Identification And Gene Mapping Of Dwarf Peanut Mutants

Plant height is an important agronomic trait that affects crop yield.Dwarf poles have the characteristics of lodging resistance and are one of the key factors that affect the structure of crop groups and increase yield.In a preliminary study in the laboratory,a dwarf peanut dwarf mutant dw1（dwarf1）,which was controlled by a recessive single gene,was found.This gene may regulate the plant height of peanut through the gibberellin（GA）signaling pathway.In this study,high-strength materials such as Luhua 14（LH14）,Tifrunner,and mutant parent DL322 were used to hybridize with dw1 respectively.Developed molecular markers between the mutant and LH14 and Tifrunner,respectively.Bulked Segregant Analysis（BSA）and map-based cloning performed preliminary and fine mapping of dwarf genes.In-depth study of dw1 gene function will help to understand the molecular mechanism of peanut plant height regulation,and lay the foundation for the study of the establishment of peanut ideal plant type and molecular-assisted breeding.The main findings are as follows:1.Dwarf mutant phenotypic analysis:The number of stems of the mutant dw1 did not change,but the internode distance was significantly shortened.Exogenous 10 mg/L GA₃was applied and sprayed once every morning.Peanuts are suitable.After 10 days of treatment,the plant height and node spacing can be partially restored to the parental level.2.Cytological analysis of dw1 internodes in dwarf mutants:Compared with the wild type,the length of the main stem cells of the mutants was significantly shortened,but the number of cells was not significantly changed.It may be that the decrease in internode length of dwarf mutants may not be due to the decrease in the number of cells,but the decrease in the length of the main stem cells.Therefore,the dwarf mutant dw1 has the same number of stem nodes and shortened internode phenotype.3.Genetic analysis of dwarf mutants:The mutant dw1,its parent DL322 and the stalk material LH14 hybrid F₁had a stalk phenotype.The chi-square test proved that the separation ratio in the F₂population was 3:1,indicating that the mutant dwarf phenotype is controlled by a recessive single gene（nuclear gene）,which is a typical quality trait.4.Construction of genetic population:The dwarf mutant dw1 was used as the female parent to cross the high rod material LH14 to construct the LA-1 population,and the reproductive inbred line（RIL）population was constructed through multiple generations of inbreds through the method of single particle transmission F₇;Similarly,the dwarf mutant dw1 was crossed with LH14 and Tifrunner to construct LA-2 and TA populations,which have now propagated to F₄.5.Molecular marker development:Utilizing the dwarf mutants dw1 and Tifrunner 30×whole genome resequencing data,and using Tifrunner genome as a reference,preliminary development of 263 Insertions or Deletions of more than 5 bp covering the entire genome of 20 peanuts between dw1 and Tifrunner Markers,196 Eco R I and other commonly used restriction site mutations in CAPS markers.Tifrunner and dw1 parents and F₁genomic DNA were used as templates for PCR amplification.After enzyme digestion（CAPS labeling）,polyacrylamide,or agarose gel electrophoresis,94 polymorphic markers were obtained,including 77 In Del and 17CAPS marks.6.Preliminary localization:The results of BSA analysis showed that the dwarf gene dw1 was initially localized on chromosome A02;a total of 459 molecular markers were uniformly covered on 20 chromosomes,of which 94 bands were clear and could be used for map-based cloning.The polymorphism was marked by In Del or CAPS.The target gene was initially located near A02 chromosome D446（5 M）using 50 TA F₂single strains.7.Fine localization:Firstly use TA F₂population to perform localization,select 50 dwarf rods to extract DNA,develop 6 clear polymorphic In Del and SSR markers near 5 M,and detect PCR and polyacrylamide gel electrophoresis Results The statistical analysis showed that the exchange rates of the six markers were relatively low.The maximum exchange rate near D434（22 M）was approximately 22%,and the exchange rates near markers D412 and D745 were the lowest.It is speculated that dw1 may be located at markers D745（6.9 M）and D412（6.4 M）;then use the LA-2 F₂population to further locate,select 200 dwarf single plants to extract DNA,develop 12clear polymorphic In Del and SSR markers near 5 M,for PCR and polyacrylamide Statistical analysis of gel electrophoresis showed that dw1 might be located in the range of 0 kb-512 kb（S91）.Based on the mapping results of the two populations,the peanut dwarf gene dw1 should be located in the 0-512 kb interval of chromosome 2.8.Functional gene prediction:Gene prediction of the sequence of the localization interval.Through gene annotation of 48 candidate genes,it was found that N16 is similar to glutaric acid-dependent dioxygenase,and this gene is related to gibberellin synthesis;N28 It is a cytochrome P450 superfamily protein and a key gene for the synthesis of GA.Therefore,we speculated that the N16 or N28 mutation might cause the peanut plant height to become shorter.