Genetic analysis and preliminary mapping of the slow-melting flesh character in peach

Abstract:【Objective】Peach is a specific seasonal supply fruit, its maturity period of fruits is concentrated in just a few days, and fruit softening goes fast in late development, affecting harvest, transportation and shelf life. Slow-melting flesh (SMF) peaches can be kept on the trees for a longer time with high firmness during the ripening period, and reduce the loss due to the fruit softening. The study aimed to understand the inheritance of the SMF character in peach.【Methods】The slow-melting flesh peach varieties Chunli and Chunrui, non- slow-melting flesh (NSMF) peach varieties Chunmei and Chunmi were used as test materials, and grown in Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences. All the fruits of the the 4 varieties were collected at normal maturity. The fruits were covered with foamed nets and shipped back to the laboratory in boxes. The samples were taken every 3 days, and 20 fruits with the same size and uniform coloring were selected for each time. The samples were fixed to determine the ethylene release rate, fruit firmness. According to the physiological changes of fruits of the SMF and NSMF, the softening characteristics of the SMF peaches were clarified. The genetic population was constructed with the slow-melting flesh varieties Chunxue and Chunrui. The fruit hardness of the offsprings was investigated every 2 days from May to August of 2021 and 2022 in the Xinxiang Experimental Base of Zhengzhou Fruit Research Institute, Chinese Academy of AgriculturalSciences. Days on the tree after fruit ripening were analyzed, and ≥ 10 days were considered as a physiological index for determining SMF fruits. Furthermore, the inheritance was analyzed and the gene was mapped by Bulked Segregant Analysis (BSA). From Chunxue selfing segregation population, 20 plants with for NSMF trait and 20 plants with SMF trait were selected. The DNA was extracted, and two DNA mixing pools (dominant pool: SMF and recessive pool: NSMF) were constructed separately. The constructed two DNA mixing pools were used for sequencing. The Raw Illumina reads were processed to remove adapter and low quality sequences. The clean paired-end reads were mapped on the reference peach Lovell genome (version 2.0_a2.1) using BWA (http://bio- bwa.sourceforge.net/bwa.shtml ). The variants calling including SNP (Single Nucleotide Polymorphism) and InDel (Insertion and Deletion) were performed using GATK (https://software.broadinstitute.org/gatk/best-practices/). The SMF trait related candidate regions were determined by the Euclidean distance (ED) method by searching markers with significant differences between the SMF and NSMF pools according to the sequencing data and the associated regions between markers were evaluated.【Results】We compared the changes of the fruit firmness and ethylene emission on the trees of two kinds of texture after fruit ripening: the SMF and NSMF. The fruit firmness of the NSMF Chunmei and Chunmi decreased to the lowest level on the 9th day after ripening (R+9), but the SMF Chunli and Chunrui fruit completely softened on the 15th day after ripening (R+15). The firmness of the NSMF Chunmei and Chunmi remained above 15 N for only 6 days at maturity stage, the SMF Chunli and Chunrui remained for at least 12 days. The SMF had very low ethylene emission (Chunl) or no ethylene (Chunrui) before the R+12 stage, and a lot of ethylene was released during the final softening. The NSMF began to produce very high ethylene release at R+6 stage. The results showed that the SMF peach remained high firmness for a long time on the tree before softening and ethylene was released late during fruit ripening. In the SMF Chunxue segregation population, the days on the tree after ripening showed a significant bimodal distribution. The segregation ratio of the phenotypes was approximately 3∶1 for the SMF∶NSMF (χ2 =0.062 5) in 88 individuals of Chunxue selfing progeny. Another population was derived from the combination of Chunxue ×99-30- 33, with a total of 58 individuals, and the segregation of the phenotypes was approximately 1∶1 for the SMF: NSMF (χ2 =0.277). This segregation ratio is indicative of a trait controlled by a single dominant gene or major effect gene. When the SMF Chunrui was crossed with the NSMF ones (04-1-91 or 04-3- 25), the F1 seedlings showed a 1∶0 segregation ratio of the SMF to NSMF ones. The genetic analysis showed that parent Chunxue was heterozygous and parent Chunrui was homozygous in the SMF gene loci. To identify the candidate gene for slow-melting flesh character, a high-throughput specific length amplified fragment was sequenced of the parent Chunxue and the SMF/NSMF pools. A total of 79 304 SNPs and 21 715 InDels were identified between the SMF and NSMF pools. The regulatory site of the SMF was associated with 4 segments of chromosome 4 with a total length of 7.87 Mb by the Euclidean Distance mapping analysis method.【Conclusion】The slow-melting flesh is controlled by a single dominant gene or major effect gene located in chromosome 4. These results could provide a basis for the SMF molecular marker assistant breeding.