Fine Mapping Of BhAPRR2,A Gene Controlling Green Or White Peel Color Of Wax Gourd

Wax gourd has a wide cultivation area and a long cultivation history in China.It is one of the important vegetables transported from south to North in China.As a fresh vegetable,wax gourd peel color has become one of the important appearance qualities affecting its commercial value.Many Cucurbitaceae crops have made in-depth research on peel color,but the research on peel color of wax gourd lags behind obviously,and the related genes are rarely cloned.In this study,two hybrid combinations of green and wax gourd（GX-71×MY-1,KX-2×YO-16）were used to construct F₂ and backcross populations,explore the genetic law of skin color of wax gourd,preliminarily locate the regulatory genes through bulked segregant analysis（BSA）,compress the candidate interval by KASP gene typing technology,and mine the candidate genes regulating skin color.It lays the foundation for deeply exploring the molecular mechanism of regulating the color of the peel of wax melon.The main results are as follows:1.The F₁,F₂ and BC₁P₂ populations were obtained by using GX-71×MY-1and KX-2×YO-16 to explore their genetic regulations.The two crosses F₁ were found to be all green,F₂,BC₁P₂ showed segregation of the trait.The ratio of green skinned winter melon（930 strains）to white skinned winter melon（314 strains）in the F₂ of the GX-71×MY-1 hybrid combination was close to the 3:1 separation ratio（χ²=0.039<χ²=3.84）,and the ratio of green skinned winter melon（238 strains）to white skinned winter melon（234 strains）in BC₁P₂ was close to the 1:1 trait segregation ratio（χ²=0.034<χ²=3.84）;The separation ratio of green skinned winter melon（180 strains）to white skinned winter melon（64 strains）in F₂ of the KX-2×YO-16 hybrid combination was close to 3:1（χ²=0.197<χ²=3.84）,and the ratio of green skinned wax gourd（118 strains）to white skinned winter melon（115strains）in BC₁P₂ was close to the 1:1 trait segregation ratio（χ²=0.039<χ²=3.84）.It can therefore be speculated that genes regulating peel color are mainly controlled by single gene.2.Two extreme admixed pools were constructed by combining 30 extreme green skinned individuals from the F₂ populations with 30 extreme white skinned individuals from GX-71×MY-1 and KX-2×YO-16,and the genotype frequencies of the two admixed pools with the parental pool were analyzed by association algorithm,and the candidate gene controlling peel color was mapped to a 26 Mb interval on Chr 5 by GX-71×MY-1,which,KX-2×YO-16 maps a gene controlling peel color to a 12.6 Mb interval on Chr 5.3.The F₂ population of GX71×MY-1 was expanded to 5000 strains,In total,17 pairs of KASP markers were used to perform a combined genotype phenotype analysis on the F₂ population,and the wax gourd skin color gene was finally mapped between marker PS13.4 and PS13.6 within the common 179 Kb region,there is only one APRR2 gene encoding a two-component response regulator,which we renamed Bh APRR2.4.Sequence analysis of the Bh APRR2 gene identified a deletion of t wo critical base GA in the coding sequence in white skinned versus gree n skinned melon,a frameshift mutation in the CDS region and a prematu re termination of protein translation were identified in white skined wax g ourd,335 amino acids deletion in white skinned versus green skinned wa x gourd resulted in the deletion of the PLN03162 domain associated with chloroplast development,and a change in the protein structure as well.Thu s,the greatly reduced chlorophyll accumulation in white skinned wax gou rd.The expression of Bh APRR2 in green skinned wax gourd was significa ntly higher than that in white skinned wax gourd,and its expression exhi bits extreme tissue specificity in pericarp.5.Based on the base sequence differences between the parents,we designed a pair of CAPS markers.Forty inbred accessions of wax gourd（25 accessions of green skins+15 accessions of white skins）were used to examine the validity of CAPS markers.The results showed that the bands of 25 accessions of green skinned winter melon were consistent with paternal GX-71 and KX-2,while the bands of 15 accessions of white skinned wax gourd were consistent with maternal MY-1 and YO-16.CAPS markers achieved 100%accuracy.