Germplasm and molecular breeding in horticultural crops

Horticulture is an important part of agricultural planting and production, which is of great significance for enriching human nutrition and beautifying and transforming the human living environment.At present, the area of horticultural crops in China is about 40 million ha, accounting for about onefourth of the national crop planting area, while the production of primary agricultural products is 1 billion tons, and the output value accounts for more than half of the total output value of the planting industry.Traditional cash crops such as fruits, vegetables, melons and tea occupy an important position in the world’s horticultural industry.In addition,the diversity of horticultural germplasm resources in China makes it convenient to exploit the great characteristics of our horticultural crops and cultivated new varieties.

In the past 40 years, the breeding of horticultural crops has mainly depended on traditional crossing methods, which has also caused many problems such as a slow breeding process, a long cycle for obtaining new varieties, and an unclear explanation of the mechanisms of superior traits.With the rapid development of science and technology in recent years, genomics, proteomics,metabolomics, transcriptomics, epigenetics and other bioinformatics techniques are now widely applied in studies on the complex traits of horticultural crops, providing unprecedented opportunities to better understand the origin and domestication of horticultural plants, as well as deciphering the genetic regulatory mechanisms of important traits, and developing highly efficient molecular breeding techniques.Taking advantage of this opportunity, it is a great honor to be invited byJournal of Integrative Agricultureto briefly review the applications of biotechnology in horticultural crop breeding, in order to provide a reference for the subsequent research on horticultural crop breeding.

Fruit

Fruit crops are an important part of the agricultural cultivation industry, and they provide a major source of nutrition for human beings.Mechanism studies in fruit crops have been difficult because of their long juvenile phase and generation cycle (Wuet al.2023).However, recent advances in genomics have shed new light on various aspects of fruit crop biology (Wang R Zet al.2023).In 2007, Jaillonet al.(2007) sequenced the whole genome of grape based on Sanger sequencing technology, and it was the first fruit tree genome sequenced.Subsequently, with the continuous development of sequencing technology and assembly methods, the whole genome sequencing of various fruits has been enriched.Currently, sequencing technology has progressed to the third generation, and 163 genomes of 91 different fruit crops have been sequenced (Wang R Zet al.2023).These large accumulations of public genomic resources have made important contributions to reveal the genetic origins and domestication of fruit trees, as well as deciphering the genetics of important horticultural traits in these fruit trees (Chenet al.2019).

Actinidia erianthahas been widely used in the hybrid breeding and genetic improvement of kiwifruit because of its high ascorbic acid (AsA) content, easy peeling of the fruit skin, heat resistance, disease resistance and storage resistance (Liaoet al.2021).Liaoet al.(2023) used the third-generation sequencing technology PacBio and Hi-C assembly technology to assemble the genome of the new kiwifruit cultivar ‘Ganlv 1’ and obtained a high-quality kiwifruit reference genome with only five gaps.Subsequent metabolome and transcriptome analyses allowed them to determine that the AsA content was highly correlated with ascorbate peroxidase genes (APX).In conclusion, the latest published genome not only provides genomic resources for kiwifruit genetic breeding research, but the regulatory network constructed will also provide a public data platform for future kiwifruit research.

As an important economic crop, the quality of the fruit produced by fruit trees directly determines the economic benefits.Color plays a crucial role in the appearance and acceptability of fruits.Anthocyanins, as important determinant of color, confer a variety of fruit colors, from red to purple and blue, which help to attract seed dispersers and protect the fruits from a variety of biotic and abiotic stresses(Landiet al.2015).Over the past few decades, a great deal of effort has been made to uncover the mechanisms by which development induces anthocyanin accumulation(Zhaoet al.2023).The results show that anthocyanin accumulation is a surprisingly complex process mediated by multiple networks that are driven by genetic, developmental,hormonal, and environmental factors (Zhaoet al.2023).Transcriptional and epigenetic regulation are the main molecular frameworks for anthocyanin biosynthesis.Among them, the MYB-bHLH-WD40 (MBW) complex plays a crucial role in the regulation of anthocyanin accumulation at the transcriptional level, in which MYB-TF is the core regulatory factor, and other ‘strengthening’ members are also involved (Liuet al.2015; Xuet al.2015; Sunet al.2020).Other transcription factors such as NAC, WRKY,and bZIP are involved in the coordinated regulation of anthocyanin accumulation by regulating the activity of the MBW complex (Zhaoet al.2023).LONG HYPOCOTYL 5(HY5), which encodes a basic leucine zipper (bZIP) TF, is a key light-inducible TF for photomorphogenesis in plants(Changet al.2008).InArabidopsisthaliana, HY5 promotes anthocyanin biosynthesis by directly regulatingCHS,CHI,F3H,F3′H,DFR, andANS(Jeonget al.2010).

The red coloring of pear fruits is mainly caused by anthocyanin accumulation.Red color bud mutation,represented by the green pear cultivar ‘Bartlett’ (BL) and the red-skinned derivative ‘Max Red Bartlett’ (MRB), is an ideal material for studying the molecular mechanism of anthocyanin accumulation in pear.Weiet al.(2023)revealed that PcHY5 methylation levels were lower in MRB than in BL and PcHY5 expression levels were higher in MRB than in BL during fruit development by transcriptomic and methylomic analyses, and these results indicated that PcHY5 is involved in the skin color changes between BL and MRB.Their analysis also identified a key differentially methylated site in the intronic region of PcHY5 that was significantly associated with the color differences between MRB and BL.In addition, the authors further verified that PcHY5 activates the promoters of the anthocyanin biosynthesis and transport genesPcUFGT,PcGST,PcMYB10, andPcMYB114using a dual-luciferase assay, thereby confirming that PcHY5 regulates not only the biosynthesis of anthocyanins, but also the transport of anthocyanins.In conclusion, the authors suggest that ‘Red Bar Pear’ PcHY5 promotes the expression of its own genes through DNA hypomethylation levels, which regulates the expression of the genes related to anthocyanin synthesis and transport, thereby promoting peel coloration.

Fruit trees are mostly perennial woody plants, and factors such as long breeding cycles and complex heritability make genetic breeding research on fruit trees slower and more difficult (Wuet al.2023).In the past, fruit breeding methods mainly relied on conventional sexual hybridization,supplemented by artificial mutation or seed selection (Wang 2018).In recent decades, some new technologies have been applied to the genetic improvement of fruit crops, which has improved the breeding efficiency.These technologies mainly include cell engineering technologies and molecular marker-assisted selection (MAS) technologies (De Mori and Cipriani 2023).

The application of MAS technology is relatively recent,and it can be roughly divided into three generations according to its development history (Songet al.2021).The first generation is based on molecular hybridization technology,such as restricted fragment length polymorphism (RFLP);while the second generation is based on PCR technology,including sequence trait loci (STS), simple repeat sequence(SSR), amplified fragment length polymorphism (AFLP),etc.With the advent of the post-genomic era, the third generation of molecular markers came into being, such as single nucleotide polymorphism (SNP) (Nadeemet al.2018).Compared with the previous two generations, SNP typing has the characteristics of a higher distribution density and better genetic stability, making it easier to achieve high throughput and automated detection.Competitive allelespecific PCR (KASP) is a novel genotyping technique based on SNPs, which can accurately type SNPs and insertiondeletion polymorphisms (Indels) at the genome level.KASP is flexible, cheap and accurate, and has been widely used in the molecular marker-assisted breeding of crops (Semagnet al.2014).

The grape (VitisL.) is one of the most important fruit crops in the world.Seedless grape cultivars are increasingly popular with consumers and producers due to their convenience, making them a key target for grape breeding programs (Varoquauxet al.2000).Among the techniques,accurate and efficient molecular marker breeding is an important way to improve grape breeding efficiency and reduce the related costs (Benniciet al.2019).At this point,several grape seedless molecular markers have been developed, such as random amplified polymorphic DNA(RAPD) markers, sequence feature amplification region(SCAR) markers, and SSR markers (Bergaminiet al.2013).However, there are still some issues with these markers, such as high false positive rates and susceptibility to experimental conditions.Wang F Qet al.(2023)constructed KASP_VviAGL11 and VviAGL11_410 markers based on a single base mutation site of theVviAGL11gene(chr18:26889437 (A/C)), and tested them on 101 grape cultivars and 81 grape F1hybrids.The results showed that both the KASP_VviAGL11 and VviAGL11_410 markers had 100% accuracy rates in detecting allele A.In conclusion,that study optimized the molecular marker-assisted selection breeding process of seedless grape with KASP_VviAGL11 as the core, providing key technical support for accelerating the breeding process of new seedless grape cultivars.

Vegetables

Vegetable breeding is essentially a process of the continuous aggregation of beneficial genes, and variation is important for breeding innovation.Since the 1970s, biotechnology has been applied to transform the genetic characteristics of vegetable crops through the discovery and selection of variations.In recent years, with the rapid development of biotechnology, omics technologies have gradually become powerful tools for precision design breeding to meet various breeding objectives.

Chinese cabbage (Brassica rapaL.ssp.pekinensis)belongs to theBrassicaspecies in the Cruciferae family and is one of the most widely cultivated vegetable crops in Asia.In recent years, clubroot, which is caused by the soil-borne protist plant pathogenPlasmodiophora brassicae, has resulted in great yield losses and economic benefit reductions in Chinese cabbage.Therefore,studies have increasingly focused on breeding work and basic research on clubroot resistance in recent years.In the process of vegetable disease resistance breeding, RNAseq has played key roles in mining relevant information on the important genes, pathways and molecular biomarkers that respond to plant diseases.InBrassicaspecies, the genes, involved in jasmonate, ethylene, hormone signaling,cell wall modification, NBS-LRR proteins, Ca2+signaling,defense-related callose deposition, chitin metabolism and other traits, have been reported to be involved in clubroot resistance by RNA-seq analysis (Chuet al.2014; Zhanget al.2016; Ninget al.2019).However, due to the short lengths of the sequencing reads and unreliable assembly results from the second-generation sequencing technology,the accuracy of transcriptome abundance calculations is greatly reduced.Therefore, these results require further verification by the third-generation sequencing techniques.In this special focus, PacBio RS II SMRT sequencing was applied to generate full-length transcriptomes of mixed roots afterP.brassicaeinfection in the clubroot-resistant line DH40R, and the key genes and metabolic pathways of clubroot resistance in Chinese cabbage were identified (Suet al.2023).These results will enrich the genome annotation and provide valuable resources for basic research on clubroot resistance in Chinese cabbage.

In recent years, nutritional composition and quality have become important breeding goals for Chinese cabbage.Carotenoids are a large group of secondary metabolites that are important nutrients, generally absorb blue and green light, and provide coloration ranging from yelloworange to red in the leaves (Rodriguez-Concepcionet al.2018).Compared with other Chinese cabbage, the orange heading Chinese cabbage is rich in carotenoids and provide higher nutritional value to consumers (Watanabeet al.2011;Zhanget al.2015).However, the regulatory mechanisms of carotenoid production are still unknown in the orange heading Chinese cabbage.Therefore, in this special focus,a paper contributed by Zhanget al.(2023) showcases the latest research on carotenoid accumulation in the orange heading Chinese cabbage by RNA sequencing.Their results showed that blue light induces significant up-regulation of the total carotenoid content and the expression levels of genes related to the carotenoid metabolic pathway in orange heading Chinese cabbage.These results provide a reference for the selection breeding and cultivation of orange Chinese cabbage.

Cucumber is a model plant in the Cucurbitaceae family for studying unisexual flowers.Unisexual flowers can effectively promote plant outcrossing and increase genetic diversity.In the process of crossbreeding, there are great advantages to use androecious plant materials as male parents.Planting androecious plants around gynoecious plants, followed by insect pollination and mechanized harvesting, can significantly reduce seed production costs.Therefore, more androecious mutants and lines with different genetic backgrounds are needed to better exploit heterosis for a wider selection of breeding traits.At the same time,parents with excellent traits can be cultivated with the help of molecular marker-assisted breeding.There are currently two reported cucumber androecious materials,namederezand406a.erezis a natural mutant material in which the mutation is a premature stop codon due to a base substitution in the CsACS11 coding region, resulting in a loss of catalytic activity and only male flowers (Boualemet al.2015).Line406ais an androecious mutant discovered in the mutant library from EMS-induced mutagenesis of cucumber inbred line ‘406’, and it carries a mutation of CsACO2 that causes only male flowers to bloom (Chenet al.2016).A new material with only male flowers was discovered in the‘406’ mutagenesis library.It is due to a base substitution in the CsACS11 coding region that leads to the loss of catalytic activity, and this mutation site differs from the one inerez(Wang Jet al.2023).This new androecious mutant was screened using molecular markers, which enriched the mutation sites that can be used in the process of breeding new androecious parents.

Cucurbita pepoL.is also a member of the Cucurbitaceae family, and female flowering time is an important yieldrelated trait.This trait will affect the time to market and yield ofC.pepo, which then directly affects its early economic benefits (Alleshet al.2019).During the flowering and fruiting period, short daylight hours, low night temperatures and mechanical stimulation can promote the differentiation of female flowers, reduce the position of the first female flower node, appropriately shorten the flowering time and increase the number of female flowers per plant, which increase the early and total yield ofC.pepo.Flowering-related traits are mostly quantitative characters that are easily influenced by the environment (Montero-Pauet al.2017; Xanthopoulouet al.2019).A QTL analysis was conducted on the F2population constructed from late-flowering and early-flowering inbred lines in zucchini, which is a type ofC.pepo, and a total of three QTL locus were identified (Quet al.2023).Molecular markers were then used to determine the major QTL,which was located in a 117 kb region on chromosome 20.Moreover, a possible candidate gene associated with early flowering was identified.Molecular markers closely linked to the major QTL were used to assist in providing technical support for further breeding of early-flowering varieties.

Melons

Melons occupy an important position in horticulture crops.The cultivation of melons in China has a history of more than 3 000 years, and China is one of the first countries to cultivate this fruit (Liuet al.2020).China also has the largest cultivation area and highest production of melons in the world (FAO 2021).

Melons have various features, especially texture, color and shape.Many economically important traits of melons,such as fruit length (FL), fruit diameter (FD) and fruit firmness (FF), are controlled by QTLs.FF is one of the key traits for melon quality, consumer acceptance and fruit transportability.Several QTLs associated with FF have been identified in melon (Pereiraet al.2020), and these QTLs are located across all 12 melon chromosomes.Next-generation sequencing (NGS) technology, including bulked segregant analysis (BSA) and genome-wide association studies(GWASs) techniques, have been widely used to detect the major QTLs in melon.Specific length amplified fragment sequencing (SLAF) is a new and efficient method for constructing high-resolution genetic maps.More recently,the QTLs of gummy stem blight (GSB) resistance genes were identified using SLAF sequencing (SLAF-seq) and BSA techniques, and flowering-related QTLs were identified in melon using SLAF-seq (Gaoet al.2020).

Chenet al.(2023) produced F2s by crossing P5 melons that have soft fruits with P10 melons that have hard fruits,and recorded the FF and fruit related traits for 2 years.By performing QTL-SLAF sequencing and molecular markerlinkage analysis, 112 844 SLAF markers were identified,and 5 919 SNPs were used to construct a genetic linkage map with a total genetic distance of 1 356.49 cM.For FF, the consistent major locus (ff2.1) was located in a 0.17 Mb candidate region on chromosome 2.Using 429 F2individuals derived from a cross between P5 and P10, the authors resolved theff2.1locus to a 28.3 kb region harboring three functional genes.These results not only provide a new candidate QTL for melon FF breeding but also a theoretical foundation for research on the mechanisms underlying melon gene function.

Tea

Tea is one of the most popular non-alcoholic beverages in the world.In addition to its pleasant aroma and attractive taste, tea has numerous health and medical benefits for humans due to its many characteristic secondary metabolites, including polyphenols, caffeine, theanine,and volatile compounds (Songet al.2018).The chemical composition of tea is highly dependent on the cultivar and environmental factors (Fanget al.2021).In recent years,targeted metabolomics combined with high-throughput transcriptomic technology has been widely used to explore tea flavor-related genes and metabolites (Fanet al.2021).Gaoet al.(2023) analyzed the non-volatile metabolites of seven tea plant cultivars using liquid chromatography–electrospray ionization–tandem mass spectrometry and determined the transcriptional regulatory network of these characteristic metabolites by using a WGCNA-based system biological strategy.Guoet al.(2023) measured the free amino acid contents in fresh leaves of 174 tea accessions using a targeted metabolomics approach and conducted a genome-wide association study based on the genotype data of these tea accessions, providing new insights into the genetic basis of variations in the amino acid contents in tea plants.

Conclusion and research prospect

Germplasm resources are formed through a long process of natural and artificial selection, and they carry a variety of genes which are indispensable materials for the breeding of new varieties.Thus, identifying the germplasms with desirable traits and useful genes will be beneficial for integrated breeding.In previous studies, although many wild, landraces and cultivars of different horticultural crops had been collected, their systematic and accurate evaluation was still limited, which affects the utility of the germplasm to some extent.In the future, with the development of high throughput phenotyping methods and compound detection techniques including metabolomics, the highly reliable phenotyping of germplasm will help us to select the right parents with elite genes for new varieties breeding.

In recent years, high-quality genomics have provided invaluable resources for sequence information, gene discovery, fine-targeting, tool development, and mutation detection.These tools have had a significant impact on our understanding of trait biology, gene function,and genetic regulation.At the same time, genome resequencing efforts have provided innovative molecular breeding approaches that can provide new knowledge about taxonomy, phylogenetic relationships, evolutionary history, domestication, and genetic diversity.With the rapid development of horticultural crop genome sequencing,genome-wide DNA markers including SNPs have been identified and developed.The availability and use of molecular markers and genomic selection can significantly help us to overcome some of the limitations of traditional breeding, providing new opportunities for the highly efficient and reliable prediction and selection of progenies with high quality and high yield, as well as disease resistance, and they can also significantly shorten the breeding cycles in horticultural crops.Meanwhile, the use of integrated multiomics approaches, including genomics, transcriptomics,epigenomics, proteomics, metabolomics, and phenomics,can not only improve our understanding of crop development and quality traits, but also facilitate the application of this knowledge into the breeding process of horticultural crops.Furthermore, gene editing is superior to other methods in terms of versatility, efficiency and specificity, and it can be adopted as a useful way to breed new varieties and improve existing varieties in the future.

Prof.WU Jun Member of Editorial Board of JIA/Guest Editor College of Horticulture, Nanjing Agricultural University,Nanjing 210095, P.R.China E-mail: wujun@njau.edu.cn

Prof.GUAN Qing-mei Member of Editorial Board of JIA/Guest Editor College of Horticulture, Northwest A&F University,Yangling 712100, P.R.China E-mail: qguan@nwafu.edu.cn

Prof.WANG Li-rong Guest Editor Zhengzhou Fruit Research Institute,

Chinese Academy of Agricultural Sciences,Zhengzhou 450009, P.R.China E-mail: wanglirong@caas.cn

Prof.LUAN Fei-shi Guest Editor College of Horticulture and Landscape Architecture,Northeast Agricultural University,Harbin 150030, P.R.China E-mail: luanfeishi@neau.edu.cn

Prof.DUAN Qiao-hong Guest Editor College of Horticulture Science and Engineering,Shandong Agricultural University,Tai’an 271018, P.R.China E-mail: duanqh@sdau.edu.cn

Prof.SONG Chuan-kui Member of Youth Editorial Board of JIA/Guest Editor State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University,Hefei 230036, P.R.China E-mail: sckfriend@163.com