The inheritance of monoterpenes in hybrid populations of Vitis vinifera cultivars

doi:10.21203/rs.3.rs-5022036/v1

Download PDF

Research Article

The inheritance of monoterpenes in hybrid populations of Vitis vinifera cultivars

https://doi.org/10.21203/rs.3.rs-5022036/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Monoterpenes were the most abundant terpenes in grapes and contribute significantly to quality control in table and wine grapes. Most inheritance research of monoterpenes in grapes was focused on wine grapes, and inheritance analyses of monoterpenes in intraspecific hybrid populations of table grapes have been limited. In this study, the composition and content of volatile monoterpenes in the parents and progeny were analysed using the Vitis vinifera table grape varieties ‘Italia’ and ‘Tamina’ and the F1 generation in two consecutive years. 25 and 24 monoterpenes were detected in each of the ‘Italia’ and ‘Tamina’ parents by headspace solid phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS), while a total of 26 monoterpene compounds were identified in the progeny and subsequently quantified. We obtained phenotypic segregation ratios of the monoterpenes isolated in the progeny and found that the segregation of 4 monoterpenes, cis-Isogeraniol, neral, linalool oxide pyranoside and cis-Rose oxide, in the progeny was consistent with Mendelian inheritance. A significant high frequency distribution of most monoterpenes was found in the region of low concentration values, and the proportion of inheritance contributing to the phenotypic total variance of the population was calculated by generalised heritability. In addition, we found significant positive correlations between most monoterpenes and negative correlations between individual monoterpenes. The results will help to understand the heredity law of grape monoterpene formation in hybrid populations, as well as provide some theoretical enlightment for grape breeding and quality enhancement.

Grape

Inheritance

Intraspecific hybrid populations

Monoterpenes

Muscat varieties

Grapes were one of the most important fruit crops and were widely cultivated throughout the world (Sabo et al. 2022). Studies report that about 7.59 million hectares of agricultural land were used for grape production, providing raw material for winemaking and for the table grape and dried grape markets (Khan et al. 2020). There were approximately 6,000-10,000 different grape varieties worldwide, of which the Vitis vinifera cultivars were the most widely used in the grape and wine industry, accounting for 71% of total grape production (Conde et al. 2007). Aroma was an important factor influencing the quality of grapes and the consumer's desire to buy them. Different grape varieties have different aromas due to differences in their aroma components (Gutierrez-Gamboa et al. 2021). Grapes were classified as Muscat, non-Muscat or neutral according to the type of aroma (Wu et al. 2020). Terpenes were an important aroma components of Muscat grapes and have received much attention in recent decades. Terpenes were mainly found in the Vitis vinifera species of grapes and were typical aroma components of Muscat grapes due to their distinctive flavour and low sensory threshold (Mele et al. 2021). Currently, there were more than 70 terpenes found in grape berries and wines, and terpenes can be classified as monoterpenes, sesquiterpenes and diterpenes based on differences in the number of structural units (Mabou and Yossa, 2021). Among them, monoterpenes were important volatile components contributing to the fruity and floral aromas of grape berries and wines, and were of great interest in varietal breeding and quality control of table and wine grapes (Chen et al. 2024). Monoterpenes can be classified into acyclic and cyclic monoterpenes, with acyclic monoterpenes being the main monoterpenes found in grape berries (Wu et al. 2016). The monoterpenes in grape berries exist in both free and glycoside-bound forms. The former can contribute directly to flavor; while the latter were a class of potential aromatic components that can be converted to odor-active forms by hydrolysis (Yue et al. 2021).

Dissecting the synthesis and regulation of monoterpenes during grapevine fruit development was important for viticulture and flavor studies of its products. Terpenes in grapes were synthesized mainly through two independent pathways, the MEP and MVA pathways (Zheng et al. 2021). The biosynthesis of monoterpenes has been shown to be mainly through the MEP pathway in grape berries (Ji et al. 2021). Several enzymatic genes contribute to the biosynthesis of monoterpenes, notably VvDXS, VvGPPS, VvTPS, VvHDR. These genes were involved in catalyzing monoterpene biosynthesis at different stages of the MEP and MVA pathways, and the expression of some of these genes has been linked to the accumulation of flavor compounds (Li et al. 2022). Previous studies have shown that monoterpene was characterized as complex quantitative traits, and their accumulation was susceptible to a variety of factors such as genetic background, growing environment and cultivation practices (Bosman and Lashbrooke, 2023). Therefore, it is difficult to study and characterize the patterns of inheritance of quantitative traits. Currently, studies on the heritability of monoterpenes in grapevine were still limited, despite the rapid breakthroughs in the inheritance patterns of monoterpene traits in grape (Mele et al. 2021; Linet al. 2020). The colocalization of quantitative trait loci on DXS and LG 5 at the level of regulating monoterpene aromatics in grape hybrid populations suggests that DXS gene plays a role in regulating metabolic fluxes through the MEP pathway (Battilana et al. 2009). Wang et al. (2020) found that 4 stable QTLs associated with muscat flavor were located on LG5 in a population of grape hybrid offspring from a Vitis vinifera species, which was in agreement with the results of a previous study (Wang et al. 2020). These results suggest that both the metabolic regulatory mechanisms and heritability guiding the expression of grape monoterpenes were controlled by critical loci and genes.

Previous studies have focused on volatile monoterpenes in wine grapes, and there have been fewer inheritance studies of monoterpenes in intraspecific hybrid populations of table grapes (Duchene et al. 2012). In this study, we analyzed the composition and content of volatile monoterpenes in offspring populations from two consecutive years of crosses between ‘Italia’ and ‘Tamina’ grapes, and explored the patterns of inheritance of these volatile monoterpenes in grape populations. The study will help us to further understand the regulatory mechanisms of monoterpene biosynthesis in grapes and provide guidelines for grape variety selection and flavor improvement.

Plant materials

The materials for the study were Vitis vinifera cultivars of grapes ‘Italia’ and ‘Tamina’ and 176 plants of F1 generation resulting from their crosses. The hybrid offspring were 153 plants collected in 2017 and 135 plants collected in 2018, respectively. ‘Italia’ and ‘Tamina’ were planted at the Institute of Forestry and Pomology, Beijing Academy of Agricultural and Forestry Sciences in China, with row spacing of 2.5 m and plant spacing of 0.75 m with 5 years self-rooted vines, the trellis system was horizontal dragon vines, with one-armed leaf curtain. Conventional cultivation and management practices were used. The grapes were collected at maturity, with a balance between shaded and sunny surfaces, and a balance between shoulder, central and top of each cluster, with a total of 100 randomly dispersed grapes, with three biological replicates. The fruit were frozen in liquid nitrogen and stored in a refrigerator at -80℃.

Qualitative and quantitative analyses of monoterpenes

Free monoterpenes were extracted and determined with reference to the method of Liu et al. (Liu et al. 2022). Retention index (RI) and mass spectrometry information were calculated using automated mass spectrometry deconvolution and identification software (AMDIS), and compounds were identified by matching the results of mass spectrometry analyses with the NIST11 library, as well as identifying compounds based on their standard retention index. Quantitative analyses of grape terpenes were performed by standard curves. 1 L of grape simulation solution containing 7 g/L tartaric acid and 200 g/L glucose was prepared based on the sugar and acid content of grape juice. Each standard was dissolved in ethanol (chromatographically pure), then all the standard solutions were mixed and the mixture was diluted to successive 15 concentration levels using a mixed matrix. For monoterpenes for which no standard curve, quantitative analyses should be carried out by means of standard curves for compounds with similar numbers of carbon atoms and similar chemical structures (Supplemental Table 1).

Statistical analysis

The phenotypic ratios of monoterpenes showing segregation in the progeny were calculated based on the presence or absence of monoterpenes in the progeny. The χ² test were calculated using the , where o is the observed value and c is the theoretical value. Box plots were plotted by R software to demonstrate the concentration range and median value of monoterpenes and to test for outliers in the monoterpene data. The upper and lower quartiles of the monoterpene data in the samples were represented by the upper and lower horizontal lines of the box plots, respectively. The analysis of variance of the monoterpene volatiles in the offspring was performed using the lme4 package in the R software, and then the broad-sense heritability was derived from the results of the calculations. Correlation analyses were performed using SPSS 27.0 (SPSS, USA) and correlation heat maps were plotted using ChiPlot (https://www.chiplot.online/).

Monoterpene composition and content of parent and offspring

For the parents and the F1 generation grape population of their crosses, free monoterpenes were detected in this study by HS-SPME-GC-MS. Through two consecutive years of study, we detected a total of 25 volatile monoterpenes in ‘Italia’ and 24 monoterpenes in ‘Tamina’ (Table 1). Volatile monoterpenes in the progeny also included trans-Isogeraniol and linalool oxide pyranoside compared to the parental line. Of these, trans-Isogeraniol was not detected in the ‘Tamina’, while linalool oxide pyranoside was not detected in either parents. In the present study, the total volatile monoterpene content of the ‘Italia’ was found to be less than that of the ‘Tamina’, with total monoterpene content of the former ranging from 900.08-1073.73 μg/kg, whereas the latter's total monoterpene content values were 1430.55- 1542.81 μg/kg (Table 1).

A total of two types of monoterpenes were detected in the study, acyclic monoterpenes and cyclic monoterpenes (Table 1). The main volatile monoterpenes detected in the ‘Italia’ parent were acyclic monoterpenes, with total content of 643.41 μg/kg and 781.73 μg/kg in the two years, respectively, accounting for an average of 73.39% of total monoterpene content during the study period; cyclic monoterpenes accounted for a relatively small proportion of total monoterpenes in this parent with a value of 26.61%, and the total cyclic monoterpene content was 430.32 μg/kg and 118.35 μg/kg in the two years, respectively. Similarly, in the ‘Tamina’ parent, we found that acyclic monoterpenes were still the dominant volatile monoterpene, accounting for 83.76% of total monoterpene content in the study period while cyclic monoterpenes occupied only 16.24%. Linalool was the most abundant monoterpene, both in ‘Italia’ and ‘Tamina’ parents (Table 1). The values of linalool content in ‘Italia’ parents were 223.49 μg/kg and 341.58 μg/kg, which accounted for 20.81%-37.95% of the total monoterpene content. In contrast, linalool accounted for 36.265-47.18% of the total monoterpene content in the ‘Tamina’ parents, with values of 727.88 μg/kg and 518.77 μg/kg, respectively. This result was higher than the value of linalool content in the ‘Italia’ parent.

We detected a total of 26 monoterpenes in the hybrid progeny and quantified them separately using standard curves (Supplemental Table 2). The average content of total volatile monoterpenes was significantly higher than the average content of total monoterpenes in the parents in both years. The total acyclic monoterpenes were more abundant in the progeny, accounting for 86.19% of the total monoterpene content, and the mean content values of total acyclic monoterpenes in the samples were 3357.16 μg/kg and 2392.30 μg/kg in the two years, respectively. The total cyclic monoterpenes had a range of concentrations of 16.63-5486.97 μg/kg and 7.01-8255.10 μg/kg, the average value of the content was 451.79 μg/kg, which accounted for 13.81% of the total monoterpene content. Similar to the parental results, the highest average monoterpene content in the progeny was linalool, with an average content value presenting 957.92 μg/kg. Geranic acid was next to linalool in terms of average content, presenting 539.51 μg/kg, and occupying 15.29% of total monoterpene content in the progeny. In addition to this, the average contents of β-Myrcene, β-cis-Ocimene and α-terpineol were similarly high, and together they constituted the main monoterpene fractions in the offspring of the Vitis vinifera hybrid grapes.

In addition, we observed the segregation of 26 monoterpenes in all hybrid offspring. Among them, the segregation of 4 monoterpenes, cis-Isogeraniol, neral, linalool oxide pyranoside and cis-Rose oxide, indicated that the presence or absence of these monoterpenes greatly conformed to Mendelian inheritance. Thus, the segregation patterns generally conformed to a 3:1 or 1:1 ratio in the progeny, but the ratio values of neral in 2018 and cis-Rose oxide in 2017 were significantly exceptional (Table 2). The accuracy of the segregation ratios was subsequently verified by a χ² test (Table 2).

Table 1 Information on the 26 monoterpenes in the ‘Italia’ × ‘Tamina’ hybrid population in two years: parental values, mid-parental values (` p ), mean progeny values (` x ), concentration ranges, and number of progeny with monoterpenes present or absent

Monoterpenes	Year	Italia	Tamina	`p	`x	Range	Presence	Absence
Acyclic monoterpenes
Linalool	2017	223.49	727.88	475.69	949.34	19.93-5808.61	153	0
Linalool	2018	341.58	518.77	430.17	966.49	8.09-20059.13	134	1
Citronellol	2017	1.55	5.24	3.39	11.97	0.82-128.34	148	5
Citronellol	2018	4.67	3.19	3.93	7.01	0.70-125.32	132	3
Geraniol	2017	11.34	13.04	12.19	298.98	21.20-2437.45	153	0
Geraniol	2018	37.18	21.14	29.16	156.66	3.40-3201.14	134	1
Geranial	2017	4.56	8.33	6.45	3.54	2.77-11.69	149	4
Geranial	2018	24.62	6.67	15.65	3.39	2.71-39.18	134	1
Geranic acid	2017	1.24	1.55	1.40	821.71	21.47-15712.56	149	4
Geranic acid	2018	1.95	8.09	5.02	257.31	12.17-4306.24	134	1
cis-Isogeraniol	2017	0.21	0.15	0.18	1.96	0.73-18.93	103	50
cis-Isogeraniol	2018	0.26	0.21	0.23	1.46	0.70-29.54	102	33
trans-Isogeraniol	2017	0.03	ND	-	0.69	0.68-5.83	54	99
trans-Isogeraniol	2018	0.10	0.10	0.10	0.32	0.68-5.84	39	96
Nerol	2017	4.22	4.80	4.51	112.91	13.94-1060.86	135	18
Nerol	2018	9.25	6.65	7.95	60.59	13.47-1033.04	112	23
Neral	2017	2.53	3.41	2.97	2.35	2.71-18.66	116	37
Neral	2018	7.68	2.63	5.16	1.85	2.71-9.69	87	48
Nerol oxide	2017	125.39	49.23	87.31	137.97	13.32-11232.96	129	24
Nerol oxide	2018	37.53	141.24	89.39	41.17	13.64-615.90	97	38
β-Myrcene	2017	102.66	350.20	226.43	261.42	3.53-2226.62	152	1
β-Myrcene	2018	166.17	205.40	185.79	325.14	1.88-9781.54	134	1
Allo-Ocimene	2017	30.63	42.61	36.62	15.86	1.47-119.44	135	18
Allo-Ocimene	2018	27.57	37.33	32.45	15.74	1.63-481.62	118	17
(E,Z)-Allo-Ocimene	2017	7.65	13.40	10.52	45.73	1.74-477.00	139	14
(E,Z)-Allo-Ocimene	2018	8.76	9.20	8.98	31.42	1.80-656.46	121	14
β-cis-Ocimene	2017	93.56	102.73	98.14	441.40	1.50-2394.60	152	1
β-cis-Ocimene	2018	80.61	119.74	100.17	410.84	4.48-5778.96	130	5
β-trans-Ocimene	2017	34.35	45.85	40.10	251.33	2.33-4670.75	153	0
β-trans-Ocimene	2018	33.80	47.17	40.49	246.25	2.42-3668.83	134	1
Subtotal	2017	643.41	1368.42	1005.92	3357.16	87.72-21723.60	-	-
Subtotal	2018	781.73	1127.53	954.63	2392.30	37.58-27985.85	-	-
Percent	2017	59.92%	88.70%	76.89%	87.69%	-	-	-
Percent	2018	86.85%	78.82%	72.97%	84.69%	-	-	-
Cyclic monoterpenes
Linalool oxide pyranoside	2017	ND	ND	-	7.62	3.58-72.95	120	33
Linalool oxide pyranoside	2018	ND	ND	-	5.97	3.55-61.64	106	29
cis-Furan linalool oxide	2017	237.92	55.74	146.83	7.98	3.55-48.45	135	18
cis-Furan linalool oxide	2018	2.98	95.65	49.31	9.23	3.56-110.79	120	15
trans-Furan linalool oxide	2017	39.79	19.89	29.84	7.07	3.54-79.04	122	31
trans-Furan linalool oxide	2018	2.33	23.51	12.92	6.30	3.56-48.05	114	21
Phellandrene	2017	12.39	13.28	12.83	4.81	1.02-44.78	145	8
Phellandrene	2018	9.71	12.62	11.17	8.90	1.02-153.39	131	4
Limonene	2017	65.55	43.85	54.70	34.45	1.43-352.24	153	0
Limonene	2018	47.41	79.93	63.67	32.58	1.25-404.88	134	1
cis-Rose oxide	2017	1.76	3.75	2.75	3.07	3.35-23.04	108	45
cis-Rose oxide	2018	1.89	2.45	2.17	2.20	3.35-9.30	74	61
trans-Rose oxide	2017	0.16	1.33	0.74	1.18	3.35-7.00	48	105
trans-Rose oxide	2018	0.34	0.45	0.40	0.82	3.35-5.10	31	104
α-Terpineol	2017	1.41	5.54	3.48	319.10	9.21-3998.58	153	0
α-Terpineol	2018	8.61	9.29	8.95	292.76	6.61-6527.58	134	1
4-Terpineol	2017	41.48	19.12	30.30	4.30	1.57-48.35	107	46
4-Terpineol	2018	32.46	50.46	41.46	3.95	1.31-66.96	124	11
Terpinolen	2017	26.42	9.62	18.02	69.90	1.66-821.00	153	0
Terpinolen	2018	10.31	25.27	17.79	59.47	1.54-880.50	134	1
γ-Terpinen	2017	3.44	2.27	2.85	11.64	1.60-78.02	153	0
γ-Terpinen	2018	2.31	3.39	2.85	10.28	1.42-158.77	129	6
Subtotal	2017	430.32	174.39	302.36	471.12	16.63-5486.97	-	-
Subtotal	2018	118.35	303.02	210.69	432.45	7.01-8255.10	-	-
Percent	2017	40.08%	11.30%	23.11%	12.31%	-	-	-
Percent	2018	13.15%	21.18%	18.08%	15.31%	-	-	-
Total	2017	1073.73	1542.81	1308.27	3828.28	104.35-24034.49	-	-
Total	2018	900.08	1430.55	1165.32	2824.75	48.90-36240.95	-	-

ND, no detected.

Table 2 Proportion of segregation and χ² test for 4 monoterpenes in the progeny population of ‘Italia’ x ‘Tamina’

Monoterpenes	2017				2018
Monoterpenes	Presence	Absence	Ratio	χ²	Presence	Absence	Ratio	χ²
cis-Isogeraniol	103	50	3:1	4.81	102	33	3:1	0.02
Neral	116	37	3:1	0.05	87	48	-	8.02
Linalool oxide pyranoside	120	33	3:1	0.96	106	29	3:1	0.89
cis-Rose oxide	108	45	-	25.94	74	61	1:1	1.25

Analysis of inheritance differences in monoterpenes

It was found that the mean total monoterpene content in the offspring showed 3828.28 μg/kg and 2824.75 μg/kg in the two years, respectively, which turned out to be significantly higher than the mean total monoterpene content of 1308.27 μg/kg and 1165.32 μg/kg in the parents (Table 1). During the two years of the study, most of the monoterpenes were found to be significantly higher in the offspring on average than in the parents. The mean content of linalool in the offspring was higher than in the parents, presenting a content of 957.52 μg/kg in the offspring and 452.93 μg/kg in the parents. It is worth noting that some monoterpenes were even hundreds of times higher in the offspring than in the parents. The average concentration of geranic acid in the offspring was 539.51 μg/kg, which was about 168 times higher than the average concentration of this compound in the parents (3.21 μg/kg) (Table 1). Besides, there were also a few monoterpenes with lower average content in the offspring than the parents. For example, the values of geranial in the offspring were lower than those of the ‘Italia’ and ‘Tamina’, where the average content of the compound was presented as 3.47 μg/kg, compared to 14.59 μg/kg and 7.5 μg/kg in the parents.

The results showed that the coefficients of variation (CV) for geranial, neral and cis-Rose oxide were in the range of 0.1-1.0, which were considered to be moderate; the CV for the other monoterpenes in the F1 population were all higher than 1.0, which were considered to be highly variable (Table 3). It was detected that most monoterpenes showed a gradual decrease in the frequency of distribution in the region from the minimum to the maximum concentration in the distribution plot, indicating that most of the monoterpenes showed a poisson distribution in the hybrid progeny, and a few of them showed a normal distribution (Table 3). Three monoterpenes showed high content in the progeny, namely linalool, geranic acid, and β-cis-Ocimene, which showed a poisson distribution, and some of the monoterpenes showed a relatively lower content in the hybrid progeny, such as trans-Isogeraniol and trans-rose oxide, which also showed poisson distribution. In addition, the concentration distributions of the two monoterpenes were different in two years. In 2017, geranial and neral were normally distributed; while in the 2018 samples both monoterpenes showed a poisson distribution (Table 3).

Table 3 Statistical analysis of monoterpene concentrations in F1 hybrid populations

Monoterpenes	2017								2018
Monoterpenes	Minimum (μg/kg)	Maximum (μg/kg)	Mean (μg/kg)	SD	CV	SK	KU	Distribution	Minimum (μg/kg)	Maximum (μg/kg)	Mean (μg/kg)	SD	CV	SK	KU	Distribution
Linalool	19.93	5808.61	949.34	1111.64	1.17	2.28	5.89	Poisson	0.00	20059.13	966.49	2258.31	2.34	6.19	43.36	Poisson
Citronellol	0.00	128.34	11.97	15.28	1.28	4.63	28.55	Poisson	0.00	125.32	7.01	11.88	1.70	7.52	71.16	Poisson
Geraniol	21.20	2437.45	298.98	391.79	1.31	2.68	8.47	Poisson	0.00	3201.14	156.66	312.33	1.99	7.37	66.10	Poisson
Geranial	0.00	11.69	3.54	1.37	0.39	2.15	10.21	Normal	0.00	39.18	3.39	3.20	0.94	10.55	115.41	Poisson
Geranic acid	0.00	15712.56	821.71	1702.46	2.07	5.44	39.03	Poisson	0.00	4306.24	257.31	612.87	2.38	4.71	24.60	Poisson
cis-Isogeraniol	0.00	18.93	1.96	2.63	1.34	3.52	17.48	Poisson	0.00	29.54	1.46	3.32	2.27	7.31	55.55	Poisson
trans-Isogeraniol	0.00	5.83	0.69	1.20	1.72	2.02	3.95	Poisson	0.00	5.84	0.32	0.67	2.09	4.61	33.13	Poisson
Nerol	0.00	1060.86	112.91	168.77	1.49	3.13	11.57	Poisson	0.00	1033.04	60.59	108.51	1.79	6.20	48.67	Poisson
Neral	0.00	18.66	2.35	1.92	0.82	3.85	32.39	Normal	0.00	9.69	1.85	1.50	0.81	0.56	3.54	Poisson
Nerol oxide	0.00	11232.96	137.97	909.32	6.59	11.99	143.78	Poisson	0.00	615.90	41.17	72.33	1.76	4.97	31.89	Poisson
β-Myrcene	0.00	2226.62	261.42	323.56	1.24	2.86	11.11	Poisson	0.00	9781.54	325.14	983.55	3.03	7.53	64.63	Poisson
Allo-Ocimene	0.00	119.44	15.86	18.67	1.18	2.65	9.17	Poisson	0.00	481.62	15.74	47.68	3.03	8.27	72.66	Poisson
(E,Z)-Allo-Ocimene	0.00	477.00	45.73	68.69	1.5	3.33	13.51	Poisson	0.00	656.46	31.42	69.10	2.20	6.82	54.07	Poisson
β-cis-Ocimene	2.33	4670.75	441.40	688.66	1.56	3.50	14.35	Poisson	0.00	5778.96	410.84	752.55	1.83	4.29	22.53	Poisson
β-trans-Ocimene	0.00	2394.60	251.33	381.20	1.52	3.31	12.46	Poisson	0.00	3668.83	246.25	470.82	1.91	4.55	24.88	Poisson
Linalool oxide pyranoside	0.00	72.95	7.62	9.82	1.29	3.60	17.38	Poisson	0.00	61.64	5.97	7.46	1.25	4.68	29.20	Poisson
cis-Furan linalool oxide	0.00	48.45	7.98	7.62	0.96	2.32	6.78	Poisson	0.00	110.79	9.23	13.66	1.48	5.18	30.92	Poisson
trans-Furan linalool oxide	0.00	79.04	7.07	10.05	1.42	3.88	19.56	Poisson	0.00	48.05	6.30	7.53	1.20	3.59	14.93	Poisson
Phellandrene	0.00	44.78	4.81	5.36	1.12	3.93	21.73	Poisson	0.00	153.39	8.90	22.70	2.55	4.93	24.88	Poisson
Limonene	1.43	352.24	34.45	58.02	1.68	3.49	13.65	Poisson	0.00	404.88	32.58	51.96	1.59	4.42	24.94	Poisson
cis-Rose oxide	0.00	23.04	3.07	2.94	0.96	2.95	16.43	Poisson	0.00	9.30	2.20	2.15	0.98	0.38	-0.62	Poisson
trans-Rose oxide	0.00	7.00	1.18	1.81	1.53	1.09	-0.12	Poisson	0.00	5.10	0.82	1.52	1.85	1.34	-0.10	Poisson
α-Terpineol	9.21	3998.58	319.10	542.87	1.70	3.90	19.00	Poisson	0.00	6527.58	292.76	658.59	2.25	7.08	60.23	Poisson
4-Terpineol	0.00	48.35	4.30	6.35	1.48	3.94	20.66	Poisson	0.00	66.96	3.95	6.62	1.68	7.21	61.71	Poisson
Terpinolen	1.66	821.00	69.90	114.86	1.64	3.82	17.68	Poisson	0.00	880.50	59.47	120.25	2.02	4.82	26.04	Poisson
γ-Terpinen	1.60	78.02	11.64	12.72	1.09	2.62	8.24	Poisson	0.00	158.77	10.28	20.68	2.01	4.88	25.91	Poisson

SD refers to the Standard Deviation of monoterpene concentration in hybrid offspring; CV refers to the coefficient of variation of monoterpene concentration in hybrid offspring; SK and KU respectively refer to the skewness coefficient and kurtosis coefficient of monoterpenes in hybrid offspring.

Genotypic variation and inheritance of monoterpenes

It was found that the total monoterpene content in the hybrid progeny was significantly higher than that of the parents and showed a wide range of continuous variation. In 2018, the mid-parental value and median value of total monoterpenes in the progeny were lower than those in 2017, suggesting that the year had a greater effect on the accumulation of total monoterpenes (Fig. 1A). Meanwhile, the concentration distribution values of total monoterpenes in the offspring were significantly different between the two years, with a wider range of total monoterpenes in 2018 than in 2017, showing 48.90-36240.95 μg/kg (Fig. 1A).

Specifically, some monoterpenes showed significant quantitative trait inheritance with continuous variation in the offspring (Fig. 1B-J, Supplemental Figure 1). Linalool had a high mean content value and the widest range of content in the offspring, presenting a concentration range of 19.93-5808.61 μg/kg and 8.09-20059.13 μg/kg of the monoterpene in 2017 and 2018, respectively (Fig. 1B). Geranic acid had the next highest mean content value in the offspring, and its distribution also showed quantitative trait inheritance (Fig. 1C); the content range of this monoterpene in 2017 (21.47-15712.56 μg/kg) was significantly higher than that in 2018 (12.17-4306.24 μg/kg). α-Terpineol accounted for 9.35% of the total monoterpene content in the offspring, it has a higher content in the cyclic monoterpenes of the offspring, occupying 67.72% of the total cyclic monoterpene content. The concentration range of α-terpineol in the progeny of both years was 9.21-3998.58 μg/kg and 6.61-6527.58 μg/kg, showing a clear continuous variation (Fig. 1D). β-Myrcene, geraniol, nerol, and citronellol demonstrated a clear tendency of the concentration distribution to lower values (Figs. 1E-G, Fig. I). In addition, the mean concentration values and median values of limonene in the parents were higher than their results in the offspring, which showed a continuous variation in the concentration distribution of limonene (Fig. 1H). cis-Rose oxide had the lowest value of content, which segregated in accordance with Mendelian inheritance, and showed quantitative trait inheritance in the offspring (Fig. 1J). Moreover, the difference between the average content of cis-Rose oxide in both parents and offspring was small, presenting 2.46 μg/kg and 2.64 μg/kg.

In addition, we analysed the 26 monoterpenes for broad-sense heritability (H²). It was found that β-Myrcene had the highest H² value, presenting 0.86 in both years (Table 4). The monoterpenes of Allo-Ocimene, (E,Z)-Allo-Ocimene, linalool oxide pyranoside, cis-Furan linalool oxide, phellandrene, and limonene had lower H² values, and their values did not exceed 0.5 in both years (Table 4).

Table 4 Broad-sense heritability (H²) of 26 monoterpenes in two years.

Monoterpenes	2017	2018
Linalool	0.77	0.86
Citronellol	0.52	0.86
Geraniol	0.50	0.50
Geranial	0.75	0.50
Geranic acid	0.77	0.50
cis-Isogeraniol	0.51	0.50
trans-Isogeraniol	0.76	0.50
Nerol	0.35	0.76
Neral	0.78	0.67
Nerol oxide	0.74	0.50
β-Myrcene	0.86	0.86
Allo-Ocimene	0.35	0.48
(E,Z)-Allo-Ocimene	0.40	0.46
β-cis-Ocimene	0.77	0.50
β-trans-Ocimene	0.77	0.86
Linalool oxide pyranoside	0.30	0.50
cis-Furan linalool oxide	0.50	0.37
trans-Furan linalool oxide	0.61	0.34
Phellandrene	0.41	0.46
Limonene	0.34	0.38
cis-Rose oxide	0.54	0.42
trans-Rose oxide	0.86	0.76
α-Terpineol	0.77	0.50
4-Terpineol	0.79	0.34
Terpinolen	0.60	0.35
γ-Terpinen	0.51	0.50
Total	0.77	0.76

Correlation analysis of monoterpenes

We performed correlation analyses for 26 monoterpenes, and the results were shown in Table 5. Overall, most of the monoterpenes showed significant positive correlations with each other, and individual monoterpenes (geranic acid and neral) showed negative correlations with phellandrene. We found that Allo-Ocimene, (E,Z)-Allo-Ocimene, β-cis-Ocimene, β-trans-Ocimene, limonene, α-terpineol, 4-terpineol, terpinolen and γ-terpinen showed strong correlations with other monoterpenes; whereas geranic acid, neral, nerol oxide and phellandrene showed weaker correlations with the other monoterpenes (Fig. 2). α-Terpineol showed the highest correlation coefficient with 4-terpineol and terpinolen of 0.963 and 0.91 (P<0.01); and the lowest correlation coefficient of 0.012 was found for phellandrene with cis-rose oxide (Supplemental Table 3). In addition, 4 acyclic monoterpenes: Allo-Ocimene, (E,Z)-Allo-Ocimene, β-cis-Ocimene and β-trans-Ocimene had high correlations with 4 cyclic monoterpenes: α-terpineol, 4-terpineol, terpinolen and γ-terpinen, with correlation coefficient values higher than 0.6 (Fig. 2).

The monoterpene compounds were the key factors affecting the quality of the grape berries, and the identification of the composition and content of monoterpenes in grape germplasm is of great significance for the development and utilization of grape germplasm resources and breeding (Xie et al. 2023). Studies have shown that ‘Italia’ and ‘Tamina’ were both Vitis vinifera grape varieties with typical muscat flavor (Yinshan et al. 2017). In this study, we identified a total of 26 monoterpenes present in both parents and offspring using headspace solid phase microextraction and gas chromatography-mass spectrometry. It was found that the total monoterpene content was higher in both parents and was better in the ‘Tamina’ than in the ‘Italia’. The monoterpene content of the progeny of the populations was correlated with the parental genotype, and the average total monoterpene content in the progeny was higher than the mid-parental value. The above results confirm the different content of monoterpenes presented in the parents and progeny in previous studies on hybrid populations (Liu et al. 2015). Many studies have identified linalool, geranic acid and geraniol as the main monoterpene constituents of Muscat grapes, and the high content and low threshold of these compounds as the main source of the characteristic aroma of muscat flavor in grape berries (Yue et al. 2020). The complex combinations and possible synergistic interactions between them can lead to successive variations in the intensity of the aroma of different Muscat grapes, as well as differences in the type of flavor. The results measured in this study revealed that geranic acid, linalool and geraniol play an important role in contributing to the muscat aroma of the Vitis vinifera grape population. Wang et al. (2021) showed that linalool, geraniol and α-terpineol were high in most of the grape populations, and the results of the present experiments were consistent with them.

Previous studies have shown that six monoterpenes, rose oxide, nerol oxide, nerol, neral, geranial and geranic acid, showed a 1:1 segregation ratio in the hybrid offspring of ‘Jingxiu’ × ‘Muscat of Alexandria’ and ‘Jingxiu’ × ‘Xiangfei’ (Wu et al. 2012). In the present study, the segregation of cis-Isogeraniol, neral, linalool oxide pyranoside and cis-Rose oxide in the progeny was in accordance with Mendelian inheritance (Table 2). Among them, the segregation ratio of cis-Isogeraniol, neral and linalool oxide pyranoside in the offspring was 3:1; while the segregation ratio of cis-Rose oxide in the offspring was 1:1. This result was different from the previous study, and it was speculated that the reason might come from different study parents and environment, among other factors. In addition, the results of the present study showed that monoterpenes exhibited continuous variation in the hybrid populations and the consistency of the trait phenotype was good in different years (Fig. 1a-j). There may be some differences in the phenotypes of this trait in different years, suggesting that grape monoterpenes belong to a complex quantitative trait, which may be influenced by factors such as genes and environment (Sun et al. 2023). Previous studies have shown that the content of volatile monoterpenes is influenced by varietal factors and that their content varies in different grape populations (Yang et al. 2024). In this study, the monoterpenes showed different distributions in different subgroups, with most of the monoterpenes belonging to a poisson distribution and some showing a normal distribution. This suggests that most of the 176 grape populations had low levels of these monoterpenes and only a few samples had high concentrations of these monoterpenes.

It was found that the different monoterpenes were strongly correlated with each other, indicating that they were not independent of each other, and it is hypothesized that these correlated compounds may be regulated by transcription factors or upstream genes. The upstream enzyme genes VvGPPS and VvTPS have been reported to play a key role in the production of monoterpenes (Wang et al. 2020). Studies have shown that acyclic monoterpenes and cyclic monoterpenes belong to the MEP pathway synthesis products, but the synthesis pathways of the two types of monoterpenes were slightly different (Lei et al. 2021). In this study, the two types of monoterpenes showed correlation, and it was hypothesized that the correlation between monoterpenes might be due to the regulation of VvGPPS and VvTPS enzyme genes in the monoterpene synthesis pathway (Kok and Bal, 2014). α-Terpineol, 4-terpineol, terpinolen and γ-terpinen showed strong correlation, and these monoterpenes were structurally similar but with a slightly different functional group, which was hypothesized to be regulated by different modifier enzyme genes in the post synthesis modification process of the monoterpenes (Mateo and Jiménez, 2000).In addition, it was shown that the strong correlation between monoterpenes may be different in different years, which is mainly due to different environmental factors such as temperature and light in different years (Martin et al. 2016). In the QTL analysis of monoterpenes in 96 grape germplasms by Yang et al. (2017), the range of aroma compound content in the 2014 and 2015 populations was not completely consistent, indicating that there may be differences in the correlation of monoterpenes in different years.

A similar phenomenon was reported by Zhang et al. (Zhang et al. 2023), suggesting that the accumulation of monoterpenes is associated with related genes and is regulated by multiple genes in the grapevine fruit. Currently, many studies have been carried out to construct population genetic maps, locate QTL and apply them to the genetic study of monoterpenes in grapes by analyzing the aroma content and inheritance patterns of grape populations. ‘Italia×Big Perlon’ and ‘Moscato Bianco×V.riparia’ as two progeny populations, reported that the candidate gene VvDXS1 located at LG5 was associated with QTL for geraniol, nerol and linalool content, and identified other QTL loci for linalool content on LG10 (Battilana et al. 2009). A recent study showed QTL localization to the presence of a geraniol synthase (VviTPS52) on chromosome 12 involved in grape monoterpene synthesis, and monoterpene-related TPS enzymes on other chromosomes (Bosman et al. 2023). The above study confirms the polygenic nature of grapevine fruit monoterpene aroma traits, which may be regulated by many gene loci on different chromosomes.

Furthermore, in the present study, we found that monoterpenes in the hybrid population exhibited a phenomenon of transgressive inheritance, the F1 progeny had monoterpene content that exceeded that of their parents. This phenomenon demonstrated the existence of genetic variation in the population (Guan et al. 2019), proving the fact of quantitative trait of monoterpenes. In agreement with previous studies, Liu et al. (2016) also found such phenomenon in ‘Beifeng’ × ‘3-34’ interspecific hybrid population (Liu et al. 2015). This transgressive inheritance of monoterpenes will have an impact on the muscat flavor of grape hybrid populations, which has important applications in plant breeding and other fields. This suggests that in the process of hybridization, despite the low level of monoterpenes in the parents, it is possible to produce F1 individuals with excellent monoterpene traits by means of rational parental selection and hybridization strategy (Wang et al. 2022), which is of great significance for genetic improvement in grape breeding and other fields. In conclusion, the inheritance study of grapevine is a complex process, and related studies on intraspecific and interspecific hybrid populations of Muscat varieties still need to be further explored.

Monoterpenes play an important role in contributing to the muscat flavor in grapes. In the present study, the determination of monoterpene content and inheritance study of a hybrid populations of table grapes ‘Italia’ × ‘Tamina’ concluded that geranic acid, linalool and geraniol were the major monoterpene components in Muscat grape varieties. Meanwhile, the segregation of the 4 monoterpenes, cis-Isogeraniol, neral, linalool oxide pyranoside and cis-Rose oxide, in the progeny was in accordance with Mendelian inheritance. Moreover, we found that the monoterpenes all showed a continuous distribution in the hybrid population, indicating that monoterpene is a quantitative trait under polygenic control. The results of correlation analyses showed that most of the monoterpenes showed significant positive correlations with each other, among which α-terpineol showed the highest correlation with 4-terpineol and terpinolen. Individual monoterpenes were significantly higher in the offspring than in the parents, demonstrating the existence of transgressive inheritance in intraspecific hybrid populations. The results provide inheritance insights into the accumulation of monoterpenes in grapes and provide a basis for the selection of high-quality grape varieties and the improvement of grape flavor.

Acknowledgements This work was supported by the BAAFS Funding for the Development of Distinguished Scientist JKZX202402; the Earmarked Fund for Modern Agro-industry Technology Research System CARS-29; Beijing Academy of Agriculture and Forestry Sciences Innovation Capability Construction Special Project (KJCX20230118).

Battilana J, Costantini L, Emanuelli F, Sevini F, Segala C, Moser S, Velasco R, Versini G, Stella Grando M (2009) The 1-deoxy-D: -xylulose 5-phosphate synthase gene co-localizes with a major QTL affecting monoterpene content in grapevine. Theor Appl Genet 118: 653-669
Bosman RN, Lashbrooke JG (2023) Grapevine mono- and sesquiterpenes: Genetics, metabolism, and ecophysiology. Front Plant Sci 14: 1111392
Bosman RN, Vervalle JA, November DL, Burger P, Lashbrooke JG (2023) Grapevine genome analysis demonstrates the role of gene copy number variation in the formation of monoterpenes. Front Plant Sci 14: 1112214
Chen T, Xu T, Wang J, Zhang T, Yang J, Feng L, Song T, Yang J, Wu Y (2024) Transcriptomic and free monoterpene analyses of aroma reveal that isopentenyl diphosphate isomerase inhibits monoterpene biosynthesis in grape (Vitis vinifera L.). BMC Plant Biol 24: 595
Conde C, Silva P, Fontes N, Dias AC, Tavares RM, Sousa MJ, Agasse A, Delrot S, Gerós H (2007) Biochemical changes throughout grape berry development and fruit and wine quality. Food, 11, 1-22
Duchene E, Butterlin G, Dumas V, Merdinoglu D (2012) Towards the adaptation of grapevine varieties to climate change: QTLs and candidate genes for developmental stages. Theor Appl Genet 124: 623-635
Guan L, Fan P, Li S-H, Liang Z, Wu B-H (2019) Inheritance patterns of anthocyanins in berry skin and flesh of the interspecific population derived from teinturier grape. Euphytica 215
Gutierrez-Gamboa G, Garde-Cerdan T, Rubio-Breton P, Perez-Alvarez EP (2021) Effects on must and wine volatile composition after biostimulation with a brown alga to Tempranillo grapevines in two seasons. J Sci Food Agric 101: 525-535
Ji X-h, Wang B-l, Wang X-d, Wang X-l, Liu F-z, Wang H-b (2021) Differences of aroma development and metabolic pathway gene expression between Kyoho and 87-1 grapes. Journal of Integrative Agriculture 20: 1525-1539
Khan N, Fahad S, Naushad M, Faisal S (2020) Grape Production Critical Review in the World. SSRN Electronic Journal
Kok D, Bal E (2014) The Response of Monoterpene Compounds of cv. Gewürztraminer Grape(Vitis vinifera L.) to Various Doses of Prohexadione-Calcium Applied at Different Periods
Lei D, Qiu Z, Qiao J, Zhao GR (2021) Plasticity engineering of plant monoterpene synthases and application for microbial production of monoterpenoids. Biotechnol Biofuels 14: 147
Li C, Chen H, Li Y, Du T, Jia J, Xi Z (2022) The Expression of Aroma Components and Related Genes in Merlot and Marselan Scion-Rootstock Grape and Wine. Foods 11
Lin H, Guo Y, Yang X, Kondo S, Zhao Y, Liu Z, Li K, Guo X (2020) QTL identification and candidate gene identification for monoterpene content in grape (Vitis vinifera L.) berries. Vitis, 591
Liu C, Fan P, He M, Zhang H, Liu X, Luo Z, Ombwara FK, Liang Z, Li S (2015) Inheritance of muscat berry volatiles in grape interspecific cross population. Euphytica 208: 73-89
Liu S, Shan B, Zhou X, Gao W, Liu Y, Zhu B, Sun L (2022) Transcriptome and Metabolomics Integrated Analysis Reveals Terpene Synthesis Genes Controlling Linalool Synthesis in Grape Berries. J Agric Food Chem 70: 9084-9094
Mabou FD, Yossa IB (2021) TERPENES : structural classification and biological activities
Martin D, Grose C, Fedrizzi B, Stuart L, Albright A, McLachlan A (2016) Grape cluster microclimate influences the aroma composition of Sauvignon blanc wine. Food Chem 210: 640-647
Mateo JJ, Jiménez M (2000) Monoterpenes in grape juice and wines. Journal of chromatography. A, 881(1-2), 557-67
Mele MA, Kang HM, Lee YT, Islam MZ (2021) Grape terpenoids: flavor importance, genetic regulation, and future potential. Crit Rev Food Sci Nutr 61: 1429-1447
Sabo J, Farkasova S, Droppa M, Ziarovska J, Kacaniova M (2022) Molecular Fingerprinting and Microbiological Characterisation of Selected Vitis vinifera L. Varieties. Plants (Basel) 11
Sun Q, He L, Sun L, Xu HY, Fu YQ, Sun ZY, Zhu BQ, Duan CQ, Pan QH (2023) Identification of SNP loci and candidate genes genetically controlling norisoprenoids in grape berry based on genome-wide association study. Front Plant Sci 14: 1142139
Wang H, Yan A, Sun L, Zhang G, Wang X, Ren J, Xu H (2020) Novel stable QTLs identification for berry quality traits based on high-density genetic linkage map construction in table grape. BMC Plant Biol 20: 411
Wang W, Feng J, Wei L, Khalil-Ur-Rehman M, Nieuwenhuizen NJ, Yang L, Zheng H, Tao J (2021) Transcriptomics Integrated with Free and Bound Terpenoid Aroma Profiling during "Shine Muscat" (Vitis labrusca x V. vinifera) Grape Berry Development Reveals Coordinate Regulation of MEP Pathway and Terpene Synthase Gene Expression. J Agric Food Chem 69: 1413-1429
Wang W, Khalil-Ur-Rehman M, Wei LL, Nieuwenhuizen NJ, Zheng H, Tao JM (2020) Effect of Thidiazuron on Terpene Volatile Constituents and Terpenoid Biosynthesis Pathway Gene Expression of Shine Muscat (Vitis labrusca x V. vinifera) Grape Berries. Molecules 25
Wang ZL, Yao F, Hui M, Wu D, Wang Y, Han X, Cao X, Li YH, Li H, Wang H (2022) Fertility analysis of intraspecific hybrids in Vitis vinifera and screening of superior hybrid combinations. Front Plant Sci 13: 940540
Wu BH, Yang CX, Liang ZC, Liu W, Wang YJ, Liu CY, Li SH (2012) Inheritance of berry volatile compounds in two half-sib grape (Vitis vinifera) populations. Euphytica 189: 351-364
Wu Y, Duan S, Zhao L, Gao Z, Luo M, Song S, Xu W, Zhang C, Ma C, Wang S (2016) Aroma characterization based on aromatic series analysis in table grapes. Sci Rep 6: 31116
Wu Y, Zhang W, Song S, Xu W, Zhang C, Ma C, Wang L, Wang S (2020) Evolution of volatile compounds during the development of Muscat grape 'Shine Muscat' (Vitis labrusca x V. vinifera). Food Chem 309: 125778
Xie S, Wu G, Ren R, Xie R, Yin H, Chen H, Yang B, Zhang Z, Ge M (2023) Transcriptomic and metabolic analyses reveal differences in monoterpene profiles and the underlying molecular mechanisms in six grape varieties with different flavors. Lwt 174
Yang C, Li Y, He L, Song Y, Zhang P, Liu S (2024) Metabolomic and transcriptomic analyses of monoterpene biosynthesis in Muscat and Neutral grape hybrids. Scientia Horticulturae 336
Yang X, Guo Y, Zhu J, Niu Z, Shi G, Liu Z, Li K, Guo X (2017) Genetic Diversity and Association Study of Aromatics in Grapevine. Journal of the American Society for Horticultural Science 142: 225-231
Yinshan G, Zaozhu N, Kai S, Jia Z, Zhihua R, Yuhui Z, Quan G, Hongyan G, Xiuwu G (2017) Composition and content analysis of sugars and organic acids for 45 grape cultivars from northeast region of China. Pak. J. Bot. 491, 155-160
Yue X, Liu S, Wei S, Fang Y, Zhang Z, Ju Y (2021) Transcriptomic and Metabolic Analyses Provide New Insights into the Effects of Exogenous Sucrose on Monoterpene Synthesis in "Muscat Hamburg" Grapes. J Agric Food Chem 69: 4164-4176
Yue X, Ren R, Ma X, Fang Y, Zhang Z, Ju Y (2020) Dynamic changes in monoterpene accumulation and biosynthesis during grape ripening in three Vitis vinifera L. cultivars. Food Res Int 137: 109736
Zhang Y, Liu C, Liu X, Wang Z, Wang Y, Zhong GY, Li S, Dai Z, Liang Z, Fan P (2023) Basic leucine zipper gene VvbZIP61 is expressed at a quantitative trait locus for high monoterpene content in grape berries. Hortic Res 10: uhad151
Zheng T, Guan L, Yu K, Haider MS, Nasim M, Liu Z, Li T, Zhang K, Jiu S, Jia H et al (2021) Expressional diversity of grapevine 3-Hydroxy-3-methylglutaryl-CoA reductase (VvHMGR) in different grapes genotypes. BMC Plant Biol 21: 279

No competing interests reported.

Download PDF

Reviewers agreed at journal
08 Nov, 2024
Reviewers agreed at journal
04 Nov, 2024
Reviewers invited by journal
27 Sep, 2024
Editor assigned by journal
03 Sep, 2024
Submission checks completed at journal
03 Sep, 2024
First submitted to journal
03 Sep, 2024

You are reading this latest preprint version

The inheritance of monoterpenes in hybrid populations of Vitis vinifera cultivars

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1