A deletion affecting an LRR-RLK gene co-segregates with the fruit flat shape trait in peach

Abstract

In peach, the flat phenotype is caused by a partially dominant allele in heterozygosis (Ss), fruits from homozygous trees (SS) abort a few weeks after fruit setting. Previous research has identified a SSR marker (UDP98-412) highly associated with the trait, found suitable for marker assisted selection (MAS). Here we report a ∼10 Kb deletion affecting the gene PRUPE.6G281100, 400 Kb upstream of UDP98-412, co-segregating with the trait. This gene is a leucine-rich repeat receptor-like kinase (LRR-RLK) orthologous to the Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) group. PCR markers suitable for MAS confirmed its strong association with the trait in a collection of 246 cultivars. They were used to evaluate the DNA from a round fruit derived from a somatic mutation of the flat variety 'UFO-4', revealing that the mutation affected the flat associated allele (S). Protein BLAST alignment identified significant hits with genes involved in different biological processes. Best protein hit occurred with AtRLP12, which may functionally complement CLAVATA2, a key regulator that controls the stem cell population size. RT-PCR analysis revealed the absence of transcription of the partially deleted allele. The data support PRUPE.6G281100 as a candidate gene for flat shape in peach.

Figure 1

Round and flat associated haplotypes in round (R), flat (F) and aborting (A) peaches. Colon represents the deletion of a nucleotide. The haplotypes consist of 13 SNPs and two INDELS, in Amplicon5 and Amplicon6 (Pp06: 26,270,679.26,271,829) (see Supplementary Table S1).

Figure 2

PCR bands reveal the deletion affecting the gene PRUPE.6G281100 in aborting and flat peaches. (a) PC1 failed to amplify the flat-associated allele. (b) Long-range PCR amplification with PC2 produced a fragment about 10 kb shorter in flat and aborting than in round peaches. (c) PCR-amplification with PC3 identified round (941pb), flat (1620/941 bp) and aborting (1620 bp) genotypes. (d) Diagrammatic representation of the position of the primers used to identify the polymorphisms associated with flat shape and the polymorphisms (SNPs and small INDELs) in PRUPE.6G281100, represented as dots and triangles (respectively).

Figure 3

Alignment of round and flat peaches reads against Pp06:26,262,400.26,264,250 region. CLC-Workbench track display including (a) Pp06:26262400.26264250 region Prupe.6G281100, (b) bulked alignment of Illumina reads from five round and (c) five flat peaches. Blue areas represent the sequence depth at each position. The reduction in the number of reads in the flat peaches reveals the deletion in heterozygosis. An increase in the number of reads in the region Pp06:26,262,400.26,264,250 (labeled in the figure with a*) is produced by the spurious alignment (confirmed by Sanger sequencing) of a highly repetitive region. (d) The CLC- InDels and structural variants tool identified the two indels in the flat varieties only.

Figure 4

RT-PCR of RNA from round, flat and aborting pistils. (a) Pistil shape observed in flower buds in stage E. On RT-PCR amplification of round, flat and aborting pistils using PC4 no amplification of the flat-associated allele was visible in both (b) agarose and (c) capillary electrophoresis.

Figure 5

Analysis of a flat variety (‘UFO-4’) and its somatic round mutant (‘UFO-4Mut’). (a) Image of the flat (left) and round (right) pistils and fruits. (b) PCR-amplification products obtained with PC4 to detect the two small INDELs associated with the flat trait. PCR reactions were carried out with DNA extracted from leaf, as well as from skin, flesh and stone fruit tissues. The peaks show that the mutation occurred in flesh tissue (meristematic layer LII) affecting the flat associated allele.