| Potato?Solanum tuberosum L.?is autotetraploid and highly heterozygous.Genetic basis of potato is narrow and the research of genetic basis is relative lag in our country.Irradiation induced mutation combined with in vitro culture,which can detect genetic resources and broaden the genetic basis,is the effective method to breeding new varieties with high quality and yield.In the present research,the efficient regeneration system of potato"Chuanyu NO.10"callus were optimized by using response surface method;The callus was treated with different doses of60CO-?irradiation,then incubated by using high efficient regeneration system to obtain mutagenesis progeny,and Mutagenic Effects of 60Co-?irradiation on potato callus was analyzed.In addition,the phenotypic mutants was screened out by morphologic method,and further researched using paraffin section,EST-SSR molecular markers,and the transcriptome technical analysis.The effects of radiation mutagenesis and the characteristics of mutant plants were analyzed.The main results are as follows:1.The response surface methodology was used to research the effect of TDZ,2,4-D,and GA3 on the differentiation of potato callus.Meanwhile,the hormone concentration was optimized.Result showed that the determination conefficient R2 of the model was 0.9921,the optimal medium for adventitious bud differentiation from stem callus of“Chuanyu NO.10”was MS+2.02mg/L TDZ+0.08 mg/L 2,4-D+2.25 mg/L GA3.With this condition,confirmed the validity of the plant growth adjustment ratio.2.The influence of callus and plant regeneration in different60CO-?irradiation dose is studied by morphological methodfor"ChuanyuNO.10”?WT?and 8 mutagenesis progeny?M1-M8?,and compared with leaf microstructure of mutations by paraffin section method,then preliminarily evaluated the stress-resistance by the principal component analysis and subordinate function Results indicated that the optimum irradiation dose of potato callus was 1024.8 Gy.Regeneration plant mutations reflected that the direction and degree of variation were uncertain.Principal component analysis selected spongy tissue,palisade tissue,upper epidermis and lower epidermis thickness as the main indicators for the preliminary assessment of drought resistance of the materials;the membership function method was used to forecast,the highest drought resistance one was mutant M1.3.To screened the potato mutants,4 of 13 pairs of primers with clear band and high diversity were selected to analyze genetic diversity by EST-SSR molecular markers for ChuanyuNO.10”?WT?and 20 mutagenesis progeny?M1-M20?.Genetic distance and cluster analysis were estimated by NTsys2.10e.The results showed that the 4 primer pairs amplified a total of 14 polymorphic sites among the 21 potato breeding resources.The average distance was 0.0498,varying from 0.0000.2057.There were genetic changes between 13 mutagenesis and WT,but the genetic distance was small.And the genetic distance between mutant M1 and WT was the largest,which was 0.2057.Using EST-SSR molecular marker technology can directly identify and screen a few mutations in DNA levels generated.with60Co-?irradiation mutation.4."ChuanyuNO.10"?WT?and mutant M1 with the greatest degree of variation and the strongest drought resistance were analysised by Illumina HiSeq x-ten sequencing.Through data filtering,genome alignment,screening of differentially expressed genes,and analysised the GO and KEGG enrichment of the differentially expressed genes.The results showed that there were a total of 2820 DEGs.1655 DEGs were annotationed to GO entries,enrichment analysis showed that the mutant M1 affected the molecular function,cell structure and biological process of the plant.1993 DEGs were annotationed to the KEGG pathway,they were mainly enriched in the biosynthesis of secondary metabolites and plant-pathogen interactions,among which ACT related to arginine and valine metabolism,ANS related to flavonoid biosynthesis,and plant effects triggering immunity-related The gene expressions related to RIN4,RPM1,RPS2,PIK1 and HSP90B were significantly different.It is speculated that the expression changes of these genes may be related to the variability of M1's ability to adapt to the environment,stress resistance and disease resistance. |
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