Agronomic Trait and Gene Analysis of Diangu 1839, a Newly Bred Fragrant and Soft Rice Variety Derived from Diantun 502 in Yunnan, China

Abstract

The fragrant and soft rice variety Diantun 502 has been a major cultivar for high-quality rice production in Yunnan Province since its approval in 1993. Over 30 years of widespread cultivation, several issues affecting this variety—including high susceptibility to rice blast, reduced yield, and declining quality—have been noted. To address these limitations, this study aimed to improve and upgrade Diantun 502. During the breeding process, parent lines with soft rice traits were selected, and molecular marker-assisted selection was employed using the soft rice gene Wx^hp and the fragrance gene badh2. Priority was given to grain quality while also considering agronomic performance. Natural induction tests were conducted in rice blast-prone regions. After 9 years of selection and evaluation, the new variety—Diangu 1839—was successfully developed. Comparative analysis of agronomic and economic traits, as well as gene loci between Diantun 502 and the improved variety Diangu 1839, revealed that the observed trait changes in Diangu 1839 corresponded with the presence of the targeted gene loci. This study involved a comparative analysis of agronomic and economic traits as well as key gene loci between Diantun 502 and the improved variety Diangu 1839. The results showed that, compared to Diantun 502, Diangu 1839 achieved a 14.1% increase in yield and a significant 25.6% reduction in chalky grain rate. Importantly, the retention of badh2 and Wx^hp genes ensured the preservation of excellent eating quality, while rice blast resistance was enhanced. Although Diangu 1839 demonstrated significant improvements in yield, rice quality, and disease resistance, its resistance to brown planthopper remains limited due to the lack of corresponding resistance genes. The technical system and breeding experience established in this study provide important technical references for the genetic improvement of high-quality, fragrant, and soft rice varieties.

1. Introduction

Rice is one of the most important staple crops, serving as the primary dietary staple for more than half of the global population [1]. It is extensively cultivated in several countries and accounts for nearly one-fifth of the world’s cereal crop production [2]. As a whole-grain cereal, rice with a strong fragrance and superior quality is typically favored by consumers and holds high commercial value [3]. With a long history of cultivation, numerous classic high-quality rice varieties have gained recognition in rice-producing countries and regions worldwide. Notable examples include jasmine rice (KDML105) from Thailand, basmati from India, Koshihikari from Japan, and Pawsan from Myanmar. Although the development of rice varieties with grain quality traits favored by consumers has emerged as a major focus of breeding programs worldwide, with some new varieties even having been developed for specific consumer preferences, improving classic high-quality rice varieties remains challenging. Developing a new generation of rice varieties that simultaneously enhance both quality and yield is a global challenge.

The jasmine rice variety KDML105 is Thailand’s most commercially valuable rice due to its strong fragrance and superior soft texture, making it a popular choice among consumers in Southeast Asia, Europe, and the United States [4,5,6]. However, it also presents notable limitations, including strong photoperiod sensitivity, extended growth duration, low yield, tall plant height prone to lodging, and weak drought tolerance, which restrict its large-scale cultivation [7,8,9,10]. Consequently, developing improved rice varieties based on KDML105 has emerged as a pressing national challenge for Thailand [11]. Considerable effort has been invested into improving KDML105, including gamma irradiation to induce mutations [12], molecular marker-assisted selection technology to breed new varieties [13,14], ion-beam-induced screening of rice blast resistance mutants [2], and trait-specific improvements such as reduced plant height and lodging resistance, resistance to brown planthopper [6], rice blast resistance [2], photoperiod insensitivity [11], and drought tolerance [15]. Although these studies have yielded numerous new varieties with improved yield and resistance, none have surpassed KDML105 in terms of its aroma and softness qualities [16].

Yunnan Province, located on the southwestern plateau of China, features elevations ranging from 72 m to over 6000 m and is characterized by a diverse ecological environment and rich rice paddy ecosystems. Yunnan possesses unique genetic resources for soft rice. The cooked grains of soft rice varieties from the region are smooth and pleasant in texture, maintaining their softness even upon cooling, rendering them ideal for direct consumption without reheating. As early as 1971, Yunnan Agricultural University began exploring and utilizing rare soft rice genetic resources. Through the process of hybridization between the traditional soft rice landrace Haomuxi and the indica variety IR64 from the International Rice Research Institute, researchers conducted selection based on grain appearance, iodine staining for low amylose content, and sensory evaluation of cooked rice to develop several soft indica rice varieties such as Dianlong 201 and Dianrui 408 [17,18]. In 1982, the aromatic soft rice variety Diantun 502 was produced through conventional hybridization using the aromatic glutinous rice variety Dianqiao 20 as the female parent and the old soft rice variety Haopi as the male parent, which generated a large pool of soft rice germplasm [19]. From the perspective of rice genetic improvement and breeding in China, these soft rice lines have served as foundational resources for developing high-quality rice varieties in Yunnan and across the country [20].

Since the 1980s, a series of soft rice varieties has been selectively bred and incorporated into agricultural production, thereby contributing to the growth of the local market for premium-quality rice. Among the numerous varieties used in the cultivation of fragrant, soft rice in Yunnan, Diantun 502 has consistently been highly popular for several years, garnering national recognition as a premium fragrant and soft rice variety. Its strong fragrance and soft texture have contributed to its popularity in the market. However, Diantun 502 exhibits notable deficiencies, including low yield, high chalkiness, poor grain appearance, high susceptibility to rice blast, and vulnerability to brown planthopper [21]. Numerous efforts have therefore been made to improve Diantun 502. According to the China Rice Data Center (https://www.ricedata.cn/variety/index.htm (accessed on 19 August 2025)), a total of 12 new varieties derived from Diantun 502 were approved between 1993 and 2021. These varieties showed improved yield, appearance, or rice blast resistance, but none fully surpassed Diantun 502 in terms of its fragrance and soft eating quality.

Due to the lack of a variety with superior overall performance, Diantun 502 has remained in widespread use for an extended period, leading to significant varietal degradation, severe susceptibility to rice blast, and marked decline in yield [21]. There is an urgent need for new varieties that can effectively function as substitutes in production. To improve Diantun 502, we analyzed its fragrance and soft texture traits. It has been verified that its fragrance is controlled by the fragrance gene badh2 and that this trait is associated with fewer grains per panicle, lower seed setting rate, and lower yield [22]. The soft rice trait is influenced by the low-amylose gene Wx^hp. Specific molecular marker technologies for both fragrance and soft rice traits were developed to support the improvement and upgrading of Diantun 502. Consequently, a new variety with improved yield and resistance, strong fragrance, and desirable soft rice texture was bred: Diangu 1839. This study details the process of improving Diantun 502 using molecular marker-assisted selection integrated with conventional breeding and quality evaluation and also analyzes the superior traits and gene loci of the newly bred variety Diangu 1839. The primary objectives of this study are to promote and introduce Diangu 1839, and to present a breeding strategy that combines field-based yield and resistance evaluation with quality-related gene identification for selection. The findings provide theoretical insights for enhancing the quality of fragrant and soft rice varieties.

2. Materials and Methods

2.1. Experimental Materials

Three indica rice varieties were used: Diantun 502, Yunhui 290, and Diangu 1839. All materials were provided by the Rice Research Institute of Yunnan Agricultural University. Diantun 502 is a famous Yunnan aromatic soft rice variety that carries the flavor gene badh2 and the low-amylose gene Wx^hp. Yunhui 290 is a soft rice variety in Yunnan that carries the low-amylose gene Wx^hp. Diangu 1839 is a newly developed fragrant, soft rice variety, bred through conventional crossing using Diantun 502 as the female parent and Yunhui 290 as the male parent. It carries both the fragrance gene badh2 and the low-amylose gene Wx^hp.

2.2. Crossing, Selection, and Breeding Methods

Diangu 1839 was developed through conventional crossing using Diantun 502 as the female parent and Yunhui 290 as the male parent. A pedigree selection method based on conventional hybridization was adopted. During the selection process, marker-assisted selection was employed using molecular markers associated with the fragrance gene badh2 and the low-amylose content gene Wx^hp.

2.3. Breeding Population Construction and Planting

Development of the breeding population: In 2012, at the greenhouse of the Rice Research Institute of Yunnan Agricultural University in Kunming, Diantun 502 was crossed with Yunhui 290 using conventional hybridization to produce the F₁ generation.

Rapid propagation of early generations: In 2013, the F₁ and F₂ generations were planted in a double-cropping indica rice area in Yuanyang County, Yunnan Province (elevation 300 m, average annual temperature 24.3 °C), enabling two generations per year and thereby facilitating rapid propagation.

Single-panicle selection in middle generations: From 2014 to 2016, F₃ to F₅ generations were planted over three consecutive years in high-quality indica rice areas in Mile County (elevation 1150 m, annual average temperature 19.4 °C) and Guangnan County (elevation 1300 m, annual average temperature 17.4 °C). Single-panicle selection was conducted based on phenotyping and genotyping for the badh2 and Wx^hp gene loci.

Line selection in advanced generations: From 2017 to 2018, F₆ and F₇ generations were planted for two consecutive years in Menghai County (elevation 1150 m, annual average temperature 18.4 °C), another high-quality indica rice region. Selection criteria included phenotypic performance, genotyping for badh2 and Wx^hp, and analyses of appearance and eating quality, agronomic and economic traits, and grain quality.

2.4. Trait Assessment Methods

2.4.1. Investigation of Major Agronomic Traits

From 2019 to 2021, ecological adaptability evaluations were conducted concurrently as part of regional and production trials for indica rice varieties of Yunnan Province. The trials were carried out in five different ecological zones: Menghai County, Yuanyang County, Guangnan County, Longchuan County (elevation 900 m, average annual temperature 18.9 °C), and Xundian County (elevation 1500 m, average annual temperature 16.5 °C). A randomized block design was used, with each plot covering 33.35 m². Comprehensive evaluations of phenotype, yield, disease resistance, and quality were conducted to assess the ecological adaptability of the variety.

Major agronomic and economic traits were examined, including full growth period (days), plant height (cm), panicle length (cm), number of effective panicles (panicles/plant), total grain number per panicle (grains/panicle), seed setting rate (%), and yield (t/ha).

2.4.2. Field Survey of Disease Resistance

The field survey focused on major rice diseases, including leaf blast, neck blast, bacterial leaf blight, sheath blight, and false smut. During the growth period, disease occurrence in the field was recorded: the absence of lesions or diseased plants was marked as “no disease occurrence,” whereas the presence of lesions or diseased plants was marked as “disease occurrence”.

2.4.3. Identification of Disease Resistance

Disease resistance identification was conducted at the Yunnan Crop Variety Resistance Identification Station, following the crop variety approval standards of the National Crop Variety Approval Committee and the rice blast resistance identification protocol for rice regional trials (NY/T 2646-2014). Both field nursery and greenhouse artificial inoculation methods were used.

2.4.4. Molecular Marker-Assisted Selection

For the identification of the aroma gene badh2 locus, a PCR-based molecular marker method was used, following the method of Bradbury et al. [23]; for the identification of the low-amylose gene Wx^hp locus, the method of Liu et al. [24] was followed.

2.4.5. Analysis of Rice Milling and Cooking Flavor Quality

Rice quality was assessed according to the method specified in NY/T 593-2013: Quality of Edible Rice Varieties. For cooking quality evaluation, 150 g of polished rice was weighed and placed in a multifunctional automatic rice cooker (MB-YN161,Midea Group Co., Ltd., Foshan, Guangdong, China). Water was added at a rice-to-water mass ratio of 1:1.2, and the rice was soaked for 20 min before cooking. After cooking, it was allowed to remain warm for 30 min before being taste-tested by eight panelists. Scoring criteria included the appearance of polished rice (20 points), cooked rice texture and color (20 points), eating quality (30 points), and retrogradation of cold rice (30 points), with a total score representing the variety’s cooking and eating quality.

2.4.6. RVA Profile Analysis of Rice Flour

The rapid viscosity analyzer (RVA) profile was measured using a Model 3-D Rapid Visco Analyzer (Newport Scientific Pty Ltd., Warriewood, NSW, Australia). A 3 g sample was mixed with 25 mL of distilled water, and its pasting properties were measured. The measurement protocol included a 2.5 min holding period. The initial stirring speed was set to 960 r/min for the first 10 s, followed by a constant 160 r/min, with readings taken at 4 s intervals. The procedure followed the guidelines of the American Association of Cereal Chemists (AACC) 1995-61-02 and RACI standard methods. Data were processed using the TWC3 software (1.2.0) package and included peak viscosity (PKV), hot paste viscosity (HPV), breakdown value (BDV = PKV − HPV), final viscosity (CPV), setback value (SBV = CPV − PKV), consistency value (CSV = CPV − HPV), pasting time, and pasting temperature. Viscosity values are expressed in centipoise (cp).

2.4.7. Determination of 2-AP Content

The 2-acetyl-1-pyrroline (2-AP) content was determined by following the method described by Grimm et al. [25] with appropriate modifications. Briefly, 1 g of rice sample was weighed into a 4 mL headspace vial and preheated in an 80 °C water bath for 30 min. After preheating, an SPME syringe was inserted into the vial, the fiber head was adjusted and fixed in position, and volatile compounds were adsorbed for 20 min. The fiber was then quickly inserted into the gas chromatograph injection port for desorption for 5 min, after which the SPME was removed for use with the next sample. An HP-5ms (30 m × 0.25 mm × 0.25 μm) capillary column (Agilent Technologies, Santa Clara, CA, USA) was used, with helium as the carrier gas, in splitless injection mode at a flow rate of 1.2 mL/min. The heating conditions were as follows: initial temperature was set at 40 °C and maintained for 4 min, then increased at a rate of 5 °C/min until reaching 150 °C and maintained for 1 min. Mass spectrometry conditions were as follows: electron impact (EI) ion source with an electron energy of 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, mass scan range (mz) 33–550, and solvent delay time of 4 min.

2.4.8. Gene Loci Determination

Healthy rice plants were selected, and fresh young leaves were collected, quickly frozen in liquid nitrogen, and stored at −80 °C for later use. The samples were used for functional gene locus identification and haplotype analysis, which were outsourced to Shuanglvyuan Chuangxin Technology Research Institute Co., Ltd., Wuhan, Hubei, China. Gene locus detection was performed using Illumina SNP chip technology with the SLYm1R chip (containing 31,753 SNP markers) for genome-wide scanning. The chip was designed based on the rice reference genome MSU7.0. SNP marker filtering criteria included GenTrain Score > 0.6, missing rate < 5%, minor allele frequency > 20%, and exclusion of Indel variations. Functional annotation of detected SNP variations was performed based on the rice functional genomics map established by Wei et al. [26]. Disease resistance gene cluster analysis was conducted using a 100 kb window SNP marker aggregation method to analyze the presence of brown planthopper resistance, blast resistance, and bacterial blight resistance genes.

2.5. Statistical Analysis

Data processing was performed using Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA) and SPSS 20.0 software (IBM Corporation, Armonk, NY, USA), and figures were generated using Origin 2021 (OriginLab Corporation, Northampton, MA, USA) and GraphPad Prism 8.02 software (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Selection and Breeding Process of the New Aromatic Soft Rice Variety Diangu 1839

Through the combination of phenotypic selection and molecular marker-assisted technology, the excellent aromatic soft rice variety Diangu 1839 was successfully developed. The breeding process is shown in Figure 1A, where conventional hybridization was used to obtain F1 hybrids, followed by generation advancement to obtain F2 populations. In the F3 to F5 generations, significant trait segregation was observed, with panicle traits, grain shape, and transparency showing obvious genetic variation, making this stage a critical period for phenotypic selection. Molecular genotyping analysis confirmed that selected individual plants carried both the aroma gene badh2 and the low-amylose gene Wx^hp. From the F6 and F7 generations onward, the agronomic traits gradually stabilized, and selected lines showed good consistency in growth period, plant architecture, and yield performance. Based on the comprehensive evaluation of appearance quality, eating quality, and molecular marker detection results, stable lines with excellent comprehensive traits were selected. Multi-location trials in the F8 and F9 generations showed that selected lines possessed moderate plant and leaf type, excellent agronomic traits, high yield levels, strong disease resistance, and superior appearance and cooking eating quality. This line participated in the Yunnan Province indica rice regional trials from 2020 to 2021 and was approved by Yunnan Province in 2022.

3.2. Changes in Major Agronomic Traits and Related Gene Loci

Based on a 2-year trial conducted in five indica rice-growing regions at altitudes of 300–1500 m (Guangnan, Longchuan, Menghai, Yuanyang, and Xundian) (

Table 1. Changes in mainly agronomic and economic traits of aromatic soft indica rice variety Diangu 1839.

Traits	Diangu 1839	Diantun 502	t-Test	p-Value
Growth duration (day)	161.4 ± 13.4	147.1 ± 15.6	2.1986	0.0412
Plant height (cm)	111.3 ± 7.5	98.4 ± 9.8	3.2753	0.0042
Panicle length (cm)	26.5 ± 2.0	26.6 ± 2.8	0.1767	0.8617
Effective panicles (panicles/plant)	10.9 ± 1.4	9.3 ± 1.4	2.4871	0.0229
Grains per panicle (grains/panicle)	149.5 ± 15.6	121.4 ± 12.8	4.3884	0.0004
Seed setting rate (%)	80.6 ± 6.6	82.9 ± 7.4	0.7118	0.4857
Yield (t/ha)	8.9 ± 1.0	7.8 ± 1.4	2.1055	0.0496

Note: t-test refers to a statistical method used to determine whether there is a significant difference between the mean values of two groups. p-value represents the significance probability, measuring the likelihood of the observed difference occurring by chance. Typically, a p-value < 0.05 indicates a statistically significant difference.

), the newly developed aromatic soft rice variety Diangu 1839 exhibited superior performance compared to Diantun 502. Diangu 1839 had a growth period of 161.1 days, extended by 14.3 days; plant height of 111.3 cm, increased by 12.7 cm; effective panicle number per plant of 10.9, increased by 1.6; grain number per panicle of 149.5, increased by 28.1; and yield of 8.9 t/ha, increased by 1.1 t/ha. These improvements were statistically significant or highly significant. Although the panicle length and seed setting rate decreased, the differences were not statistically significant.

The detection of 16 functional gene loci related to growth period, plant type, yield, and stress resistance (

Table 2. Changes in functional gene loci between aromatic soft indica rice varieties Diangu 1839 and Diantun 502.

Serial Number	Gene	Type	Reference Variety	Phenotype	Diangu 1839	Diantun 502
1	Gn1a	Yield	Habataki,9311	Increased grains per panicle	√	√
2	OsSPL16	Yield	HJX74	High yield	√	√
3	SKC1	Resistance to abiotic stress	Nona Bokra	Salt tolerance	√
4	qUVR-10	Resistance to abiotic stress	Kasalath	Reduced photorepair activity	√	√
5	TOND1	Resistance to abiotic stress	Teqing	Nitrogen deficiency tolerance	N	N
6	NRT1.1B	Resistance to abiotic stress	9311	Enhanced nitrogen uptake	√
7	Rymv1	Resistance to biotic stress	Nipponbare	Resistance to yellow mottle virus disease	√	√
8	STV11	Resistance to biotic stress	Kasalath	Durable resistance to rice stripe virus		√
9	OsAAP6	Quality	9311	High protein	√	√
10	GW2	Quality	WY3, Oochikara	Large grain
11	GS3	Quality	Minghui 63	Long grain	√	√
12	OsCYP704A3	Quality	IR 24	Low chalkiness	√	√
13	Chalk5	Quality	H94	Increased amylopectin content in non-glutinous rice	√
14	Wxhp	Quality	Nipponbare	Low amylose content	√	√
15	ALK	Quality	Minghui 63	Increased medium- and long-chain amylopectin content	√	√
16	badh2	Quality	Suyunuo	Fragrance	√	√
17	Hd1	Growth period	Tadukan, Kasalath, Ma Sho	Photoperiod insensitivity, promoting rice heading
18	Hd16	Growth period	Koshihikari	Delayed flowering stage	N	N
19	Hd17	Growth period	Koshihikari	Delayed flowering	√	√
20	Sd-1	Plant type	DGWG-type	Semi-dwarf plant	√	√
21	Sdt97	Plant type	Y98149	Semi-dwarf mutant		√
22	qNGR9	Plant type	O. rufipogon	Upright panicle	√	√
23	TAC1	Plant type	IR24	Increased tiller angle	√
24	sh4	Other	Nipponbare	Non-shattering	√	√

Note: √ indicates the presence of the gene in the variety; N indicates no signal detected.

) showed that both Diangu 1839 and Diantun 502 carried genes for yield (Gn1a and OsSPL16), semi-dwarfism (Sd-1), moderate seed shattering (sh4), and stress resistance (qUVR-10). However, Diangu 1839 additionally carried genes for high nitrogen uptake efficiency (NRT1.1B), salt tolerance (SKC1), and increased tiller angle (TAC1). The presence of these gene loci indicates that the new variety Diangu 1839 has improved performance in terms of plant type, yield, and stress resistance. The function of the nitrogen efficient uptake gene NRT1.1B improves soil nutrient use efficiency and increases yields [27].

3.3. Changes in Rice Quality Traits and Gene Loci

The results of the rice quality test showed that the appearance, milling, physicochemical, and cooking taste qualities of Diangu 1839 were improved (

Table 3. Changes in rice quality of aromatic soft indica rice varieties Diangu 1839 and Diantun 502.

Characteristics	Diangu 1839	Diantun 502	t-Test	p-Value
Brown rice rate (%)	80.9 ± 0.2	77.6 ± 1.1	4.8439	0.0083
Milled rice rate (%)	70.8 ± 5.5	67.9 ± 1.3	1.9267	0.1263
Amylose content (%)	13.9 ± 0.4	13.0 ± 0.2	2.1019	0.1034
Alkali spreading value (grade)	4.4 ± 0.3	4.7 ± 1.0	0.4126	0.7011
Gel consistency (mm)	76.0 ± 4.0	70.6 ± 1.3	4.0000	0.0161
Grain length (mm)	7.7 ± 0.0	7.2 ± 0.0	10.607	0.0004
Length-to-width ratio	3.2 ± 0.0	3.1 ± 0.0	0.7071	0.5185
Transparency (grade)	2.0	2.0	---	---
Chalkiness degree (%)	0.7 ± 0.1	7.2 ± 0.1	26.429	0.0000
Chalky grain rate (%)	3.7 ± 0.3	29.3 ± 1.1	37.654	0.0000
2-AP (ng/g)	16.1 ± 0.9	16.7 ± 0.7	0.8690	0.4339
Cooking and eating quality score	90.9 ± 1.6	85.3 ± 3.6	6.9794	0.0000

Note: t-test refers to a statistical method used to determine whether there is a significant difference between the mean values of two groups. p-value represents the significance probability, measuring the likelihood of the observed difference occurring by chance. Typically, a p-value < 0.05 indicates a statistically significant difference.

). Compared with the classic aromatic soft high-quality variety Diantun 502, Diangu 1839 showed improvements in appearance quality traits: grain length (7.7 mm) and length/width ratio (3.2) increased, whereas chalkiness degree (0.7%) and chalky grain rate (3.7%) decreased significantly; milling quality also improved, with an increased brown rice rate (80.9%) and milled rice rate (70.8%); and the physicochemical quality showed a significant increase in gel consistency (76 mm). The cooked rice taste score was 90.9 points, indicating improved soft rice quality. It also consistently exhibited traits such as level 2 grain transparency, low amylose content (13.9%), and aroma compound 2-AP content (16.1 ng/g).

The testing of eight functional gene loci related to quality (Table 2) showed that both Diangu 1839 and Diantun 502 carry genes for aroma (badh2), long-grain shape (GS3 and OsCYP704A3), and increased intermediate- and long-chain amylopectin (ALK). Additionally, Diangu 1839 carried the low chalkiness gene Chalk5. The presence of multiple quality-related genes in the new variety supports its superior qualities.

RVA characteristic values measured with a 3D rapid viscosity tester indicated that, compared with Diantun 502, Diangu 1839 showed significantly increased gelatinization temperature, peak viscosity, and breakdown value, whereas the setback value decreased significantly (Figure 2). Studies have reported that rice varieties with superior eating quality generally have breakdown values above 100 RVU and setback values below 25 RVU, often even negative [28,29]. Diangu 1839 showed a breakdown value of 2524 RVU and a setback value of −1678 RVU, supporting its superior rice eating quality. This finding is consistent with the sensory evaluation of cooked rice taste, demonstrating that Diangu 1839 possesses exceptional characteristics in terms of softness and eating quality.

3.4. Changes in Disease Resistance Levels and Related Gene Loci

From the results of regional trials and resistance characterization, the level of rice blast resistance of Diangu 1839 was improved (

Table 4. Disease resistance characteristics of Diangu 1839 and Diantun 502 rice varieties against major rice diseases.

Variety	Rice Blast					Bacterial Leaf Blight		Sheath Blight
	Blast Loss Rate	Comprehensive Resistance Index	Disease Grade	Resistance Category		Disease Grade	Resistance Category	Disease Grade	Resistance Category
Diangu 1839	5	5.07		7	S	3	R	3	R
Diantun 502	9	8.3		9	HS	3	R	5	MR

Note: R = Resistant, MR = Moderately resistant, S = Susceptible, HS = Highly susceptible.

). Over 2 consecutive years of planting trials in five different indica rice areas, mild occurrences of blast disease, and bacterial leaf blight in Diangu 1839 were observed only at the Longchuan test site. In contrast, Diantun 502 showed severe blast disease outbreaks at the Guangnan, Longchuan, and Xundian test sites. Field and greenhouse artificial inoculation resistance evaluations at the Yunnan Crop Variety Resistance Identification Station indicated that Diangu 1839 had a blast disease severity rating of 7 (susceptible), sheath blight rating of 3 (resistant), and bacterial leaf blight rating of 3 (resistant). In comparison, Diantun 502 showed a blast disease severity rating of 9 (highly susceptible), sheath blight rating of 5 (moderately resistant), and bacterial leaf blight rating of 3 (resistant). As shown in Figure 3, under the same planting conditions, Diangu 1839 (left) exhibited better growth performance, while Diantun 502 (right) showed more severe rice blast disease occurrence, posing potential risks that could affect yield.

The detection of 21 resistance gene loci related to blast, bacterial leaf blight, and rice planthopper (

Table 5. Changes in disease resistance loci of aromatic soft indica rice varieties Diangu 1839 and Diantun 502.

Serial Number	Gene	Variant Site	Phenotype	Diangu 1839	Diantun 502
1	Bph14	29SNP	Brown planthopper resistance	0.48	0.48
2	Bph15	56SNP	Brown planthopper resistance	0.16	0.16
3	Bph18	30SNP	Brown planthopper resistance	0.72	0.72
4	Bph26	24SNP	Brown planthopper resistance	0.52	0.52
5	Bph6	26SNP	Brown planthopper resistance	0.62	0.38
6	Bph9	26SNP	Brown planthopper resistance	0.44	0.44
7	Pi1	10SNP	Blast resistance	0.3	0.3
8	Pi2	99SNP	Blast resistance	0.66	0.66
9	Pi5	33SNP	Blast resistance	1	0.79
10	Pi9	80SNP	Blast resistance	0.6	0.6
11	Pia	13SNP	Blast resistance	1	0.77
12	Pid2	66SNP	Blast resistance	1	0.85
13	Pid3	64SNP	Blast resistance	1	1
14	Pigm	90SNP	Blast resistance	0.58	0.58
15	pikh	34SNP	Blast resistance	0.53	0.53
16	Pita	32SNP	Blast resistance	0.16	0.12
17	xa13	61SNP	Bacterial leaf blight resistance	0.08	0.08
18	Xa21	12SNP	Bacterial leaf blight resistance	1	0.83
19	Xa23	40SNP	Bacterial leaf blight resistance	0.07	0.1
20	xa5	48SNP	Bacterial leaf blight resistance	0.35	0.33
21	Xa7	18SNP	Bacterial leaf blight resistance	0.17	0.17

Note: Haploid analysis: Probe types of “haplotype” belong to this type of analysis. Based on the genotype published in this article and specific phenotype validation data, a specific set of polymorphic markers within the gene and the upstream and downstream 100 kb regions were selected to determine whether the sample contains a certain resistance gene. The “number + SNP” in the table refers to the number of polymorphic markers corresponding to each gene, and the decimal below the sample number represents the similarity between the genotype of each sample in this set of polymorphic markers and the standard sample.

) revealed that Diantun 502 carried only the blast resistance gene pid3, whereas Diangu 1839 carried three additional blast resistance genes pi5, pia, and pid2, as well as the bacterial leaf blight resistance gene Xa21. Neither variety carried rice planthopper resistance genes. This aligns with practical field observations, indicating that Diantun 502 is seriously affected by blast and planthopper damage, whereas Diangu 1839 shows some level of resistance to blast but remains vulnerable to planthopper damage.

4. Discussion

4.1. Selection of Parental Soft Rice Traits and Their Early Accurate Identification

The aromatic soft rice variety Diantun 502 is a famous high-quality specialty rice from Yunnan, characterized by long and thick grains, semi-translucent endosperm (grade 2), a soft and elastic cooked rice texture, and a strong aroma. However, it exhibits the notable drawback of high chalkiness, which adversely affects its appearance, quality, and consumer acceptance [30], thereby influencing market preference. Soon after its development, breeding efforts prioritized improving this defect by selecting for low chalkiness and enhanced grain transparency. Prior to 2014, nine derived lines were developed with level 1 endosperm transparency and low chalkiness, resulting in improved appearance quality. However, these lines generally exhibited higher amylose content and lacked the desirable soft texture or strong aroma traits (Figure 4). Therefore, in the early phase of improvement, we clearly identified the key genes controlling soft rice and aroma traits in Diantun 502 as Wx^hp and badh2 [22], and established corresponding molecular marker-assisted selection techniques for accurate identification of these traits. Both the female parent Diantun 502 and the male parent Yunhui 290 are classic soft rice varieties carrying the soft rice gene, increasing the probability of selecting progeny with soft rice traits. During the F₃ to F₅ generations, single-panicle identification was conducted in segregating populations, and molecular markers for low-amylose Wx^hp and fragrance badh2 loci were used, combined with phenotypic screening of semi-translucent endosperm, low chalkiness, and grain size, ensuring that selected panicles retained aroma and soft rice characteristics. Quality trait and gene loci analyses confirmed that the new variety Diangu 1839 retained the excellent characteristics of Diantun 502, including soft texture, aroma, and long grain while additionally carrying genes for nitrogen-efficient uptake (NRT1.1B), salt tolerance (SKC1), and low chalkiness (Chalk5), thereby improving yield and appearance quality. Regarding the functional genes not detected in the parents, we believe this may be attributed to the technical characteristics of functional gene detection [26]: functional gene identification is based on haplotype patterns of multiple SNP marker loci, and materials are only considered positive when their genotypes at these key loci highly match known functional alleles. The sequences of these functional genes may have already existed in the parents, but the genotype combinations at key marker loci were incomplete. Through hybridization between parents and multiple generations of genetic recombination, functional loci fragments originally dispersed in different parents were assembled into the same haplotype through chromosomal exchange and genetic recombination, enabling the formation of complete functional gene haplotypes that could be detected and identified [31]. This phenomenon reflects the important role of gene complementation effects and epistatic interactions in hybrid breeding, and also explains why Diangu 1839 exhibited transgressive segregation in multiple traits.

4.2. Naturally Induced Stress by Field Diseases to Select for Resistant Plants

The high-quality variety Diantun 502 is highly susceptible to rice blast and leaf blight. According to evaluations of the Yunnan Provincial Crop Variety Resistance Identification Station, its resistance levels were rated grade 9 for blast and grade 3 for bacterial leaf blight. In the absence of disease control, the variety had a 100% incidence of rice blast in the field, reducing yields by 41.9% [21]. Almost every strain of the blast pathogen infected this variety in inoculation tests [32]. Therefore, increasing the level of disease resistance is also an important objective in the improvement of the fragrant, soft variety Diantun 502. Yunnan has the richest rice germplasm resources and the most diverse rice ecotypes in China. Guangnan is a high-quality rice production area, yet it is also recognized as a hotspot for blast and bacterial leaf blight. We planted segregating F₃ to F₅ populations in this high-disease-pressure area without disease control, applying natural infection stress screening over several years to effectively eliminate susceptible lines. The disease resistance evaluation results showed that the new variety Diangu 1839 exhibited a rice blast resistance rating of 7 and a bacterial blight resistance rating of 5. This improvement may be attributed to the additional incorporation of three rice blast resistance genes (Pi5, Pia, and Pid2) and one bacterial blight resistance gene (Xa21), compared to its female parent Diantun 502 (Figure 5). Although Diangu 1839’s rice blast resistance remains at a susceptible level, it represents a significant improvement compared to the highly susceptible rating of 9 observed in the parent Diantun 502 (Figure 3). In rice disease resistance breeding practice, the improvement from highly susceptible to susceptible levels is equally valuable. This enhanced resistance can effectively reduce disease severity, decrease pesticide application, and lower production costs. As a material that aggregates multiple resistance genes, Diangu 1839 could also serve as an intermediate material for fragrant rice resistance improvement in the future. However, neither this variety nor Diantun 502 carried brown planthopper resistance genes; therefore, enhanced management of brown planthopper control remains necessary in production applications.

4.3. Yield Trait Selection Prioritizing Grain Quality

From global rice breeding practices, many studies have reported a trade-off between yield and quality [33]. Our study also found that aroma traits correlate with fewer spikelets per panicle, a lower seed setting rate, and lower yield [22]. Therefore, during field selection, we chose both lines with excellent agronomic traits and lines with moderate traits. Subsequent indoor quality testing prioritized quality traits while also considering agronomic traits, ensuring that the improved variety Diangu 1839 combined excellent soft rice eating quality and aroma with acceptable yield traits (Figure 5). Agronomic traits such as plant height, spikelet number, effective panicles, and yield are readily observable and, as a result, were prioritized in the selection process. Quality traits, including softness, starch properties, and aroma, require instrumental detection and were selected later. Due to the trade-off between agronomic and quality traits, field selection during the improvement of high-quality rice often prioritizes agronomic performance, resulting in the loss of important quality characteristics. Data from the China Rice Data Center show that, from 1993 to 2024, 15 improved varieties derived from Diantun 502 were approved (Figure 4). Although these varieties improved yield and resistance levels, it is noteworthy that only Diangu 1839 surpassed Diantun 502 in terms of soft rice eating quality. In the process of improving fragrant and soft rice varieties, it is crucial to prioritize the selection of quality traits and take into account the selection of economic traits. This strategy is essential for improving and enhancing high-quality-characteristic varieties.

5. Conclusions

Analysis of the agronomic, economic, and quality traits of the improved and newly bred variety Diangu 1839 showed that it retained the desirable aromatic soft rice characteristics of Diantun 502, while analysis of loci further revealed that it carries the soft rice gene Wx^hp and the flavor gene badh2. Its resistance to rice blast was improved through the incorporation of the resistance genes Pi5, Pia, Pid2, and Pid3. Improvements in appearance and yield may be attributed to the presence of Chalk5, the nitrogen efficient uptake gene NRT1.1B, and the salt tolerance gene SKC1. Overall, the new variety Diangu 1839 matched the classic soft fragrant variety Diantun 502 in terms of aroma and soft rice taste quality, while surpassing it in terms of appearance, rice blast resistance, and yield-related traits (Figure 5). The fragrant, soft variety Diangu 1839 has been adopted for production and demonstration purposes, representing a successful model for enhancing and upgrading fragrant, soft varieties.