarity coefficient 0.690.All of the accessions could be effectively distinguished by specific bands and different primer combinations.The specific molecular identity cards for the 15 accessions of Narenga porphyrocoma(Hance)Bor germplasm collections were established based on the 104 bands amplified with 3 AFLP primer combinations.The results showed that a high level of genetic diversity exists in Narenga porphyrocoma(Hance)Bor germplasm.2.Using chromosome counting,the chromosome inheritance was analyzed among the F1 and BC1 population hybrids between sugarcane and Narenga porphyrocoma(Hance)Bor.It was deduced that the chromosomal transfer models were "n+n" and "n+2n" in F1 population,and "n+" in BC1 population.3.Using AFLP and CE techniques,the genetic genes from the wild parent of Narenga porphyrocoma(Hance)Bor were analyzed in the F1 and BC1 progeny.24 pairs of AFLP primer combinations were used for amplification for 4 individuals of F1,9 individuals of BC1 and their parents.A total of 1774 bands were detected,of which 1690 were polymorphic and the polymorphic rate was 95.26%.The polymorphism of each primer combination ranged from 87.04%to 100%.Further analysis showed that the proportion of the specific loci from the maternal parent GT05-3256 to individuals in F1 population ranged 63.92%-71.65%,and the ratio of the specific loci from the paternal parent GXN1 to individuals in F1 popuplation ranged 8.66%-12.85%.The proportion of specific loci from the parental YC94-46 to individuals in BC1 population ranged 35.19%-48.50%,and the proportion of specfic loci from the paternal parent T6-3 to individuals in BC1 population ranged 33.97%-59.33%.The ratio of specific loci in GXN1 inherited to individuals in Fi population ranged from 8.66%to 12.85%,but to indiviaduals in BC1 population,the ratio ranged from 1.12%to 3.35%only.The genes from wild Narenga porphyrocoma(Hance)Bor inherited to the BC1 offspring are at a very low level.In addition,the specific loci of F1 and BC1 maintain a high level.4.The level and difference pattern of the DNA methylation in the F1 and BC1 hybrids and their parents were analyzed using MSAP and CE.MSAP ratio in hybrid F1 was 56.4%-59.0%,which is lower than that in both parents.The ratio of MSAP in BC1 was 56.9%-69.8%,and the average was 62.8%,higher than that in both parents.The total methylation level of the BC1 generation is slightly higher than the level in the F1 generation.The methylation of CCGG loci in sugarcane genome of the individuals in F1 and BC1 was mainly based on internal cytosine double-strand methylation.71 types of methylation were detected in F1 and BC1 populations,and were further divided into 5 types:type A,the methylation patterns in hybrids were the same to their parents;type B,demethylation,the methylation of the hybrids was weaken compared to the methylation of the parents;type C,hypermethylation,the methylation of the hybrid was enhanced corresponding to its parents;type D:hypomethylation,the degree of methylation of the hybrid was lower than that of both parents;type E:uncentain type.Among of them,type A is the genetic type from the parent to the hybrid,and types B,C,D and E are the methylation variants of the hybrids.The results showed that the methylation type of individuals in F1(A)was significantly lower than that in BC1,but the variant types B,C,D,and E were higher than those in BC11.During the F1 and BC1 hybridization,the hypennethylation occurred mainly in the whole genome.5.Narenga porphyrocoma(Hance)Bor was selected and treated with drought stress.Leaves +1 from drought treated(DTS)and control(CK)plants were obtained for deep sequencing.Paired-end sequencing enabled us to assemble 104,644 genes(N50=1605 bp),of which 38,721 were aligned to other databases,such as UniProt,NR,GO,KEGG and Pfam.Single-end and paired-end sequencing identified 30,297 genes(>5 TPM)in all the samples.Compared to CK,3,389 differentially expressed genes(DEGs)were identified in DTS samples,comprising 1,772 up-regulated and 1,617 down-regulated genes.Functional analysis showed that the DEGs were involved in biological pathways like response to blue light,metabolic pathways,and plant honnone signal transduction.The expression patterns of several important gene families were found,including aquaporins,late embryogenesis abundant proteins,auxin related proteins,transcription factors(TFs),heat shock proteins,light harvesting chlorophyll a-b binding proteins,disease resistance proteins,and ribosomal proteins.Interestingly,the regulation of the genes varied in different subfamilies of aquaporin and ribosomal proteins.In addition,LHCB,DIVARICATA and heat stress TFs in response to water deficit were first found in Narenga porphyrocoma(Hance)Bor leaves. |
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