西瓜少侧枝性状的分子标记开发与辅助育种研究OA

[Objective]This study attempted develop a high-throughput kompetitive Allele-Specific PCR(KASP)marker targeting the sparse lateral branching in watermelon(Citrullus lanatus),systemati-cally validated its specificity,stability and applicability,and applied it to marker-assisted breeding(MAS)to create elite lines with reduced lateral branches and high quality,as well as to breed new variet-ies suitable for industrial cultivation.This study aims to reduce the high cost of manual pruning and im-prove the low efficiency of traditional breeding,both of which are caused by excessive lateral branch-ing,and thereby accelerate the breeding of watermelon varieties with an ideal plant architecture.[Meth-ods]Donor ZXG1361(homozygous mutant AA,reduced lateral branches)and recipients FLW03Z01/FLW03Z02(homozygous wild-type CC,normal lateral branches)were used to construct F1,F2,F3,and F4 populations,grown at two experimental stations(2023-2025)under uniform protected cultivation.Lateral branch number(≥2 cm),plant height,and internode number were investigated at 30 and 45 days after transplanting;chi-square test verified F2 segregation ratio,and T-test(α=0.05)analyzed paren-tal phenotypic differences.Genomic DNA was extracted from cotyledons via CTAB method.Based on the C/A SNP at position 1334 of the Clbl gene,KASP marker FLB-K1 was developed,comprising FAM-labeled primer for CC genotype,HEX-labeled primer for AA genotype,and a common reverse primer.PCR was performed in 384-well plates;fluorescence signals were detected with a Pherastar scanner,and genotype-phenotype concordance was analyzed via Fisher's exact test.A multi-generation-al MAS system was established:F2 seedlings with AA genotype were selected by FLB-K1,followed by phenotypic(lateral branch number≤7 per plant)and quality(central soluble solids≥10%)screening;F3/F4 populations were further screened for stable traits(lateral branch number≤5 per plant,central solu-ble solids≥10.5%).Two elite F4 lines(SC-3711-③and SC-5352-①)were crossed to breed hybrid Shao-cha No.1,with Zhongmi No.1 as control for trait comparison.[Results]F1 plants exhibited normal branching,confirming dominant inheritance of the normal trait.The F2 populations of AF2(693 plants)and BF2(390 plants)showed segregation ratios of 3.03∶1(χ2=0.012,P=0.913)and 3.11∶1(χ2=0.085,P=0.770)for normal vs.reduced lateral branches plants,respectively,indicating control by a single reces-sive gene.FLB-K1 achieved clear and distinct genotyping in F2 populations,with blue clusters for CC genotype,red for AA genotype,green for CA genotype,and no cross-contamination with blank con-trols.Genotype-phenotype concordance rates reached 98.28%(681/693)in AF2 and 98.97%(386/390)in BF2,with un-typable samples accounting for only 0.8%and 0.5%respectively,demonstrating high specificity and stability.Compared with traditional dCAPS markers,FLB-K1 eliminated restriction en-zyme digestion and electrophoresis steps,avoiding misjudgment caused by incomplete digestion,and shortened the screening cycle from over 30 days after transplanting to a few days within the 1-leaf stage,and improved detection throughput by 4 times via 384-well plates compared with 96-well plates for dCAPS markers.Multi-generational MAS maintained 100%AA genotype homozygosity across F2 to F4 generations.F4 lines showed stable traits of sparse lateral branching(2-5 per plant)and significant-ly improved quality,with central soluble solids of 10.5%-13%,which was 2.5%-4%higher than donor ZXG1361(8%-9%).Five elite homozygous lines were successfully created,among which SC-3711-③had 2-4 lateral branches per plant,a fruit weight of 5.4-6.5 kg,a peel thickness of 0.6-0.8 cm,and cen-tral soluble solids up to 13%;SC-5352-① had 2-5 lateral branches per plant,a fruit weight of 5.3-5.9 kg,and central soluble solids 11%-12%;Shaocha No.1 had 2-5 lateral branches per plant at 45 days af-ter transplanting,which was significantly fewer than the control Zhongmi No.1(19-25 branches per plant,P＜0.001),completely eliminating the need for manual pruning.Its central soluble solids reached≥12%,fruit weight 4.0-6.5 kg.It had tender flesh with medium fiber content,and a peel thick-ness of 0.7-1.0 cm,with comprehensive agronomic traits comparable to the control,while showing ob-vious advantages in simplified cultivation.[Conclusion]The developed FLB-K1 marker enables accu-rate,high-throughput genotyping for the watermelon with sparse lateral branch trait at the seedling stage,displaying superior performance to traditional dCAPS markers in operation simplicity,detection efficiency and stability.Application of FLB-K1 in multi-generational MAS successfully yielded five elite homozygous lines integrating traits of sparse lateral branching and high quality,solving the prob-lem of poor quality in existing reduced lateral branch materials.The bred hybrid Shaocha No.1 com-bines excellent quality traits and simplified cultivation advantages,meeting the demand for industrial watermelon production.This marker and the corresponding multi-generational MAS system effectively enhanced the breeding efficiency of watermelon ideal plant architecture,providing important variety support and technical guarantee for simplified cultivation of watermelon.