Identification of S-RNase genotype and analysis of its origin and evolutionary patterns in Malus plants

Identification of the S genotype of Malus plants will greatly promote the discovery of new genes,the cultivation and production of apple,the breeding of new varieties,and the origin and evolution of self-incompatibility in Malus plants.In this experiment,88 Malus germplasm resources,such as Aihuahong,Xishuhaitang,and Reguanzi,were used as materials.Seven gene-specific primer combinations were used in the genotype identification.PCR amplification using leaf DNA produced a single S-RNase gene fragment in all materials.The results revealed that 70 of the identified materials obtained a complete S-RNase genotype,while only one S-RNase gene was found in 18 of them.Through homology comparison and analysis,13 S-RNase genotypes were obtained:S_(1)S_(2)(Aihuahong,etc.),S_(1)S_(28)(Xixian Haitang,etc.),S_(1)S_(51)(Hebei Pingdinghaitang),S_(1)S_(3)(Xiangyangcun Daguo,etc.),S_(2)S_(3)(Zhaiyehaitang,etc.),S_(3)S_(51)(Xishan 1),S_(3)S_(28)(Huangselihaerde,etc.),S_(2)S_(28)(Honghaitang,etc.),S_(4)S_(28)(Bo 11),S_(7)S_(28)(Jiuquan Shaguo),S_(10)S_e(Dongchengguan 13),S_(10)S_(21)(Dongxiangjiao)and S_(3)S_(51)(Xiongyue Haitang).Simultaneously,the frequency of the S gene in the tested materials was analyzed.The findings revealed that different S genes had varying frequencies in Malus resources,as well as varying frequencies between intraspecific and interspecific.S_(3) had the highest frequency of 68.18%,followed by S_(1)(42.04%).In addition,the phylogenetic tree and origin evolution analysis revealed that the S gene differentiation was completed prior to the formation of various apple species,that cultivated species also evolved new S genes,and that the S_(50) gene is the oldest S allele in Malus plants.The S_(1),S_(29),and S_(33) genes in apple-cultivated species,on the other hand,may have originated in M.sieversii,M.hupehensis,and M.kansuensis,respectively.In addition to M.sieversii,M.kansuensis and M.sikkimensis may have also played a role in the origin and evolution of some Chinese apples.

Analysis of genetic diversity and structure across a wide range of germplasm reveals genetic relationships among seventeen species of Malus Mill,native to China

China is a center of diversity for Malus Mill,with 27 native species including 21 wild species and six domesticated species.We applied a set of 19 simple sequence repeat markers to genotype 798 accessions of 17 species(12 wild species and five cultivated species)of Malus originating from 14 provinces in China.A total of 500 alleles were detected.Diversity statistics indicated a high level of genetic variation as quantified by the average values of the effective allele number(N_(e)).expected heterozygosity(H_(e)),and Shannon's Information Index(I)(10.309,0.886,and 2.545,respectively).Malus sieversii(MSR;H_(e)=0.814,I=2.041,N_(e)=6.054),M.baccata(MBB;H_(e)=0.848,/=2.350,N_(e)=8.652),M.toringoides(MTH;He=0.663,I=1.355,N_(e)=3.332),and M.hupehensis(MHR;H_(e)=0.539,I=0.912,N_(e)=0.579)showed a higher level of genetic diversity in this study than the previous studies.MSR and MBB contributed to the origin and evolution of some accessions of M.domestica subsp.chinensis(MDC).However,other accessions of MDC showed a closer genetic distance with MBB and cultivated species,especially M.robusta(MRB),M.asiatica(MAN),and M.prunifolia(MPB).Not all accessions of MDC were descended from MSR in Xinjiang Uygur Autonomous Region of China.This research provides novel insights into the genetic relationships of Malus native to China,which will be useful for genetic association studies,germplasm conservation,and breeding programs.

MdAIL5 overexpression promotes apple adventitious shoot regeneration by regulating hormone signaling and activating the expression of shoot development-related genes

Adventitious shoot(AS)regeneration is a significant factor in the genetic transformation of horticultural plants.It is also a noteworthy approach to their vegetative propagation.AS regeneration remains highly dependent on the genotype or maturity of explants.We here found that the AS regeneration abilities of apple leaves were positively correlated with MdAIL5 expression.MdAIL5 overexpression dramatically increased AS regeneration efficiency.Notably,MdAIL5 overexpression could restore the AS formation ability of explants to a certain extent,which was lost with an increase in maturity.Endogenous hormone detection revealed that MdAIL5 overexpression changed the contents of auxin,cytokinin(CK),and other hormones in apple leaves.Transcriptome analysis revealed that many genes related to auxin,CK,and brassinolide signaling pathways were significantly and differentially expressed between MdAIL5-overexpressing transgenic apple and wild-type apple plants.Yeast one-hybrid assays,the electrophoretic mobility shift assay,and the dual-luciferase reporter assay revealed that MdAIL5 directly binds to MdARF9 and MdHB14 promoters and positively affects their expression.We here established a model of MdAIL5 regulating AS formation,which acts as a theoretical basis for facilitating genotype-or explant maturity-independent AS regeneration in the future.

A 14 nucleotide deletion mutation in the coding region of the PpBBX24 gene is associated with the red skin of“Zaosu Red”pear(Pyrus pyrifolia White Pear Group):a deletion in the PpBBX24 gene is associated with the red skin of pear

Red skin is an important quality trait for pear fruits and is determined by the concentration and composition of anthocyanins.The regulatory mechanism underlying anthocyanin accumulation is a popular topic in fruit research.Red mutants are ideal materials for studying the molecular mechanism of color diversity in pear.Although several red pear mutants have been cultivated and are in production,no exact locus containing the responsible genetic mutation has been identified.In this study,by combining the bulked segregant analysis with whole-genome sequencing,we identified a 14 nucleotide deletion mutation in the coding region of the PpBBX24 gene from the red pear mutant“Zaosu Red”.We further verified that the deletion was present only in the red mutant of“Zaosu”and in its red offspring,which was different from that which occurred in other red pear fruits.This deletion results in a coding frame shift such that there is an early termination of the PpBBX24 gene and loss of key NLS and VP domains from PpBBX24.The lost domains may reduce or alter the normal function of PpBBX24.In addition,we found that the transcript levels of the PpMYB10 and PpHY5 genes in red samples were significantly higher than those in green samples,whereas the results for the normal-type PpBBX24 gene were the opposite.We ultimately revealed that the 14 nucleotide deletion mutation in the coding region of the PpBBX24 gene is associated with the red skin of the“Zaosu Red”pear.This finding of somatic mutational events will be helpful for breeding new red pear cultivars and for understanding the regulatory mechanisms involved in pear skin pigmentation.