Cytological Observations and Bulked-Segregant Analysis Coupled Global Genome Sequencing Reveal Two Genes Associated with Pollen Fertility in Tetraploid Rice

Nabieu Kamara,Xiangdong Liu,Jinwen Wu,Kelvin Dodzi Aloryi,Muhammad Qasim Shahid,Zijun Lu,Yamin Jiao

doi:10.3390/ijms22020841

Nabieu Kamara, Xiangdong Liu + Show 5 more

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https://doi.org/10.3390/ijms22020841

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Abstract

Neo-tetraploid rice with high fertility is a useful germplasm for polyploid rice breeding, which was developed from the crossing of different autotetraploid rice lines. However, little information is available on the molecular mechanism underlying the fertility of neo-tetraploid rice. Here, two contrasting populations of tetraploid rice, including one with high fertility (hereafter referred to as JG) and another with low fertility (hereafter referred to as JD), were generated by crossing Huaduo 3 (H3), a high fertility neo-tetraploid rice that was developed by crossing Jackson-4x with 96025-4x, and Huajingxian74-4x (T452), a low fertility autotetraploid rice parent. Cytological, global genome sequencing-based bulked-segregant (BSA-seq) and CRISPR/Cas9 technology were employed to study the genes associated with pollen fertility in neo-tetraploid rice. The embryo sacs of JG and JD lines were normal; however, pollen fertility was low in JD, which led to scarce fertilization and low seed setting. Cytological observations displayed low pollen fertility (25.1%) and approximately 31.3 and 27.2% chromosome lagging at metaphase I and II, and 28.8 and 24.8% chromosome straggling at anaphase I and II in JD, respectively. BSA-seq of F2–3 generations and RNA-seq of F4 generation detected a common fragment, i.e., 18,915,234–19,500,000, at chromosome 7, which was comprised of 78 genes associated with fertility. Among 78 genes, 9 genes had been known to be involved in meiosis and pollen development. Two mutants ny1 (LOC_Os07g32406) and ny2 (LOC_Os07g32040) were generated by CRISPR/Cas9 knockout in neo-tetraploid rice, and which exhibited low pollen fertility and abnormal chromosome behavior. Our study revealed that two unknown genes, LOC_Os07g32406 (NY1) and LOC_Os07g32040 (NY2) play an important role in pollen development of neo-tetraploid rice and provides a new perspective about the genetic mechanisms of fertility in polyploid rice.

Highlights

Polyploid species are widely found in plants and had higher economic value and confers greater stress resistance than diploid plants [1,2,3]
A high fertility neo-tetraploid rice line, Huaduo3 (H3), which was developed by crossing Jackson-4x with 96025-4x, was used to generate the F1 hybrid by crossing with a low fertility autotetraploid rice line, Huajingxian74-4x (T452) that was developed from a diploid rice cultivar (Huajingxian74-2x)
bulked segregant analysis (BSA)-seq of F2–3 generations, and RNA-seq of F4 detected a common fragment on chromosome No.7, which is comprised of 78 genes associated with fertility

Summary

Introduction

Polyploid species are widely found in plants and had higher economic value and confers greater stress resistance than diploid plants [1,2,3]. Autotetraploid rice was derived from diploid rice by chromosome doubling through colchicine treatment to enrich the breeding germplasm of rice, which showed great biological advantages, stress resistance and high heterosis [6,7,8,9,10]. Two important tetraploid rice germplasms with high seed set and high pollen fertility, named as PMeS and Neo-tetraploid rice, have been developed [9,15]. These two fertile tetraploid germplasms were derived from the progenies of crosses between two intersubspecific autotetraploid rice lines.

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