西瓜少侧枝性状的分子标记开发与辅助育种研究

Development of molecular markers and marker-assisted breeding for sparse lateral branching in watermelon

摘要

【目的】基于已知的西瓜少侧枝 Clbl基因及其关键C/A SNP位点，开发高通量实用型KASP标记，建立多世代分子辅助育种体系，实现少侧枝优质材料创制与新品种培育。【方法】以少侧枝材料ZXG1361（供体）和正常侧枝优良材料FLW03Z01、FLW03Z02（受体）为亲本，构建F1、F2遗传群体，通过表型鉴定、基因测序、KASP标记开发及F2→F4多世代筛选开展研究。【结果】开发的KASP标记FLB-K1在2个F2群体中表型-基因型吻合率达98.28%~98.97%，通过FLB-K1辅助筛选获得5份纯合材料，核心材料SC-3711-③与SC-5352-①农艺性状突出，组配的少杈1号定植45 d单株侧枝数2~5条，中心可溶性固形物含量（ w，后同）为12%，与对照品质相当，但节省整枝成本。【结论】成功开发针对西瓜少侧枝性状的KASP标记FLB-K1，该标记精准高效、适配高通量筛选；利用该标记快速创制5份优良纯合材料，培育出少侧枝新品种少杈1号，达成了分子标记技术与育种实践的深度结合。

Abstract

【Objective】This study attempted develop a high-throughput kompetitive Allele-Specific PCR (KASP) marker targeting the sparse lateral branching in watermelon ( Citrullus lanatus), systematically 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 varieties suitable for industrial cultivation. This study aims to reduce the high cost of manual pruning and improve the low efficiency of traditional breeding, both of which are caused by excessive lateral branching, and thereby accelerate the breeding of watermelon varieties with an ideal plant architecture. 【Methods】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 parental 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-generational 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 soluble solids ≥10.5%). Two elite F4 lines (SC-3711-③ and SC-5352-①) were crossed to breed hybrid Shaocha 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 ( χ ²=0.012, P=0.913) and 3.11∶1 ( χ ²=0.085, P=0.770) for normal vs. reduced lateral branches plants, respectively, indicating control by a single recessive 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 controls. 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 enzyme 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 significantly 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-3711-③ had 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 central 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 after 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 thickness of 0.7-1.0 cm, with comprehensive agronomic traits comparable to the control, while showing obvious advantages in simplified cultivation. 【Conclusion】The developed FLB-K1 marker enables accurate, 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 problem of poor quality in existing reduced lateral branch materials. The bred hybrid Shaocha No.1 combines 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.

关键词

西瓜 / 少侧枝性状 / Clbl基因 / KASP标记 / 分子辅助育种

Key words

Citrullus lanatus / Sparse lateral branching trait / Clbl gene / KASP marker / Molecular marker-assisted breeding

参考文献

[1] Drummond R S M, Janssen B J, Luo Z W, Oplaat C, Ledger S E, Wohlers M W, Snowden K C . Environmental control of branching in petunia[J]. Plant Physiology, 2015, 168(2): 735-751.

[2] Zhang L, Yu H, Ma B, Liu G F, Wang J J, Wang J M, Gao R C, Li J J, Liu J Y, Xu J, Zhang Y Y, Li Q, Huang X H, Xu J L, Li J M, Qian Q, Han B, He Z H, Li J Y . A natural tandem array alleviates epigenetic repression of IPA1 and leads to superior yielding rice[J]. Nature Communications, 2017, 8: 14789.

[3] Zhao T C, Wang J B, Li R Y, Zhang P F, Guo X Y, Qi Y C, Li Y S, Cheng S H, Ji J C, He A B, Ai Z Y . Effects of nitrogen fertilizer types and planting density on the yield and nitrogen use efficiency of salt-tolerant rice under salt stress conditions[J]. Plants, 2025, 14(4): 501.

[4] Schmitz G, Theres K . Shoot and inflorescence branching[J]. Current Opinion in Plant Biology, 2005, 8(5): 506-511.

[5] Van Es S W, Muñoz-Gasca A, Romero-Campero F J, González-Grandío E, De Los Reyes P, Tarancón C, Van Dijk A D J, Van Esse W, Pascual-García A, Angenent G C, Immink R G H, Cubas P . A gene regulatory network critical for axillary bud dormancy directly controlled by Arabidopsis BRANCHED1[J]. New Phytologist, 2024, 241(3): 1193-1209.

[6] Ai L, Qiu J, Wang J G, Qian M Y, Liu T T, Cao W, Xing F Y, Gul H, Zhang Y Y, Gong X L, Li J, Duan H, Xiao Q L, Liu Z Z . A naturally occurring 31 bp deletion in TEOSINTE BRANCHED1 causes branched ears in maize[J]. Journal of Integrative Agriculture, 2025, 24(9): 3322-3333.

[7] Liu Y T, Gao X, Liu H J, Yang X R, Liu X, Xu F, Zhu Y Z, Li Q Y, Huang L L, Yang F, Lai J S, Shi J P . Constraint of accessible chromatins maps regulatory loci involved in maize speciation and domestication[J]. Nature Communications, 2025, 16: 2477.

[8] Song X G, Lu Z F, Yu H, Shao G N, Xiong J S, Meng X B, Jing Y H, Liu G F, Xiong G S, Duan J B, Yao X F, Liu C M, Li H Q, Wang Y H, Li J Y . IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice[J]. Cell Research, 2017, 27(9): 1128-1141.

[9] Ikeda K, Nagasawa N, Nagato Y . ABERRANT PANICLE ORGANIZATION 1 temporally regulates meristem identity in rice[J]. Developmental Biology, 2005, 282(2): 349-360.

[10] Ikeda-Kawakatsu K, Yasuno N, Oikawa T, Iida S, Nagato Y, Maekawa M, Kyozuka J . Expression level of ABERRANT PANICLE ORGANIZATION1 determines rice inflorescence form through control of cell proliferation in the meristem [J]. Plant Physiology, 2009, 150(2): 736-747.

[11] Dou J L, Yang H H, Sun D L, Yang S, Sun S R, Zhao S J, Lu X Q, Zhu H Y, Liu D M, Ma C S, Liu W G, Yang L M . The branchless gene Clbl in watermelon encoding a TERMINAL FLOWER 1 protein regulates the number of lateral branches[J]. Theoretical and Applied Genetics, 2022, 135(1): 65-79.

[12] Kalendar R, Shustov A V, Akhmetollayev I, Kairov U . Designing allele-specific competitive-extension PCR-based assays for high-throughput genotyping and gene characterization [J]. Frontiers in Molecular Biosciences, 2022, 9: 773956.

[13] Potts J, Michael V N, Meru G, Wu X B, Blair M W . Dissecting the genetic diversity of USDA cowpea germplasm collection using kompetitive allele specific PCR-single nucleotide polymorphism markers[J]. Genes, 2024, 15(3): 362.

[14] Suo W, Shi X J, Xu S, Li X, Lin Y . Towards low cost, multiplex clinical genotyping: 4-fluorescent Kompetitive Allele-Specific PCR and its application on pharmacogenetics[J]. PLoS One, 2020, 15(3): e0230445.

[15] Rehman S U, Ali Sher M, Saddique M A B, Ali Z, Khan M A, Mao X G, Irshad A, Sajjad M, Ikram R M, Naeem M, Jing R L . Development and exploitation of KASP assays for genes underpinning drought tolerance among wheat cultivars from Pakistan[J]. Frontiers in Genetics, 2021, 12: 684702.

[16] Kaur R, Dhillon G S, Kaur A, Kaur S, Toor P I, Kaur D, Kumar A, Mavi G S, Grewal S K, Sharma A, Srivastava P, Chhuneja P, Kaur S . Marker-assisted introgression of genes into rye translocation leads to the improvement in bread making quality of wheat (Triticum aestivum L.) [J]. Heredity, 2022, 128(6): 531-541.

[17] Miedaner T, Korzun V . Marker-assisted selection for disease resistance in wheat and barley breeding[J]. Phytopathology®, 2012, 102(6): 560-566.

[18] Ren Y, Li M Y, Guo S G, Sun H H, Zhao J Y, Zhang J, Liu G M, He H J, Tian S W, Yu Y T, Gong G Y, Zhang H Y, Zhang X L, Alseekh S, Fernie A R, Scheller H V, Xu Y . Evolutionary gain of oligosaccharide hydrolysis and sugar transport enhanced carbohydrate partitioning in sweet watermelon fruits[J]. The Plant Cell, 2021, 33(5): 1554-1573.

[19] Deng Y, Liu X, Liu S C, Li X N, Xue L F, Bai T, Xu B H, Li G Q, Sun Y D, Zhang X P . Fine mapping of ClLOX, a QTL for powdery mildew resistance in watermelon (Citrullus lanatus L.) [J]. Theoretical and Applied Genetics, 2024, 137(3): 51.

基金资助

国家西瓜甜瓜育种联合攻关任务(农种发〔2022〕5号)

2023年安徽省西瓜甜瓜良种联合攻关项目(合农〔2023〕41号)

合肥市农业行业首席专家工作室项目(202319)