Fine Genetic Mapping of Dwarf Trait in Cucumber (Cucumis sativus L.)Using a RIL Population
2020-11-03QinZhiWeiYangJingZhouXiuYanWangLeiandXinMing
Qin Zhi-Wei, Yang Jing, Zhou Xiu-Yan, Wang Lei, and Xin Ming*
1 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture, Northeast Agricultural University, Harbin 150030, China
2 College of Resources and Environmental Sciences, Northeast Agricultural University, Harbin 150030, China
Abstract: Plant height in cucumber is not only an important trait for breeding, but also one of the model traits suitable for the study of developmental biology.Amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers techniques were applied for the construction of genetic linkage maps in cucumber.To understand the dwarf trait genetic basis in cucumber, the quantitative trait loci (QTL) were identified using F6 recombinant inbred line populations (RILs) comprising 336 lines from the cross between the two cultivars D0462 (the dwarf) and DN129 (the vine).In total, six SSR markers and 15 AFLP markers were detected on the five linkage groups covering 152.8 cM with a mean marker interval of 7.28 cM.Only one QTL was found to be linked with plant height and the dwarf trait locus remained in the fourth linkage group.The contributory percentage of the single QTL to plant height was 11.39%.
Key words: cucumber (Cucumis sativus L.), dwarf, quantitative trait locus (QTL), recombinant inbred line (RIL)
Introduction
Cucumber (Cucumis sativusL.) is one of the most economically important horticultural crops, because cucumber fruit production is the second largest of all the cucurbits in the world.China is the largest cucumber grower in the world and represented over 62% of the world production in 2005 (Staubet al.,2008).There are vine and dwarf species in terms of plant height of cucumber and the vine is the main cultivar in China.But the disease, lodging and manual harvest have severely disrupted fruit quality and production (Xinet al., 2015).
It is well known that the plant height is an important economical character of cucumber (Zhanget al., 2011).The dwarf stature is prone to lodging and diseases resistance (Liet al., 2011; Renet al., 2010).Meanwhile, reductions in cucumber height can significantly influence earlier flowering, accumulation and metabolize of nutrients, early-ripe, shorter growth period, and therefore gains greater and stability yield (Hedden, 2003).With the need of mechanical automatic harvesting, food processing and cost reduction, more and more researchers pay close attention to the dwarf trait, which has become a key objective of cucumber breeding programs worldwide from the period as "Green Revolution" (Zenget al.,2011).
Quantitative trait loci (QTL) mapping is an effective tool for genetic dissection of quantitative factors in major plants, such as agronomic traits, and increasing numbers of QTL studies have been conducted to dissect the genetic basis of the plant height and its components.To utilize QTL effectively, suitable mapping procedures for detecting QTL with various genetic effects are needed.The recombinant inbred line (RIL), one of the most powerful mapping populations, has a great effect on the QTL analysis and genetic effect (Ashrafiet al., 2009; Capelet al., 2015).By cross-breeding with homozygous parents and then multigeneration inbred, the RIL population has a stable and homozygous source of germplasm under various environmental conditions (Simonet al., 2008; Kimet al., 2014).The RIL population not only provides equivalent information about dominant and codominant marker systems as F2plants from the same parents, but also improves precision and efficiency of QTL detection (Cuiet al., 2012; Burr and Burr, 1991).Furthermore, RIL has been successfully employed to map QTLs for many agronomic traits since they can be replicated and evaluated in multiple over space and time, providing more accurate data in various laboratories and environments (Knapp and Bridges,1990).However, QTLs for the height of cucumber through RIL populations are rarely reported.
In the present study, QTL mapping analysis for the dwarf-related traits were detected using 336 RIL populations derived from the cross between two cultivated cucumber inbred lines, the dwarf type D0462 and the vine type 129.The objective of this study was to further reveal the genetic mechanism with SSR and AFLP markers in controlling dwarf-related traits and identifying molecular markers for related-genes.
Materials and Methods
Plant materials
The parent lines were described in the previous report(Xinet al., 2015).The early-ripe, shorter stem line D0462 (the dwarf cultivar) was crossed with the normal line 129 (the vine cultivar) during the spring of 2005, and F1plants were self-pollinated to obtain 413 individuals of F2plants.F2populations were then self pollinated by single-seed descent to obtain 336 F2S6RILs.All of these plants were developed in the greenhouse under controlled conditions with 16 h day temperature at (25±2)℃ and 8 h night temperature at(15± 2)℃ and 60%-70% relative humidity.
The young leaves were collected from individual cucumber plants, and genomic DNA extraction was performed according to the procedures described by Yuanet al(2008).DNA concentrations and qualities were estimated with a Biophotometer Plus (Expander,Germany) and by electrophoresis in 0.8% agarose gels.
Field evaluation and trait evaluation
These RILs, two parents and F1hybrid progeny, were evaluated in the Experimental Station of Horticulture,Northeast Agricultural University, Heilongjiang Province, China in 2010.The RILs were arranged in a randomized complete block design with five replications per location.Each replication had 10 plants,and individual plants were spaced 30 cm apart in rows,with an interval of 45 cm between the rows.
At maturity, 10 plants random selected from the middle of each plot were evaluated for plant height from the ground to the tip of cucumber.Basic statistical analysis for the phenotypic data in D0462, 129, F1and RILs populations was performed using SPSS 19.0 software package with analysis of variance (ANOVA)and the significance difference test atP=0.05.
SSR marker analyses
A total of 116 pairs of SSR primers were screened,according to Fazioet al.(2002) for polymorphisms between two parents.Out of these, 15 polymorphic markers (12.93%) were polymorphic.Moreover,molecular markers linked to the dwarf gene were further indentified by bulk segregant analysis (BSA),which was constructed by randomly selected 10 plants of each phenotypic class (extremely short plants andextremely tall plants) from the F2S6RILs.Eventually,13 markers were equally distributed across the genome and were also used for genotyping the RILs.The PCR for SSR was carried out on a volume of 25 μL in a Peltier Thermal cycler.The reaction mixture contained 60 ng template DNA, 1 UTaqpolymerase, 10 μmol · L-1of each primer, 6.5 mmol· L-dNTPs, 2.5 μL 10×Taqbuffer (containing Mg2+) and 17 μL ddH2O.
The DNA amplification reaction conditions were as the followings: initial denaturation of 5 min at 94℃,followed by 35 cycles of 30 s denaturation at 94℃,60 s annealing at either 45℃-60℃ (depending on the individual microsatellite marker), 60 s extension at 72℃, and a final extension at 72℃ for 4 min.The PCR products were denaturated at 94℃ in PCR instrument,and then separated on 6% polyacrylamide gels for 1 h at 100 W, then visualized by a silver staining procedure and photographed.
AFLP marker analyses
The DNA samples (60 ng · μL-1) were double-digested byEcoRⅠandMseⅠcompletely.Specific doublestranded adapters were subsequently ligated to the restriction fragment ends by T4DNA ligase.After five fold dilutions, the adapter-ligated DNA was preamplified with AFLP primers in 20 μL solution.A set of 64EcoRⅠandMseⅠselective primer combinations were used to detect the polymorphisms for AFLP between the two parents, following the protocol of Buerstmayret al.(2007), of which 25 markers(39.06%) showed polymorphic.After screened further among DNA samples of BSA, 11 primer pairs were applied to genetic analysis and linkage mapping.
Linkage analyses
The genotype data for 336 RILs were used to perform linkage analyses in the MAPMAKER/EXP3.0 software package (Landeret al., 1987) with a likelihood ratio statistic (LOD) threshold >3.0 and the maximum genetic distance of 50 cM.The linkage groups were created using MapChart software for Windows (Voorrips, 2002).For each marker locus,χ2goodness-of-fit test was used to check the segregation pattern from expected Mendelian segregation ratio of 1 : 1 at 0.05 in the RILs.Markers deviating from expected ratios were deleted from the linkage analysis.Of the putative polymorphic bands (loci) observed,segregation of 36 bands was applicable to map construction and QTL analyses.
QTL analyses
The genotype data for 336 RILs were used to perform linkage analyses by the composite interval mapping(CIM) of WinQTLCart (Wanget al., 2012), using Model 6 of QTL Cartographer (version 1.21) with a walking speed of 2 cM, a window size parameter of 10 cM and the inclusion of 11 maximum background marker loci in a stepwise forward regression procedure.The likelihood-ratio statistic (LOD) was applied to test the significance of each QTL interval.The LOD threshold for significant QTLs of each trait atP=0.05 was determined using a permutation test with 1 000.TheLODvalue of 2.5 was used as the threshold significance.If the distance between the two neighboring QTLs (LOD) was more than 2.5 cM with a contribution rate>10%, then they were considered as major QTLs, which showed significance in the dwarf and the vine cucumber as stable QTLs.
Results
Phenotypic variations of plant height
The mean phenotypic values, standard derivation and range of the plant height trait from the parents, F1and RILs are presented in Table 1, respectively.Significant differences of plant height were detected between the two parents, 129 and D0462 (P=0.05).Among the RILs, the height of plants was significantly segregate(p<0.001), with higher variation.RILs all exhibited a normal distribution typical (Fig.1) for quantitative traits with the two parents, in general, representing the phenotypic extremes and F1value as the intermediate.It indicated that the height trait of cucumber was controlled by the multiple genes.
Table 1 Plant height of parental lines (129 and D0462) and RILs (cm)
Fig.1 Frecency distribution of F6 isolated population
Development of a genetic linkage map based on RILs
In order to generate a genetic map of RILs, a total of 36 codominant markers were chosen according with Mendelian segregation ratios.When LOD=3.0,21 markers were screened, which included six SSRs and 15 AFLPs (Fig.2).The genetic linkage map consisted of five linkage groups, and on the average,21 markers were evenly distributed in each chromosome, providing a uniform coverage of the genome.The genetic map obtained with these data covered a genetic length of 158.2 cM, and the chromosome length varied from 1.2 cM (chromosome LG1) to 9.1 cM (chromosome LG5), with an average length of 7.28 cM.
Fig.2 Molecular genetic maps of QTL related to plant height in F6 RILs from cross D0462 and 129
Linkage analyses
The primer pairs mapped with RILs were applied to linkage analyses.The resulting genetic map was illustrated in Fig.3, and a majority of the 36 marker loci were detected with derivation from expected Mendelian segregation ratio of 1 : 1 among the 148 RILs.While eight markers (20.4 %) showed distorted segregation onχ2tests (p<0.05), of which four loci (68%) favored the female parent (D0462) and four (32%) favored the male parent (129).Eventually,six marker loci involved in the construction of linkage maps.
QTL analyses
Plant materials in this study were evaluated for the height trait of cucumber.These data affected the height was used to detect QTLs in comparison to the map developed herein.Only one QTL was associated with the height trait based (Fig.3), which was located in linkage groups LG4 with the distance to CSWACC02 peak of 1.4 cM.It explained 11.39 % of phenotypic variation and showed a negative significant additive effect, with LOD scores of 2.14.
Fig.3 Map location of QTL associated with dwarf trait in cucumber
Discussion
Plant height is a significant agronomic factor that highly influences yield, disease and lodging resistance and machinery harvest.Moreover, the dwarfing trait has therefore been an important breeding target for many decades in the majority of the crops (Leeet al.,2014).Therefore, it is essential to explore the genetic basis of plant height traits.
There were some previous studies reported the dwarfness in cucumber decided by a recessive gene.Kauffman and Lower (1976) first identified a recessive gene cp as major donor produce the dwarf phenotype.Kubickiet al.(1986) confirmed thecp-2 gene was related to reduce cucumber plant length.In studying the genome-wide molecular mapping on dwarf architecture of cucumber, Liet al.(2011)considered the compact phenotype was decided by only one single recessive gene cp.By contrast, it could be identified one QTL for cucumber height which explained 11.39% of the genotypic variance and exhibited a negative significant additive effect(Fig.3).In accordance with numerous researches performed on plant height in various plants, the plant height was generally considered to be a complex trait and controlled by both qualitative and quantitative genes.For example, five QTL were identified to play an important role in regulating plant height of Switchgrass (Serbaet al., 2015).Katharinaet al.(2014)identified 12 main-effect QTL controlling plant height which contributed an average of 59.6% of the total phenotypic variation.Therefore, the plant height had been supposed to be controlled by major genes and many minor genes with small effects.In addition, it was reported that network of genes and environment were responsible for plant height (Wanget al., 2010).A similar conclusion could be drawn from the QTL mapping for plant height in RILs-by Cuiet al.(2012)that environment had an important effect on plant height by two RILs.The plant height could be affected by the growth environment.
To date, no report has examined QTL for the dwarf traits using RILs in cucumber.In the present study,RILs were employed to determine the chromosomal locations of QTL for dwarf trait in cucumber.Of the two RILs parents, the seed parent D0462 was known as a typical dwarf cultivar with an average height around 100 cm across seasons, while the male parent DN129 was a vine type with an average height over 250 cm.The RILs had many advantages over other populations and been found particularly suitable for mapping quantitative traits to explore QTLs (Ashrafiet al.,2009).Besides providing more equivalent information,RILs with large enough samples allowed to raise the recombinant frequency, reduce experimental error(Yanget al., 2015), accurately predicted the phenotype(Zouet al., 2005) and increased authenticity and accuracy of the QTL detection in different laboratories and environments.In order to compare thirteen maize morphological traits, Brownet al.(2011) exploited a nest association mapping (NAM) population consisting of 4 892 RILs to enhance accuracy and precision and decrease experimental error of QTL estimates.The fruit quality traits of tomato were detected in 169 RILs from the TO-937×moneymaker cross, as well as the epistatic relationships between QTL and the QTL ×environment interaction effects (Capelet al., 2015).The present research used the mapping population including 336 F6RILs, which might reduce error variances and enhance the accuracy and precision of QTL detection of dwarfness in cucumber.
In the present study, 24.32% (36 of 148) of markers fitted the derivation from expected 1 : 1 Mendelian segregation.And among the 36 markers with Mendelian segregation of 1 : 1 ratio, it could be found more than 22% (8/36) markers with segregation distortion (SD).SD ratio was widespread in chromosome of plants and animals and most of SDRs were different in mapping populations, and therefore SD seemed to arise anyplace of cucumber genome (Zhanget al., 2012).It seemed to be a common phenomenon that SD appeared in cucumber genetic mapping studies.For example, Zhouet al.(2015) found 13 segregation distortion regions (SDRs) on the high-resolution cucumber map by using a RIL population derived from a gynoecious line 9110Gt and a monoecious line 9930.Previous studies of QTL mapping study with 148 RILs derived from 9110Gt (European greenhouse background cucumber) and 9930 (a typical northern China fresh market cucumber) had detected that 46 out of 255 mapped markers (18.0%) with SD (Miaoet al., 2011).Yuanet al.(2008) reported the 75 markers according to expected 1 : 1 Mendelian segregation favor to one parent, of which 51 loci (68%) biased toward the female parent and 24 (32%) biased toward the male parent (S06) by the 224 RILs derived from S94 (northern China type)×S06 (northern European type).In the present study, among the eight markers with segregation distortion, four (50%) favored the female parent (D0462) and four (50%) favored the male parent (DN 129).
It was still not clear the biased distribution of SD markers in genome.In research of genetic and cytogenetic map, Renet al.(2009) considered the wild alleles had more competition than the domesticated ones.However, Renet al.(2015) found 513 (83.3%)biased toward the cultivated female parent K3 and 103 (8.9%) favored the wild female parent PI 189225 among the 616 SD markers of the watermelon.Previous studies suggested that SD was arisen by selective transmission of the parental genomes, and the SD might occur more frequently as the genetic incompatibilities was bigger between the parents (Habuet al., 2015).Moreover, physiology and environment were also important factors influencing SD ratio(Hashemiet al., 2015).
Conclusions
A total of 336 F2S6RILs were obtained from shorter stem line D0462 (the dwarf cultivar) crossing with the normal line 129 (the vine cultivar).RILs exhibited a normal distribution typical for quantitative traits with the two parents, which indicated the height trait of cucumber was controlled by multiple genes.The genetic linkage map consisted of five linkage groups,was constructed by Mendelian segregation ratios, and 21 markers were found to evenly distribute in each chromosome, providing a uniform coverage of the genome.The genetic map covered a genetic length of 158.2 cM and the chromosome had an average length of 7.28 cM.According to the linkage analyses, 68%markers showing distorted segregation favored thefemale parent (D0462) and 32% favored the male parent (129).Eventually, one QTL, locating in linkage groups LG4 with the distance to CSWACC02 peak of 1.4 cM, was detected to associate with the height trait.It explained 11.39 % of phenotypic variation and showed a negative significant additive effect, with LOD scores of 2.14.
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