Pollen parent affects rutin content of seeds of buckwheat (Fagopyrum esculentum)

doi:10.21203/rs.3.rs-4702901/v1

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Pollen parent affects rutin content of seeds of buckwheat (Fagopyrum esculentum)

https://doi.org/10.21203/rs.3.rs-4702901/v1

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Buckwheat (Fagopyrum esculentum) is a heterostylous self-incompatible crop that requires outcrossing for seed formation. Pollen parents influence the traits of seeds in many plants, but their influence in buckwheat is unknown. Here, we crossed self-incompatible (SI) and self-compatible (SC) lines with different rutin contents. The average rutin contents of SI leading cultivars were all 0.15–0.21 mg/g, and a SI high rutin content line that we had developed was 0.6 mg/g, although it has wide range SDs (0.12–0.41). On the other hand, the average rutin contents of SC lines were 0.01–0.06 mg/g, with stable SDs (0.02–0.03). In crosses between high- and low-rutin parents, the average rutin content of F₁ seeds was significantly lower than that of the high-rutin parent and higher than that of the low-rutin parent, indicating that the pollen parent influences the rutin content in seeds of buckwheat. RNA-seq analysis confirmed that alleles of several genes encoding enzymes involved in rutin synthesis derived from pollen parents were expressed during seed formation.

buckwheat

rutin

pollen

xenia

RNA-seq

self-incompatibility

Buckwheat is a pseudocereal crop in the Polygonaceae and is widely grown, notably in Russia, China, France, and Japan (FAOSTAT, 2023). It is used to make bread, noodles, and ethnic foods in combination with wheat, rice, or maize in many countries (Krkosková and Mrázová, 2005). In Japan, buckwheat noodles have been eaten for more than 400 years and are considered a traditional food (Krkosková and Mrázová, 2005; Qu et al., 2013). Known for their health benefits (Krkosková and Mrázová, 2005), buckwheat grains contain abundant starch, vitamins, minerals, an well-balanced amino acid composition, fiber (Huda et al., 2021), rutin (Matsui and Walker, 2020), a flavonoid with antioxidant, anti-inflammatory, anti-diabetic, anti-cancer, and pro-lipid-metabolism effects (Bhatt et al., 2022; Chu et al., 2014; Lee et al., 2016; Qu et al., 2013).

Since rutin is not present in other major crops, new buckwheat lines with a high rutin content are desired. However, it is difficult to develop new lines, because buckwheat is an outcrossing plant on account of heterostylous self-incompatibility (Matsui and Yasui, 2020). Buckwheat has two flower types, pin and thrum: pin flowers have a long style and short stamens, whereas thrum flowers have a short style and long stamens (Darwin, 1897). It is possible to cross only between plants with different flower types, so all resultant seeds are F₁s with high heterozygosity (Matsui and Yasui, 2020).

Self-compatible buckwheat lines have been developed from an interspecific cross between F. esculentum and F. homotropicum (Aii et al., 1998; Campbell, 1995; Matsui et al., 2003; Wang et al., 2005; Woo et al., 1999). We developed the self-compatible line ‘Kyushu PL4’ (Matsui et al., 2008), which has been used as a maternal line to introduce self-compatibility into other lines, such as ‘Kyukei SC7’ (Hara et al., 2020; Takeshima et al., 2021, 2022). A PL4 genome database recently developed by a research group including ourselves (Fawcett et al., 2023) has provided much genetic information.

Flavonoids, including rutin, also known as quercetin-glycoside-rhamnoside, are synthesized via the flavonoid biosynthesis pathway in several sequential steps within the phenylpropanoid biosynthesis pathway (Matsui and Walker, 2020). Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:CoA ligase (4CL) convert phenylalanine into p-coumaroyl-CoA. Chalcone synthase (CHS), chalcone isomerase (CHI), and flavone 3-hydroxylase (F3H) catalyze p-coumaroyl-CoA into dihydrokaempferol. From dihydrokaempferol, flavonoid 3′-hydroxylase (F3′H), flavonoid 3′5′-hydroxylase (F3′5′H), and flavonol synthase (FLS) produce quercetin (Matsui and Walker, 2020; Zhang et al., 2017). The quercetin is then glycosylated by glycosyltransferases (GTRs) including GT (glucosyltransferase) and RT (rhamnosyltransferase) to produce rutin (Matsui and Walker, 2020; Zhang et al., 2017) (Fig. 1).

It is well known that pollen can influence the character of seeds or fruits, a phenomenon called xenia (effect on endosperm and embryos) or metaxenia (effect on surrounding tissues) (Denney, 1992). For example, the pollen parent affects the fruit set, size, and mass of grapes and peonies (Sabir, 2015; Xie et al., 2017); the mass and ripeness of highbush blueberries (Doi et al., 2021); the color of the seed coat of Trifolium alexandrinum (Malaviya et al., 2019); and the contents of chemical components in peonies, almonds, rapeseed, and Siraitia grosvenorii (Kodad et al., 2009; Sánchez-Pérez et al., 2012; Wang et al., 2010; Xie et al., 2017; Yan et al., 2019).

Although buckwheat requires cross-pollination, little is known about the influence of pollen parents. Here, by measuring the rutin contents of F₁ seeds produced by crosses between lines with high and low rutin contents, we clarified that the pollen parent influences the rutin content of F₁ seeds. RNA-seq analysis of maturing F₁ seeds detected several alleles related to rutin synthesis derived from parental lines.

Plant materials

We used five self-incompatible (SI) lines and two self-compatible (SC) lines (Table S1). Four of the SI lines—‘Kitamitsuki’ (KTM), ‘Kitawasesoba’ (KTW), ‘Reranokaori’ (RRN), and ‘Kitayuki’ (KTY)—are leading cultivars in Hokkaido, Japan (Ohsawa, 2020). By recurrent repeated individual selection for rutin content over 12 generations, we bred the SI ‘High Rutin content line No. 8’ (HR8) from ‘Botansoba’, which has high heterogeneity. We bred the SC line ‘Kyushu PL4’ (PL4) from a cross between F. esculentum and F. homotropicum (Matsui et al., 2008; Matsui and Yasui, 2020). We also selected a low-rutin-content SC line (LoR) from an F₄ segregating line produced from a cross between SC ‘Kyukei SC2’ (Matsui et al., 2003) and SC ‘C0408-0RP’, which was bred at Kade Research Ltd. (Hara et al., 2011).

Production of F₁ seeds in a field for measurement of rutin content

Lines were crossed at the Hokkaido Agricultural Research Center (42°88.3′N, 143°05.5′E) in the combinations listed in Table S2. Each cross was performed in a mesh-netted plot of two rows 1 m long and 60 cm between rows, with 10 to 20 plants of one line per row. For SI × SI crosses, pin plants of one line were grown in one row and thrum plants from the other line were grown in the other row. For SI × SC crosses, pin plants of the SI line were planted in one row and plants of the SC line were planted in the other row (Matsui and Yasui, 2020). Plants were pollinated by flies. Seeds were harvested at maturity, dried, and stored in a refrigerator.

Production of F₁ seeds in a glasshouse with hand-pollination for RNA sequencing

To clarify whether genes related to rutin synthesis derived from pollen parents are expressed in F₁ seeds, we crossed HR8 × PL4 and HR8 × HR8. The plants were grown in pots in a glasshouse at the Institute of Crop Science (36°03.0′N, 140°09.9′E). As HR8 is SI, each plant had a different genotype. To detect alleles from the pollen parent, we prepared two HR8 pin plants (HR8-pin-A and HR8-pin-B) as maternal plants; and two HR8 thrum plants (HR8-thrum-C and HR8-thrum-D) and two PL4 long-homostyle plants (PL4-LH-A and PL4-LH-B) as pollen parents. Using different branches, we crossed HR8-pin-A × PL4-LH-A, HR8-pin-A × HR8-thrum-C, HR8-B × HR8-thrum-D, and HR8-B × PL4-LH-B (Fig. S1). Each cross was hand pollinated and then bagged to prevent crosses with other plants. Immature seeds were harvested 10–20 days after crossing and frozen quickly in liquid nitrogen. Four seeds derived from each cross combination were used for RNA extraction.

Measurement of rutin contents by high-performance liquid chromatography (HPLC)

One seed was placed in a 2.0-mL tube with a small bead and crushed (2500 rpm for 60 s) in a Micro Smash MS-100 cell disruptor (Tomy Seiko, Tokyo, Japan). Then 1 mL of 80% ethanol was added, and the samples were incubated at 37°C for 3 h. They were centrifuged at 21 000 ×g for 5 min at 4°C and the supernatant was analyzed for rutin content by HPLC (Ishiguro et al., 2016). The HPLC system consisted of two pumps (LC-20AD), an autoinjector (SIL-20AC), and a column oven (CTO-20AC, all from Shimadzu, Kyoto, Japan). Into a reversed-phase column (3 µm, 150 mm × 2 mm i.d., Cadenza CD-C18, Imtakt Co., Ltd., Kyoto, Japan) at 40°C was injected 2 µL of extract. The mobile phase was composed of phase A (7.5% v/v acetonitrile containing 0.1% v/v trifluoroacetic acid [TFA]) and phase B (50% v/v acetonitrile containing 0.1% v/v TFA). Samples were eluted with a 35% solution of phase B in phase A at 0.3 mL/min for 18 min. Rutin was identified from the retention time and UV-vis spectra of a standard, and was quantified against an external standard on a calibration curve based on detection at 360 nm.

Maternal and pollen effects on rutin content

Five plants were selected from each row (Table S2). The rutin content of each of 15 seeds per plant was measured. Means were compared by Fisher’s Least Significant Difference (LSD) test at P = 0.05, 0.01, or 0.001 in BellCurve for Excel software (Social Survey Research Information Co., Ltd. Tokyo, Japan).

Maternal and pollen effects on the rutin content were calculated based on the method of Wang et al. (2010). The rutin content of F₁ seeds, F₁, was calculated as:

F ₁ = mP_m + (1 – m)P_p

where P_m and P_p are the rutin contents of the maternal and pollen parents, and m is the maternal effect, calculated as m = (F₁ – P_p) / (P_m – P_p), and therefore the pollen parent effect = 1 – m. The mid-parental value (MP) = (P_m + P_p) / 2.

RNA sequencing analysis for identifying alleles derived from pollen parent and calculation of the ratio of alleles derived from pollen parent

We investigated the expression of genes related to rutin synthesis in the phenylpropanoid and flavonoid biosynthesis pathways in maturing seeds of HR8 pin plants × PL4 pollen parent and of HR8 pin plants × HR8 thrum plants to confirm that any allelic differences are not caused by natural variation in HR8.

Maturing seeds were frozen in liquid nitrogen and homogenized with a mortar and pestle. Total RNA was extracted from the seeds with a Maxwell RSC Plant RNA Kit in a Maxwell RSC Instrument (Promega) according to the manufacturer’s protocol. A Next Generation Sequencing library was constructed with a TruSeq stranded mRNA Library Preparation Kit (Illumina), and RNAs were sequenced on a NovaSeq 6000 sequencer to generate 150-bp paired-end reads. Short reads were cleaned in fastp software (Chen et al., 2018), and the first and last 6 bases of each locus were trimmed because the reliability of the ratio of nucleotide in each SNP was low due to low quality of base-call in this region. The cleaned short reads were mapped to the cDNA sequence of PL4 (Fawcett et al., 2023) in Bowtie 2 software (Langmead and Salzberg, 2012). The mapped data were visualized in Integrative Genomics Viewer (IGV) software (Robinson et al., 2011).

Genes encoding enzymes involved in rutin synthesis were selected based on the report by Fawcett et al. (2023). After the RNA-seq data were mapped to the reference sequence, loci of expressed genes were determined and SNPs were searched in IGV. Alleles derived from pollen parents were detected by comparison of the results between HR8 × HR8 and HR8 × PL4.

We also estimated the ratio of expression of alleles derived from pollen parent by calculating the ratio of nucleotides derived from pollen parent on each SNP indicated by IGV to determine the relationship between the ratio of expression of alleles derived from pollen parent and rutin content in seed (Fig. S2).

Rutin contents among materials

The average rutin contents of KTM, KTW, KTY and RRN were all 0.15–0.21 mg/g, and that of HR8 was 0.6 mg/g. Because these are all SI and thus heterozygosity is high, SD had a wide range (0.12–0.41; Table 1). On the other hand, the average rutin contents of SC lines PL4 and LoR were both 0.01–0.06 mg/g, with stable SDs (0.02–0.03; Table 1). From these results, we classified these lines into three groups by rutin content—low (< 0.1 mg/g), medium (0.1–0.5 mg/g), and high (> 0.5 mg/g)—and investigated the effects of the pollen parent with crosses among these groups (Table S1).

Table 1

Parental lines and seed rutin contents.
Parent	Average rutin content (mg/g)	SD
HR8	0.60	0.41
PL4	0.06	0.03
LoR	0.01	0.02
KTM	0.18	0.09
KTW	0.21	0.13
RRN	0.15	0.12
KTY	0.17	0.14
Flower type and self-compatibility of these lines are shown in Table S2.

Effect of pollen parent on rutin content

Because only HR8 had a high rutin content, we used this line in all crossing combinations. When HR8 as the maternal parent was crossed with KTM, KTW, KTY, and RRN as the pollen parents, the rutin contents of the seeds (0.31–0.40 mg/g; Table 2) were significantly lower than that of HR8 (0.60 mg/g; Table 1), indicating that the rutin content was influenced by the pollen parent (Figs. 2a–d).

Table 2

Parental effects on the rutin contents of F₁ seeds.
Cross combination by rutin contents	Cross combination of lines	Cross No. ¹⁾	Mid-parental value	Rutin content (mg/g)	SD	Significance ²⁾		Maternal effect value	Pollen parent effect value
Cross combination by rutin contents	Cross combination of lines	Cross No. ¹⁾	Mid-parental value	Rutin content (mg/g)		P_m vs. F₁	P_p vs. F₁	Maternal effect value	Pollen parent effect value
High (♀) × medium or low (♂)	HR8 × KTM	a1	0.39	0.38	0.26	***	***	0.49	0.51
	HR8 × KTW	b1	0.40	0.36	0.36	***	**	0.40	0.60
	HR8 × RRN	c1	0.37	0.40	0.26	***	***	0.56	0.44
	HR8 × KTY	d1	0.38	0.31	0.26	***	**	0.32	0.68
	HR8 × PL4	e	0.33	0.44	0.28	**	***	0.71	0.29
	HR8 × LoR	f	0.30	0.45	0.27	**	***	0.74	0.26
	average	–	–	–	–	–	–	0.54	0.46
Medium (♀) × high (♂)	KTM × HR8	a2	0.39	0.32	0.28	**	***	0.65	0.35
	KTW × HR8	b2	0.40	0.22	0.19	0.87	***	0.98	0.02
	RRN × HR8	c2	0.37	0.21	0.16	0.16	***	0.86	0.14
	KTY × HR8	d2	0.38	0.28	0.20	*	***	0.75	0.25
	average	–	–	–	–	–	–	0.81	0.19
Flower type and self-compatibility of these lines are shown in Table S2.
1) Cross No. with the same letter indicates that they were grown and crossed in the same plot.
2) Means were compared by LSD test. P₁, maternal parent; P₂, pollen parent. Significant differences at 0.05, 0.01, and **0.001 probability levels.

Similarly, when KTM and KTY as the maternal parents were crossed with HR8 as the pollen parent, the rutin contents of the seeds (0.32, 0.28 mg/g; Table 2) were significantly higher than that of each maternal parent (0.18, 0.17 mg/g; Figs. 2a, d; Table 1). On the other hand, when KTW and RRN as the maternal parents were crossed with HR8 as the pollen parent, the rutin content of the seeds (0.22, 0.21 mg/g; Table 2) was only marginally higher than that of the seed parents (0.21, 0.15 mg/g; Figs. 2b, c; Table 1).

In the crosses between HR8 and PL4 or LoR, the rutin contents of the seeds (0.44 and 0.45 mg/g) were significantly lower than those of HR8 (0.60 mg/g; Table 2) and higher than those of PL4 or LoR (0.06, 0.01 mg/g; Figs. 2e, f; Table 1), indicating that the rutin content was influenced by the pollen parent.

Identification of expressed genes derived from pollen parents

Genes encoding enzymes in the rutin biosynthesis pathway were expressed in maturing seeds (Table 3), indicating the synthesis of rutin during seed development. Approximately 35% of involved genes were expressed (Table 3). Allele sequences derived from the pollen parent PL4 were recognized at 14 loci (Fig. 3; Table 3).

Table 3

Gene families encoding enzymes involved in the rutin biosynthesis pathway and the number of genes expressed during seed formation.
Enzyme	Number of loci
Enzyme	Detected in reference	Expressed	Pollen parent alleles detected
Phenylalanine ammonia-lyase (PAL)	4	4	3
Cinnamate-4-hydroxylase (C4H)	6	2	1
4-Coumarate CoA ligase (4CL)	2	2	1
Chalcone synthase (CHS)	13	5	3
Chalcone isomerase (CHI)	3	1	1
Flavanone-3-hydroxylase (F3H)	2	2	1
Flavonoid-3′-hydroxylase (F3′H)	2	2	1
Flavonoid-3′-5′-hydroxylase (F3′5′H)	16	3	3
Flavonol synthase (FLS)	2	1	0
Glycosyltransferase (GTR)¹⁾	12	(1)	(1)
Total ¹⁾	62	22	14
1) Gene for GTR was expressed in only one plant. The total does not include GTR.

Paternal and maternal effects on the rutin content of F₁ hybrid seeds

Pollen parents have a direct genetic influence on F₁ seeds in some plants (Denney, 1992; Kanade et al., 2024). They influence sugar content and enzymes for secondary metabolites in palm fruit (Shahsavar and Shahhosseini, 2022) and yield and anthocyanin content in highbush blueberry (Doi et al., 2021). However, to our knowledge, there are no reports in buckwheat.

We clarified the effects of the pollen parent on the rutin content of F₁ seeds through the use of the SC lines PL4 and LoR and of the high-rutin-content line HR8. As most buckwheat cultivars are SI, it is difficult to develop lines in which rutin content is fixed, especially at a high level, because of the absence of genetic information. Using only common cultivars, it would be difficult to determine whether rutin content is influenced by the pollen parent or simply varies within a line. The low rutin contents of PL4 and LoR are fixed. So when they were crossed with HR8, the effect of the pollen parent was clearly detected (Fig. 2e, f).

HR8 was developed by long-term recurrent selection for high rutin content and has a significantly higher content than the other SI lines, although variation is still large. Significant differences in the rutin content between the F₁ seeds and the maternal parent lines were found when HR8 as the maternal parent was crossed with the other SI lines as the pollen parents and when KTM and KTY as the maternal parents were crossed with HR8, but not when KTW and RRN as the maternal parents were crossed with HR8 as the pollen parent. The lack of significant differences in the rutin content between F₁ seeds and the maternal parents in some cross combination would be probably caused by the broad range of both lines. HR8 can help clarify the effect of the pollen parent on the rutin content of seeds, but the development of better-fixed lines would be more useful.

In crosses between medium-rutin-content lines KTM, KTW, KTY, and RRN as the maternal parents and HR8 as the pollen parent, the effect value of the pollen parent on rutin content averaged 0.19 (range, 0.02–0.35; Table 2), lower than that of the opposite crosses including crosses between HR8 and PL4 or LoR (0.46; range, 0.26–0.68). The reason for the difference is not evident, and the development of an SC line with high rutin content would help.

Detection of alleles derived from pollen parents and relationship between the ratio of alleles and rutin contents

If rutin contents are influenced by pollen parents, alleles related to rutin synthesis of the pollen parent should be expressed during seed development. We detected the expression of pollen parent alleles in F₁ seeds for several genes encoding enzymes involved in rutin synthesis. Approximately 60% of the alleles derived from the pollen parents were recognized in the expressed loci (Table 3). Approximately 40% of the loci, the ratio of pollen alleles was 20–40% (Table 4), comparable to the influence of the pollen parent on the rutin content of PL4 × HR8 (0.29; Table 2). However, some genes lay outside this range, in which the lowest influence of pollen parent on genes was 9.6% and the highest was 74.2% (Table 4). Buckwheat seeds are composed of a diploid embryo, a triploid endosperm, and a diploid testa derived from the maternal parent, with different rutin contents, which are highest in the embryo (Suzuki et al., 2002). The expression levels of genes involved in rutin synthesis seem to depend on maturity stages (Penin et al., 2021). Thus, a larger embryo size and a larger proportion of embryo in the seed would mean a higher rutin content. The expression of genes related to rutin synthesis may increase as the embryo grows larger, but the embryo’s size in the seed may be regulated by a number of genes. Further study will be needed to clarify the relationships between the ratio of each allele and rutin content.

Table 4

SNPs detected in expressed genes related to flavonoid biosynthesis and the ratio of alleles derived from pollen parent.
Enzyme	Locus	Total number of nucleotides	HR8-pin-A × PL4-LH-A				HR8-pin-B × PL4-LH-B
			Number of SNPs		Ratio of pollen parent allele		Number of SNPs		Ratio of pollen parent allele
			Pattern I¹⁾	Pattern II¹⁾	Average	± SE	Pattern I¹⁾	Pattern II¹⁾	Average	± SE
PAL	FesPL4_r1.1_Chr3.g195460.1	2173	22	3	30.3	1.94	15	10	16.9	4.02
	FesPL4_r1.1_Chr4.g269240.1	2110	20	18	22.5	9.80	43	5	14.6	5.60
	FesPL4_r1.1_Chr8.g155630.1	2113	4	12	44.4	6.63	8	7	47.1	6.55
C4H	FesPL4_sc0109.1.g001280.1	1516	39	5	20.7	2.05	22	7	26.7	5.11
4CL	FesPL4_r1.1_Chr4.g271010.1	1642	13	1	38.5	-	35	2	18.0	2.00
CHS	FesPL4_r1.1_Chr4.g217000.1	1183	3	7	68.0	3.15	3	7	66.1	5.61
	FesPL4_r1.1_Chr7.g094080.1	1180	5	0	-	-	4	1	74.2	0.00
	FesPL4_r1.1_Chr7.g094660.1	1180	5	0	-	-	4	1	9.6	0.00
CHI	FesPL4_r1.1_Chr3.g000530.1	772	11	0	-	-	10	0	-	-
F3H	FesPL4_r1.1_Chr5.g258370.1	1105	10	3	21.4	0.91	13	3	20.0	2.52
F3′H	FesPL4_r1.1_Chr8.g248260.1	1588	9	16	54.9	8.55	8	16	53.9	7.66
F3′5′H	FesPL4_r1.1_Chr4.g265140.1	1834	13	1	36.4	-	15	0	-	-
	FesPL4_r1.1_Chr4.g265180.1	1876	31	1	11.8	-	18	0	-	-
	FesPL4_r1.1_Chr4.g265190.1	1576	5	0	-	-	5	0	-	-
1) Two different SNP patterns are recognized because HR8 is SI and keeps heterozygosity in many loci (Fig. S2).

We confirmed that the rutin content of buckwheat is influenced by the pollen parent in both combinations of crossing a high-rutin line with low- or medium-rutin lines. The pollen parent alleles of several genes encoding enzymes involved in rutin synthesis were expressed. Since the pollen parent affects multiple traits in many species, it is likely that the pollen parent also affects traits other than rutin content in buckwheat. As buckwheat requires outcrossing, this result could be important knowledge for breeding and growing buckwheat.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by NARO and partly supported by the Toyo Suisan Foundation.

Author Contribution

SO and KM conceived and designed the experiments. SO and KM crossed the plants. SO, TH, and KI measured rutin by HPLC. TH and KIM developed plant materials. SO, YY, and KM performed RNA sequencing analysis. SO and KM wrote the manuscript. All authors edited and approved the final manuscript.

Acknowledgement

We thank Kiku Fujii and Mayumi Hayashida for technical assistance. We also thank Tomohiro Takakura and Kazaha Nagashima for growing the plants.

Data Availability

The datasets generated during this study are available from the corresponding author on reasonable request. The raw RNA-Seq data were submitted to the DDBJ Sequence Read Archive (https://www.ddbj.nig.ac.jp/dra/index-e.html) under accession numbers DRA018730, DRA018757, DRA018758, and DRA018772.

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No competing interests reported.

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Pollen parent affects rutin content of seeds of buckwheat (Fagopyrum esculentum)

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Plant materials

Production of F₁ seeds in a field for measurement of rutin content

Production of F₁ seeds in a glasshouse with hand-pollination for RNA sequencing

Measurement of rutin contents by high-performance liquid chromatography (HPLC)

Maternal and pollen effects on rutin content

Results

Rutin contents among materials

Effect of pollen parent on rutin content

Identification of expressed genes derived from pollen parents

Discussion

Paternal and maternal effects on the rutin content of F₁ hybrid seeds

Conclusion

Declarations

Competing interests

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

Pollen parent affects rutin content of seeds of buckwheat (Fagopyrum esculentum)

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Plant materials

Production of F1 seeds in a field for measurement of rutin content

Production of F1 seeds in a glasshouse with hand-pollination for RNA sequencing

Measurement of rutin contents by high-performance liquid chromatography (HPLC)

Maternal and pollen effects on rutin content

Results

Rutin contents among materials

Effect of pollen parent on rutin content

Identification of expressed genes derived from pollen parents

Discussion

Paternal and maternal effects on the rutin content of F1 hybrid seeds

Conclusion

Declarations

Competing interests

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

Production of F₁ seeds in a field for measurement of rutin content

Production of F₁ seeds in a glasshouse with hand-pollination for RNA sequencing

Paternal and maternal effects on the rutin content of F₁ hybrid seeds