Rice blast, caused by the Magnaporthe oryzae, is the most detrimental disease to rice. Yield losses caused by this disease were from 10% to 30% in rice planting areas (Skamnioti and Gurr, 2009); severe cases may even lead to complete cessation of production (Parker et al., 2008). Cloning rice blast resistant genes and applying them to cultivate resistant varieties is a practical and effective method for controlling rice blast disease. So far, more than 70 disease resistance genes and QTLs have been identified, of which at least 25 have been cloned and used in disease resistance breeding (Luo et al., 2017). The Pi2 gene encodes a protein with a nucleotide-binding site and leucine-rich repeat (LRR) domain (Zhou et al., 2006), and is one of the most broad-spectrum and efficient resistance genes to rice blast, exhibiting resistance to 36 out of 43 rice blast strains from 13 countries (Liu et al., 2002). However, many elite varieties do not carry Pi2 gene, which seriously hinders the widespread and sustainable promotion of these varieties.

Marker-assisted selection (MAS) is one of the important technologies in modern breeding. It uses molecular markers to quickly detect individual plants carrying target genes, greatly improving breeding efficiency and saving breeding costs. Previously, Jiang et al. (2015) developed blast-resistant thermosensitive genic male sterile (TGMS) lines by employing MAS. High-density whole genome single nucleotide polymorphism (SNP) chips are also a technology that accelerates the breeding process. Therefore, combining MAS and SNP chips can make breeding improvement faster and more accurately. For example, Guo et al. (2024) used MAS and Green Super Rice 40K (GSR40K) detection to screen improved strains with high genetic similarity to the receptor in BC₁F₂. In this study, we successfully generated an improved rice germplasm with blast-resistant by integrating MAS and GSR40K detection technology.

F₁ plants obtained from the cross of Zhonghui261 (ZH261; a good quality restoring line of Huazheyou 261 [HZY261], which was selected as a demonstration and promotion variety of super rice in 2024) and Yuenongsimiao (YNSM; carrying Pi2), were backcross with ZH261 to generate BC₁F₁. Among 1025 BC₁F₁ plants, we selected 70 strains that similar to ZH261 and 26 individual plants were identified to carry Pi2 (Heterozygous) using the marker Pi2-CM1. Then, the single plant out of 26 with 89.72% of genetic background reversion was selected by detecting with GSR40K. The single plant was backcrossed again with ZH261 to generate 964 BC₂F₁ plants. We selected 10 strains that are similar to ZH261 and three individual plants were identified to carry Pi2 (Heterozygous) using the marker Pi2-CM1. The single plant out of three with 97.86% of genetic background reversion was selected to selfing and generate BC₂F₂. The single plant named ZH261-Pi2 was selected from BC₂F₂ by employing MAS and detecting with GSR40K (Figure 1a). ZH261-Pi2 exhibited 97.81% genetic similarity to ZH261 and 99.97% purity (Figure 1b). Based on 48 simple sequence repeats (SSR) markers followed by the protocol NY/T 1433-2014, only one pair of SSR marker (RM176) was different between ZH261 and ZH261-Pi2 (Figure 1c), indicating that ZH261 and ZH261-Pi2 were the same variety. Finally, we hybridised with Huazhe2A using ZH261 and ZH261-Pi2, respectively, to produce HZY261 and HZY261-Pi2, and evaluated their rice blast resistance, yield and quality traits (Figure 1a).

We found that ZH261-Pi2 and HZY261-Pi2 significantly increased resistance to blast compared to ZH261 and HZY261 (Figure 1d–g). ZH261-Pi2 showed a similar grain numbers per panicle, but higher tiller numbers per plant, 1000 grain weight and grain yield per plant than that of ZH261 (Figure 1h–k). On the other hand, HZY261-Pi2 displayed lower 1000 grain weight, but higher grain numbers per panicle, tiller numbers per plant and grain yield per plant compared with HZY261 (Figure 1h–k). Together, our results indicate that introduction of Pi2 in ZH261 by integrating MAS and GSR40K detection can simultaneously enhance rice blast resistance and yield.

Amylose content is the most critical parameter determining rice eating and cooking quality (Wang et al., 2024). ZH261-Pi2 showed a similar length–width ratio, alkali spreading value and amylose content, but higher gel consistency than that of ZH261 (Figure 1l–o). HZY261-Pi2 had higher length–width ratio, but lower amylose content and gel consistency than that of HZY261 (Figure 1l–n). As for alkali spreading value, no significant difference was observed between HZY261-Pi2 and HZY261 (Figure 1o).

Balancing the yield, quality and resistance to disease is a daunting challenge in crop breeding due to the negative relationship among these traits (Xiao et al., 2021). However, it is feasible to improve the rice blast resistance, yield and quality of existing rice varieties through molecular design breeding (Mao et al., 2021). Our work provides a molecular design strategy to rapidly improve rice blast resistance, yield and quality by integrating MAS and GSR40K detection. In particular, the improved varieties can be directly applied to production, providing an important guarantee for food security.

由稻瘟病菌引起的稻瘟病是对水稻最有害的疾病。在水稻种植区，这种疾病造成的产量损失为 10% 至 30%（Skamnioti 和 Gurr，2009 年）;严重的情况下甚至可能导致完全停止生产（Parker et al.， 2008）。克隆稻瘟病抗性基因并应用于培养抗性品种是防治稻瘟病的一种实用有效的方法。到目前为止，已经鉴定出 70 多个抗病基因和 QTL，其中至少 25 个已被克隆并用于抗病育种（Luo et al.， 2017）。Pi2 基因编码具有核苷酸结合位点和富含亮氨酸重复序列 （LRR） 结构域的蛋白质（周 et al.， 2006），是对稻瘟病最广谱和最有效的抗性基因之一，对来自 13 个国家的 43 种稻瘟病菌株中的 36 种表现出抗性（Liu et al.， 2002）。然而，许多优良品种并不携带 Pi2 基因，严重阻碍了这些品种的广泛和可持续推广。

标记辅助选择 （MAS） 是现代育种中的重要技术之一。它使用分子标记快速检测携带目标基因的个体植物，大大提高了育种效率，节省了育种成本。以前，江 et al.（2015） 通过使用 MAS 开发了抗爆炸的热敏基因雄性无菌 （TGMS） 线。高密度全基因组单核苷酸多态性 （SNP） 芯片也是一种加速育种过程的技术。因此，结合 MAS 和 SNP 芯片可以使育种改良更快、更准确。例如，Guo 等 人。（2024） 使用 MAS 和 Green Super Rice 40K （GSR40K） 检测筛选与 BC₁F₂ 受体具有高遗传相似性的改良菌株。在这项研究中，我们通过整合 MAS 和 GSR40K 检测技术，成功生成了具有抗稻瘟病能力的改良水稻种质。

中会261（ZH261，华哲优261[HZY261]的优质恢复系，2024年被选为超级稻示范推广品种）和月农思苗（YNSM，携带Pi2）杂交获得的F₁植株与ZH261回交，生成BC₁F₁。在 1025 株 BC₁F₁ 植物中，我们选择了 70 株与 ZH261 相似的菌株，并使用标记 Pi2-CM1 鉴定了 26 株携带 Pi2 （杂合子） 的单株植物。然后，通过 GSR40K 检测从 26 株中筛选出 89.72% 遗传背景回归的单株。单株植物再次与 ZH261 回交，生成 964 株 BC₂F₁ 株植物。我们选择了 10 个与 ZH261 相似的菌株，并使用标记 Pi2-CM1 鉴定了 3 个携带 Pi2 （杂合子） 的单个植物。选择具有 97.86% 遗传背景回归的 3 株植物中的一株进行自交并产生 BC₂F₂。通过使用 MAS 并使用 GSR40K 检测，从 BC₂F₂ 中选择名为 ZH261-Pi2 的单一植物（图 1a）。ZH261-Pi2 与 ZH261 的遗传相似性为 97.81%，纯度为 99.97% （图 1b）。基于方案 NY/T 1433-2014 遵循的 48 个简单序列重复 （SSR） 标记，ZH261 和 ZH261-Pi2 之间只有一对 SSR 标记 （RM176） 不同（图 1c），表明 ZH261 和 ZH261-Pi2 是同一品种。最后，我们分别使用 ZH261 和 ZH261-Pi2 与华浙 2A 杂交，生产 HZY261 和 HZY261-Pi2，并评价它们的抗稻瘟病性、产量和品质性状（图 1a）。

我们发现，与 ZH261 和 HZY261 相比，ZH261-Pi2 和 HZY261-Pi2 显着提高了抗爆炸性（图 1d-g）。ZH261-Pi2 显示出每穗的粒数相似，但与 ZH261 相比，每株植物的分蘖数、1000 粒重和每株产量更高（图 1h-k）。另一方面，与 HZY261 相比，HZY261-Pi2 的 1000 粒重较低，但每穗粒数、每株分蘖数和每株产量较高（图 1h-k）。总之，我们的结果表明，通过整合 MAS 和 GSR40K 检测在 ZH261 中引入 Pi2 可以同时提高稻瘟病抗性和产量。

直链淀粉含量是决定大米食用和蒸煮品质的最关键参数（Wang et al.， 2024）。ZH261-Pi2 显示出相似的长宽比、碱铺展值和直链淀粉含量，但凝胶稠度高于 ZH261（图 1l-o）。HZY261-Pi2 具有较高的长宽比，但比 HZY261 低直链淀粉含量和凝胶稠度（图 1l-n）。至于碱扩散值，HZY261-Pi2 和 HZY261 之间没有观察到显着差异（图 1o）。

由于这些性状之间的负相关关系，平衡产量、质量和抗病性是作物育种中一项艰巨的挑战（Xiao et al.， 2021）。然而，通过分子设计育种提高现有水稻品种的抗稻瘟病性、产量和品质是可行的（毛等 人，2021）。我们的工作提供了一种分子设计策略，通过整合 MAS 和 GSR40K 检测来快速提高稻瘟病抗性、产量和品质。特别是改良品种可直接应用于生产，为粮食安全提供了重要保障。