Full-length Transcriptome Analysis Of Allopolyploid MTP And Genetic Study Of A Dwarf Mutant From MTP's Backcross Offspring With Maize

Expanding germplasm resources has always been one of the important research objectives of crop breeding.In the previous study,the allopolyploid material MTP(Maize-Tripsacum-Teosinte),which combines the genetic material of a variety of distant species,has been developed,and its agronomic traits have shown superior advantages.In this study,the MTP full-length transcriptome was obtained by real-time single-molecule sequencing technology,and the gene function annotation,transcriptome remodeling,and the available parental(Maize,Tripsacum,and Teosinte)databases were used to further study the difference.Laying the foundation for the composition of the source polyploid and advancing its application in breeding.At the same time,using the data obtained from the transcriptome,45 SSR molecular markers were developed to reveal the genetic diversity of the derived forage material with MTP as the parent.In addition,dwarf mutants were found in high-generation backcross inbred lines with MTP as a donor and cultivar inbred line B73 as a receptor.Plant dwarfing is an important goal of lodging resistance and the green revolution of food.Exploring new dwarf gene resources,clarifying their genetic characteristics and dwarfing mechanism are of great significance for crop breeding and production applications.In this study,the newly obtained dwarf mutation d MTPB73 was comprehensively identified by trait determination,hormone test,cell section,and map cloning,and the candidate genes regulating maize dwarfing were predicted.The main results obtained in this study are as follows:1.Full-length transcriptome sequencing of allopolyploid MTP.1)To obtain more comprehensive MTP transcriptomics data,we generated transcriptome data from four different MTP tissues using Pac Bio Iso-seq sequencing technology.A total of 9,422,711raw reads were obtained from allopolyploid MTP,resulting in 227,375 Unigenes after assembly and quality control;in seven databases(Nr,Nt,GO,KEGG,KOG,Swiss Prot,and Pfam)94.73%of unigenes were annotated,showing a higher rate of annotations.Through structural prediction,12,982 Lnc RNAs and 9,508 TFs were identified.At the same time,based on the Cogent reconstruction of MTP transcriptome,1,673 Uni Trans Model-based AS events were identified,and the obtained reference transcriptome will provide reference for the study of MTP at the subsequent transcription level.Through Blast with the NCBIRef database,7,137 MTP unique new transcripts were inferred.Comparing the 24,521 identified MTP CDS sequences with the available databases of Tripsacum,teosinte,and the maize reference genome,the results showed that MTP was more likely to retain the genetic information of maize after fusion of the parental genome.In addition,potential unigenes involved in stress-resistant,salt-tolerant,cold-tolerant and other stress-related key genes were re-characterized and identified.As a result of this study,MTP transcriptome data can help to explore useful genomic resources related to stress resistance in MTP,thus promoting the application of MTP in breeding.2)The MTP EST-SSR molecular marker was developed based on the results of Iso-seq sequencing data.The genetic diversity of MTP,maize(two materials),4 Tripsacum(four materials),teosinte(6 materials),and 17 MTP-derived forage materials were used to identify the applicability of the developed EST-SSR primers.From 227,375 unigenes of MTP,a total of 222,110 SSR loci were identified in 92,983 unigenes with a frequency of40.89%.The success rate of 300 pairs of primers developed was 76%.Among them,45were polymorphic primers,and polymorphic primers obtained 397 bands in 30 materials.The average polymorphic content was 0.7558;the genetic distance ranged from 0.5678 to0.9096.The results of cluster analysis showed that the same species of materials were clustered into one class,and the clustering between materials was correlated with the material source.These results confirm the applicability of SSR markers developed using MTP transcriptome data and reveal the genetic diversity between the tested materials.It provides a basis for future research on the genetic diversity of MTP in forage cultivation and lays a foundation for molecular marker-assisted breeding.2.Identification of dwarf mutant characters and analysis of their genetic effects.In the backcrossing planting carp of MTP and B73,the dwarf mutant d MTPB73 was found.Compared with the normal plant MTPB73,the difference in the plant height,ear height,ear length was extremely significant.The difference in ear thickness reached a significant level.The difference in tassel branch number,cob diameter,row number per ear,grain thickness(five grains)and growth period was not significant.The results of grain quality test showed that except for the high lysine content(0.37%),it was significantly higher than the dwarf plant(0.33%)(P=0.0077),and there was no significant difference between other nutrients.The plant height dynamic test results showed that the plant height difference between d MTPB73 and MTPB73 was extremely significant after 35 days of sowing,and the plant height increased gradually with the growth period.Sensitivity test of exogenous hormones showed that d MTPB73 was insensitive to GA₃ and IAA,and it responded to BR but the plant height of d MTPB73could not be restored to the height of MTPB73 at the adult stage its effect needs further study.There was no significant difference in the number of stem stalks.The internode length was measured separately,and the length of each intenode of d MTPB73 was extremely significantly shorter than the internode length corresponding to MTPB73.the comparison of cell sections showed that the vascular bundle arrangement of d MTPB73was tighter than that of MTPB73.The d MTPB73 showed that the internode length shortened,resulting in even stunting between the nodes,and the plant height was reduced.The dwarf mutant d MTPB73 was positively and negatively crossed with five inbred lines.After t-test,the difference in plant height between the positive and negative crosses was not significant,and the possibility that the plant height was affected by the cytoplasm was excluded.The statistical analysis of the F₂ and BC₁ population showed that the separation ratio of high and dwarf plants was 3:1 and 1:1,respectively.This indicates that the dwarf trait is controlled by a recessive single gene.3.d MTPB73 dwarf gene localization.First,a homozygous dwarf mutant and a normal plant DNA were used to construct a high and dwarf mixed pool.After sequencing,the maize B73-V4 was used as the reference genome.The mapping rates of high and dwarf gene pools were 97.95%and 97.75%,respectively.Comparing the SNP and Indel difference sites,selecting the window larger than the threshold at the 95%confidence level as the candidate interval,and initially positioning the dwarf gene on the short arm of maize chromosome 3.Simultaneously,mapping the reads that cannot be mapped to the maize reference genome with the MTP reference transcriptome library,the mapping rates of the high and dwarf gene pools were 0.77%and 1.04%,respectively.The results showed that the dwarf mutant had a high background recovery rate,but the dwarf mutant had more MTP-specific sequences than the normal one.In addition,the hybrid F₂ of SW1611 and d MTPB73 was used as a fine mapping group.From the SSR markers uniformly distributed on the 10 chromosomes of maize,22 pairs of codominant markers with polymorphism between the parents and the high and dwarf plant were screened;Linkage analysis showed that the dwarf gene was linked to the chromosome 3 marker,so the dwarf gene was mapped to the short arm of chromosome 3,which is consistent with the sequencing analysis.In order to fine mapping the dwarf gene,the co-dominant markers were continued to be screened in the candidate interval to amplify the recessive individual in the F₂ population.According to the band type statistics,the target gene candidate interval was narrowed between Indel markers Chr3-5580580 and Chr3-5847200,and 11 genes were included in the interval.4.Mark development and candidate gene identification.After cloning the gene Zm0001d039456,sequence analysis was performed to design and develop the Indel marker.The dwarf gene was finally mapped between the markers 5580580 and Indel-1,and the physical distance was 95 kb.The two maize genes contained in this interval are different from the previously reported dwarf genes.Among them,Zm00001d039454encodes nucleotide pyrophosphatase/phosphodiesterase 1,and Zm00001d039455encodes an allylated rab receptor family protein.There is a single base mutation in both d MTPB73 and MTPB73 in Zm00001d039454 and Zm00001d039455,but the variation in Zm00001d039455 is a synonymous mutation that does not cause amino acid variation,whereas in Zm00001d039454,a guanine on the protein coding region(G)A single base mutation that becomes adenine(A)results in an amino acid variation in the amino acid sequence of the gene from alanine(Ala)to threonine(Thr).Therefore,the results of this study indicate that the gene Zm00001d039454 is a potential dwarf gene,and the functional and allelic identification of this gene remains to be further verified and studied.